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Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions., When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface. IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae. In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.Ticks are important vectors of infectious emerging diseases and tick-borne phleboviruses represent a growing threat to humans globally. We employed here a high-resolution, label-free mass spectrometry and RNA interference screen approach to reveal the host cell protein GBF1 as a proviral factor, not only for tick-borne phleboviruses, but also for many other important zoonotic RNA viruses. This study lays the basis for the development of innovative antiviral strategies against a broad range of human pathogenic viruses.

PurposeSix-19% of critically ill COVID-19 patients display circulating auto-antibodies against type I interferons (IFN-AABs). Here, we establish a clinically applicable strategy for early identification of IFN-AAB-positive patients for potential subsequent clinical interventions.MethodsWe analysed sera of 430 COVID-19 patients with severe and critical disease from four hospitals for presence of IFN-AABs by ELISA. Binding specificity and neutralizing activity were evaluated via competition assay and virus-infection-based neutralization assay. We defined clinical parameters associated with IFN-AAB positivity. In a subgroup of critically ill patients, we analyzed effects of therapeutic plasma exchange (TPE) on the levels of IFN-AABs, SARS-CoV-2 antibodies and clinical outcome.ResultsThe prevalence of neutralizing AABs to IFN-α and IFN-ω in COVID-19 patients was 4.2% (18/430), while being undetectable in an uninfected control cohort. Neutralizing IFN-AABs were detectable exclusively in critically affected, predominantly male (83%) patients (7.6% IFN-α and 4.6% IFN-ω in 207 patients with critical COVID-19). IFN-AABs were present early post-symptom onset and at the peak of disease. Fever and oxygen requirement at hospital admission co-presented with neutralizing IFN-AAB positivity. IFN-AABs were associated with higher mortality (92.3% versus 19.1 % in patients without IFN-AABs). TPE reduced levels of IFN-AABs in three of five patients and may increase survival of IFN-AAB-positive patients compared to those not undergoing TPE.ConclusionIFN-AABs may serve as early biomarker for development of severe COVID-19. We propose to implement routine screening of hospitalized COVID-19 patients according to our algorithm for rapid identification of patients with IFN-AABs who most likely benefit from specific therapies.

Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.

Autoantibodies against IFN-α and IFN-ω (type I IFNs) were recently reported as causative for severe COVID-19 in the general population. Autoantibodies against IFN-α and IFN-ω are present in almost all patients with autoimmune polyendocrine syndrome type 1 (APS-1) caused by biallelic deleterious or heterozygous dominant mutations in AIRE. We therefore hypothesized that autoantibodies against type I IFNs also predispose patients with APS-1 to severe COVID-19. We prospectively studied 6 patients with APS-1 between April 1, 2020 and April 1, 2021. Biobanked pre–COVID-19 sera of APS-1 subjects were tested for neutralizing autoantibodies against IFN-α and IFN-ω. The ability of the patients’ sera to block recombinant human IFN-α and IFN-ω was assessed by assays quantifying phosphorylation of signal transducer and activator of transcription 1 (STAT1) as well as infection-based IFN-neutralization assays. We describe 4 patients with APS-1 and preexisting high titers of neutralizing autoantibodies against IFN-α and IFN-ω who contracted SARS-CoV-2, yet developed only mild symptoms of COVID-19. None of the patients developed dyspnea, oxygen requirement, or high temperature. All infected patients with APS-1 were females and younger than 26 years of age. Clinical penetrance of neutralizing autoantibodies against type I IFNs for severe COVID-19 is not complete.

Cytosolic DNA detection via the DNA sensor cGAS initiates a major cell-intrinsic response to infection and malignancies. In this issue of Immunity, Zhou et al. (2020) report that the catalytic product of cGAS, cGAMP, can alert bystander cells over large distances through its cell-to-cell transmission via volume-regulated anion channels.

In COVID-19, immune responses are key in determining disease severity. However, cellular mechanisms at the onset of inflammatory lung injury in SARS-CoV-2 infection, particularly involving endothelial cells, remain ill-defined. Using Syrian hamsters as model for moderate COVID-19, we conducted a detailed longitudinal analysis of systemic and pulmonary cellular responses, and corroborated it with datasets from COVID-19 patients. Monocyte-derived macrophages in lungs exerted the earliest and strongest transcriptional response to infection, including induction of pro-inflammatory genes, while epithelial cells showed weak activation. Without evidence for productive infection, endothelial cells reacted, depending on cell subtypes, by strong and early expression of anti-viral, pro-inflammatory, and T cell recruiting genes. Recruitment of cytotoxic T cells as well as emergence of IgM antibodies preceded viral clearance at day 5 post infection. Investigating SARS-CoV-2 infected Syrian hamsters can thus identify cell type-specific effector functions, provide detailed insights into pathomechanisms of COVID-19, and inform therapeutic strategies.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

Summary Background The COVID-19 agent, SARS-CoV-2, is conspecific with SARS-CoV, the causal agent of the severe acute respiratory syndrome epidemic in 2002–03. Although the viruses share a completely homologous repertoire of proteins and use the same cellular entry receptor, their transmission efficiencies and pathogenetic traits differ. We aimed to compare interferon antagonism by SARS-CoV and SARS-CoV-2. Methods For this functional study, we infected Vero E6 and Calu-3 cells with strains of SARS-CoV and SARS-CoV-2. We studied differences in cell line-specific replication (Vero E6 vs Calu-3 cells) and analysed these differences in relation to TMPRSS2-dependent cell entry based on inhibition with the drug camostat mesilate. We evaluated viral sensitivity towards type I interferon treatment and assessed cytokine induction and type I interferon signalling in the host cells by RT-PCR and analysis of transcription factor activation and nuclear translocation. Based on reverse genetic engineering of SARS-CoV, we investigated the contribution of open reading frame 6 (ORF6) to the observed phenotypic differences in interferon signalling, because ORF6 encodes an interferon signalling antagonist. We did a luciferase-based interferon-stimulated response element promotor activation assay to evaluate the antagonistic capacity of SARS-CoV-2 wild-type ORF6 constructs and three mutants (Gln51Glu, Gln56Glu, or both) that represent amino acid substitutions between SARS-CoV and SARS-CoV-2 protein 6 in the carboxy-terminal domain. Findings Overall, replication was higher for SARS-CoV in Vero E6 cells and for SARS-CoV-2 in Calu-3 cells. SARS-CoV-2 was reliant on TMPRSS2, found only in Calu-3 cells, for more efficient entry. SARS-CoV-2 was more sensitive to interferon treatment, less efficient in suppressing cytokine induction via IRF3 nuclear translocation, and permissive of a higher level of induction of interferon-stimulated genes MX1 and ISG56. SARS-CoV-2 ORF6 expressed in the context of a fully replicating SARS-CoV backbone suppressed MX1 gene induction, but this suppression was less efficient than that by SARS-CoV ORF6. Mutagenesis showed that charged amino acids in residues 51 and 56 shift the phenotype towards more efficient interferon antagonism, as seen in SARS-CoV. Interpretation SARS-CoV-2 ORF6 interferes less efficiently with human interferon induction and interferon signalling than SARS-CoV ORF6. Because of the homology of the genes, onward selection for fitness could involve functional optimisation of interferon antagonism. Charged amino acids at positions 51 and 56 in ORF6 should be monitored for potential adaptive changes. Funding Bundesministerium fur Bildung und Forschung, EU RECOVER project.

In COVID-19, the immune response largely determines disease severity and is key to therapeutic strategies. Cellular mechanisms contributing to inflammatory lung injury and tissue repair in SARS-CoV-2 infection, particularly endothelial cell involvement, remain ill-defined. We performed detailed spatiotemporal analyses of cellular and molecular processes in SARS-CoV-2 infected Syrian hamsters. Comparison of hamster single-cell sequencing and proteomics with data sets from COVID-19 patients demonstrated inter-species concordance of cellular and molecular host-pathogen interactions. In depth vascular and pulmonary compartment analyses (i) supported the hypothesis that monocyte-derived macrophages dominate inflammation, (ii) revealed endothelial inflammation status and T-cell attraction, and (iii) showed that CD4+ and CD8+ cytotoxic T-cell responses precede viral elimination. Using the Syrian hamster model of self-limited moderate COVID-19, we defined the specific roles of endothelial and epithelial cells, among other myeloid and non-myeloid lung cell subtypes, for determining the disease course.

Interferon-induced transmembrane (IFITM) proteins restrict infection by enveloped viruses through interfering with membrane fusion and virion internalisation. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies remain largely unexplored. Here, we characterized the impact of IFITM proteins and variants on entry and spread of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in human cells, and provide first evidence for a CHIKV-mediated antagonism of IFITM proteins. IFITM1, 2 and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and during cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in the loss of its antiviral activity. rs12252-C, a naturally occurring variant ofIFITM3that has been proposed to associate with severe influenza in humans, restricted CHIKV, MAYV and influenza A virus infection as efficiently as wild-typeIFITM3. Finally, all antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several non-structural protein(s) of CHIKV.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) restrict numerous viral pathogens and are thought to prevent infection by severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudoparticle infection of cells artificially overexpressing IFITMs. Here, we confirmed that overexpression of IFITMs blocks pseudoparticle infections mediated by the Spike proteins of β-coronaviruses including pandemic SARS-CoV-2. In striking contrast, however, endogenous IFITM expression promoted genuine SARS-CoV-2 infection in human lung cells both in the presence and absence of interferon. IFITM2 was most critical for efficient entry of SARS-CoV-2 and enhanced virus production from Calu-3 cells by several orders of magnitude. IFITMs are expressed and further induced by interferons in the lung representing the primary site of SARS-CoV-2 infection as well as in other relevant tissues. Our finding that IFITMs enhance SARS-CoV-2 infection under conditions approximating the in vivo situation shows that they may promote viral invasion during COVID-19. HIGHLIGHTS Overexpression of IFITM1, 2 and 3 restricts SARS-CoV-2 infection Endogenous IFITM1, 2 and 3 boost SARS-CoV-2 infection of human lung cells IFITM2 is critical for efficient entry of SARS-CoV-2 in Calu-3 cells

In recent years, (re-)emerging arboviruses including Chikungunya virus (CHIKV) and Mayaro virus (MAYV) have caused growing concern due to expansion of insect vector ranges. No protective vaccine or specific antiviral strategies are currently available. Long-term morbidity after CHIKV infection includes debilitating chronic joint pain, which has associated health and economic impact. Here, we analyzed the early cell-intrinsic response to CHIKV and MAYV infection in primary human synovial fibroblasts. This interferon-competent cell type represents a potential source of polyarthralgia induced by CHIKV infection. Synovial fibroblasts from healthy and osteoarthritic donors were similarly permissive to CHIKV and MAYV infectionex vivo. Using RNA-seq, we defined a CHIKV infection-induced transcriptional profile with several hundred interferon-stimulated and arthralgia-mediating genes upregulated. Type I interferon was both secreted by infected fibroblasts and protective when administered exogenously. IL-6 secretion, which mediates chronic synovitis, however, was not boosted by infection. Single-cell RNA-seq and flow cytometric analyses uncovered an inverse correlation of activation of innate immunity and productive infection at the level of individual cells. In summary, primary human synovial fibroblasts serve as bona-fideex vivoprimary cell model of CHIKV infection and provide a valuable platform for studies of joint tissue-associated aspects of CHIKV immunopathogenesis.

Shock-and-kill is one of the conceptually most advanced strategy towards establishment of an HIV-1 cure. Treatment with latency-reversing agents (LRAs), including histone deacetylase inhibitors with chromatin-remodeling capabilities, combined with anti-retroviral therapy, reactivates HIV-1 transcriptionin vivo. However, LRA treatment fails to significantly reduce the HIV-1 reservoir in HIV-1-positive individuals, indicating that it is probably insufficient to eliminate latently infected cells. The global and T-cell subset-specific impact of individual LRAs on the transcriptome of CD4+T-cells, the main HIV-1 reservoir containing cell typein vivo, remains understudied. Here, using single cell RNA-sequencing, we characterize LRA treatment-induced alterations of CD4+T-cell subset composition and of subpopulation-specific transcriptomes, using Vorinostat and Panobinostat as two prototypic HDAC inhibitors.Ex vivoexposure of CD4+T-cells from an aviremic HIV-1-positive individual to Panobinostat markedly reduced the percentage of TREGcells. Furthermore, it altered expression of a multitude of interferon-regulated genes, resulting in suppression of several well-characterized antiviral genes, and in enhancement of selected interferon-regulated genes with proviral activities. These changes were most pronounced in TN, TCM, TTMand TEM, and less pronounced in TREG. Exposure to Vorinostat resulted in a comparably mild change of cellular transcriptomic profile, regarding both the number of deregulated genes and their fold change of expression. Nevertheless, selected interferon-regulated genes exhibited a subset-specific expression profile upon Vorinostat treatment. Finally, some genes were deregulated by both treatments in a subset-specific manner. We conclude that treatment by both individual HDAC inhibitors induces an overall proviral milieu in CD4+T-cells subsets. While this proviral state might be favorable for efficient HIV-1 reactivation, we hypothesize that it may impede the instruction of activation of cellular and adaptive immunity required for effective killing of reactivated cells.

The clinical course of COVID-19 is highly variable, however, underlying host factors and determinants of severe disease are still unknown. Based on single-cell transcriptomes of nasopharyngeal and bronchial samples from clinically well-characterized patients presenting with moderate and critical severities, we reveal the different types and states of airway epithelial cells that are vulnerable for SARS-CoV-2 infection. In COVID-19 patients, we observed a two- to threefold increase of cells expressing the SARS-CoV-2 entry receptorACE2within the airway epithelial cell compartment.ACE2is upregulated in epithelial cells through Interferon signals by immune cells suggesting that the viral defense system may increase the number of potentially susceptible cells in the respiratory epithelium. Infected epithelial cells recruit and activate immune cells by chemokine signaling. Recruited T lymphocytes and inflammatory macrophages were hyperactivated and showed a strong interaction with epithelial cells. In critical patients, increased expression ofCCL2, CCL3, CCL5, CXCL9, CXCL10, IL8, IL1BandTNFin macrophages was identified as a likely cause of a hyperinflammatory lung pathology. Moreover, we observed exacerbated epithelial cell death, likely leading to lung injury and respiratory failure in fatal cases. Our study provides novel insights into the pathophysiology of COVID-19 and suggests an immunomodulatory therapy along the CCL2, CCL3/CCR1 axis as promising option to prevent and treat critical course of COVID-19.

A subset of intracellular mono-ADP-ribosyltransferases diphtheria toxin-like (ARTDs, aka mono-PARPs) is induced by type I interferons. Some of these mono-ARTDs feature antiviral activity while certain RNA viruses, including Chikungunya virus (CHIKV), encode mono-ADP-ribosylhydrolases, suggesting a role for mono-ADP-ribosylation (MARylation) in host-virus conflicts. CHIKV expresses four non-structural proteins (nsP1-nsP4), with nsP3 containing a macrodomain that hydrolyzes and thereby reverses protein MARylation in vitro and in cells. This de-MARylation activity is essential as hydrolase inactivating mutations result in replication defective virus. However, the substrates of MARylation during CHIKV infection are unknown and thus it is unclear how the macrodomain contributes to virus replication and how mono-ARTD-dependent MARylation confers antiviral immunity. We identified ARTD10 and ARTD12 as restriction factors for CHIKV replication in a catalytic activity-dependent manner. CHIKV replication requires processing of the non-structural polyprotein nsP1-4 by the nsP2-encoded protease and the assembly of the four individual nsPs into a functional replication complex. Expression of ARTD10 and ARTD12 resulted in a reduction of processed nsPs. Similarly, MAR hydrolase inactive CHIKV replicon mutants revealed a decrease in processed nsPs, comparable to an nsP2 protease defective mutant. This suggested that the macrodomain contributes to nsP2 protease activity. In support, a hydrolase-deficient virus was complemented by a protease-deficient virus. We hypothesized that MARylation regulates the proteolytic function of nsP2. Indeed, we found that nsP2 is MARylated by ARTD10. This inhibited nsP2 protease activity, thereby preventing polyprotein processing and consequently virus replication. This inhibition was antagonized by the MAR hydrolase activity of nsP3. Together, our findings provide a mechanistic explanation for the need of the viral MAR hydrolase for efficient replication of CHIKV.Author SummaryInfectious diseases still pose major health threats. Especially fast evolving viruses find ever new strategies to manipulate the immune response. With climate warming and increased human mobility vector-borne pathogens like Chikungunya virus (CHIKV) spread and cause world-wide epidemics. Beyond the acute phase, CHIKV patients regularly suffer from chronic rheumatism. This entails a decline in life quality and an economic burden. To date no drugs are approved and the mode of pathogenesis remains elusive. Here we describe a mechanistic function of the CHIKV nsP3 macrodomain. We found that the viral nsP2 is mono-ADP-ribosylated interfering with its auto-proteolytic function. The nsP3 macrodomain removes this modification and restores the protease activity that is essential for replication. Because macrodomains are highly conserved they might represent broad antiviral targets.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections. Andreas Meyerhans and Javier P. Martinez were supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER grant no. SAF2016-75505-R (AEI/MINEIC/FEDER, UE). Mark Brönstrup, Andreas Meyerhans and Javier P. Martinez would like to acknowledge a networking contribution from the COST Action CM1407 “Challenging organic syntheses inspired by nature – from natural products chemistry to drug discovery”. Martin Messerle and Thomas F. Schulz were supported by funding from DZIF (project 07.802 TTU IICH). Christine Goffinet, Thomas Pietschmann and Mark Brönstrup were supported by a grant provided by “Innovationsfonds der Helmholtz-Zentren”. Christine Goffinet was supported by a DFG grant within German African Cooperation Projects in Infectiology (GO2153/3-1) and by funding of the Helmholtz Center for Infection Research (HZI) and of Berlin Institute of Health (BIH). Sergej Franz was supported by the Infection Biology international PhD program of Hannover Biomedical Research School

Summary Coronavirus Disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progresses to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19, associated with increased neutrophil counts and dysregulated immune responses, remains unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole blood and peripheral blood mononuclear cells to determine changes in immune cell composition and activation in mild vs. severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and it reveals profound alterations in the myeloid cell compartment associated with severe COVID-19., Highlights ● SARS-CoV-2 infection induces profound alterations of the myeloid compartment ● Mild COVID-19 is marked by inflammatory HLA-DRhiCD11chi CD14+ monocytes ● Dysfunctional HLA-DRloCD163hi and HLA-DRloS100Ahi CD14+ monocytes in severe COVID-19 ● Emergency myelopoiesis with immature and dysfunctional neutrophils in severe COVID-19

Endogenous and heterologpusly expressed human IFITM3 restricts early and late replication steps of chikungunya and Mayaro virus infection and is downregulated at the protein level in infected cells., Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3. Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation., Author summary The accessory protein Nef promotes primate lentiviral replication and enhances the pathogenicity of HIV-1 by mechanisms of immune evasion and enhancing viral infectivity and replication. Here, we show that the evolutionarily most isolated primate lentivirus SIVcol lacks several otherwise conserved Nef functions. Nevertheless, SIVcol Nef potently antagonizes SERINC5, a recently discovered inhibitor of viral infectivity, by down-modulating it from the cell surface and inducing its proteasomal degradation. We identified Y86 in SIVcol Nef as a key determinant of SERINC5 antagonism. Efficient counteraction of SERINC5 did not increase HIV-1 replication in preactivated CD4+ T cells and in ex vivo infected lymphoid tissue but had modest enhancing effects when resting PBMCs were first infected and activated six days later. Evolution of high anti-SERINC5 activity by SIVcol Nef supports a relevant role of this antagonism in vivo, for instance by enhancing virion infectivity under conditions of limited T cell activation.

Cellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent human immunodeficiency virus type 1 (HIV-1) virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce the particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered the fact that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient, for 90K-mediated antiviral activity. Rather, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single “humanizing” amino acid exchange in the BTB (broad-complex, tramtrack, and bric-à-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue the antiviral activity of a shortened version of rhesus macaque 90K, but not that of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domains of 90K from rhesus macaques and humans point toward a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein. IMPORTANCE The cellular 90K protein has been shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear smaller amounts of the HIV-1 Env glycoprotein, which is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but, surprisingly, not from rhesus macaques, inhibit HIV-1. The introduction of a single amino acid exchange into a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.

Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC.In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation.We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

Background Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. Results In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. Conclusions We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC. Electronic supplementary material The online version of this article (doi:10.1186/s12977-017-0358-1) contains supplementary material, which is available to authorized users.

Background In the past decade, the identification and characterization of antiviral genes with the ability to interfere with virus replication has established cell-intrinsic innate immunity as a third line of antiviral defense in addition to adaptive and classical innate immunity. Understanding how cellular factors have evolved to inhibit HIV-1 reveals particularly vulnerable points of the viral replication cycle. Many, but not all, antiviral proteins share type I interferon-upregulated expression and sensitivity to viral counteraction or evasion measures. Whereas well-established restriction factors interfere with early post-entry steps and release of HIV-1, recent research has revealed a diverse set of proteins that reduce the infectious quality of released particles using individual, to date poorly understood modes of action. These include induction of paucity of mature glycoproteins in nascent virions or self-incorporation into the virus particle, resulting in poor infectiousness of the virion and impaired spread of the infection. Conclusion A better understanding of these newly discovered antiviral factors may open new avenues towards the design of drugs that repress the spread of viruses whose genomes have already integrated.

Human CD317 is an intrinsic immunity factor that restricts the release of enveloped viruses, including the major pathogens HIV and Lassa virus, from infected cells in culture. Its importance for infection control in humans is unclear, due in part to its incompletely defined in vivo expression pattern. CD317 also has been proposed as a selective target for immunotherapy of multiple myeloma. To provide a framework for studies of the biological functions, regulation, and therapeutic potential of CD317, we performed microarray-based expression profiling in 468 tissue samples from 25 healthy organs from more than 210 patients. We found that CD317 protein was expressed to varying degrees in all organs tested and detected in a number of specialized cell types, including hepatocytes, pneumocytes, ducts of major salivary glands, pancreas and kidney, Paneth cells, epithelia, Leydig cells, plasma cells, bone marrow stromal cells, monocytes, and vascular endothelium. Although many of these cell types are in vivo targets for pathogenic viruses, restriction by CD317 or virus-encoded antagonists has been documented in only some of them. Limited cell type–dependent coexpression of CD317 with the IFN biomarker MxA in vivo and lack of responsive stimulation in organ explants suggest that interferons may only partially regulate CD317. This in vivo expression profiling sheds light on the biology and species-specificity of CD317, identifies multiple thus far unknown interaction sites of viruses with this restriction factor, and refutes the concept of its restricted constitutive expression and primary IFN inducibility. CD317's widespread expression calls into question its suitability as a target for immunotherapy.

Human CD317 (BST-2/tetherin) is an intrinsic immunity factor that blocks the release of retroviruses, filoviruses, herpesviruses, and arenaviruses. It is unclear whether CD317 expressed endogenously in rodent cells has the capacity to interfere with the replication of the retroviral rodent pathogen murine leukemia virus (MLV) or, in the context of small-animal model development, contributes to the well-established late-phase restriction of human immunodeficiency virus type 1 (HIV-1). Here, we show that small interfering RNA (siRNA)-mediated knockdown of CD317 relieved a virion release restriction and markedly enhanced the egress of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) in rat cells, including primary macrophages. Moreover, rodent CD317 potently inhibited MLV release, and siRNA-mediated depletion of CD317 in a mouse T-cell line resulted in the accelerated spread of MLV. Several virus-encoded antagonists have recently been reported to overcome the restriction imposed by human or monkey CD317, including HIV-1 Vpu, envelope glycoproteins of HIV-2 and Ebola virus, Kaposi's sarcoma-associated herpesvirus K5, and SIV Nef. In contrast, both rat and mouse CD317 showed a high degree of resistance to these viral antagonists. These data suggest that CD317 is a broadly acting and conserved mediator of innate control of retroviral infection and pathogenesis that restricts the release of retroviruses and lentiviruses in rodents. The high degree of resistance of the rodent CD317 restriction factors to antagonists from primate viruses has implications for HIV-1 small-animal model development and may guide the design of novel antiviral interventions.

SummaryMammals encode proteins that inhibit viral replication at the cellular level. In turn, certain viruses have evolved genes that can functionally counteract these intrinsic restrictions. Human CD317 (BST-2/HM1.24/tetherin) is a restriction factor that blocks release of human immunodeficiency virus type 1 (HIV-1) from the cell surface and can be overcome by HIV-1 Vpu. Here, we show that mouse and rat CD317 potently inhibit HIV-1 release but are resistant to Vpu. Interspecies chimeras reveal that the rodent-specific resistance and human-specific sensitivity to Vpu antagonism involve all three major structural domains of CD317. To promote virus release, Vpu depletes cellular pools of human CD317, but not of the rodent orthologs, by accelerating its degradation via the 20S proteasome. Thus, HIV-1 Vpu suppresses the expression of the CD317 antiviral factor in human cells, and the species-specific resistance to this suppression may guide the development of small animal models of HIV infection.

SummarySexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.

The current testing of anti-HIV drugs is hampered by the lack of a small animal that is readily available and easy to handle; can be infected systemically with HIV type 1 (HIV-1); harbors the major HIV-1 target cells in a physiological frequency, organ distribution, and activation state; and is established as a pharmacological model. Here, we explored the potential of outbred Sprague–Dawley rats that transgenically express the HIV-1 receptor complex on CD4 T cells and macrophages as a model for the preclinical evaluation of inhibitors targeting virus entry or reverse transcription. The concentrations of the peptidic fusion inhibitor enfuvirtide or the nonnucleoside reverse transcriptase inhibitor efavirenz required to inhibit HIV-1 infection of cultured primary CD4 T cells and macrophages from human CD4 and CCR5-transgenic rats differed by no more than 3-fold from those required for human reference cultures. Prophylactic treatment of double-transgenic rats with a weight-adapted pediatric dosing regimen for either enfuvirtide (s.c., twice-daily) or efavirenz (oral, once-daily) achieved a 92.5% or 98.8% reduction, respectively, of the HIV-1 cDNA load in the spleen 4 days after i.v. HIV-1 challenge. Notably, a once-daily dosing regimen for enfuvirtide resulted in a ≈5-fold weaker inhibition of infection, unmasking the unfavorable pharmacokinetic characteristics of the synthetic peptide in the context of an efficacy trial. This work provides proof of principle that HIV-susceptible transgenic rats can allow a rapid and predictive preclinical evaluation of the inhibitory potency and of the pharmacokinetic properties of antiviral compounds targeting early steps in the HIV replication cycle.

A rapid method for efficient gene delivery into primary rodent lymphocytes would greatly facilitate the study of signaling and metabolic pathways in untransformed hematopoietic cells as well as the validation of gene expression and targeting strategies before the generation of knockout or knock-down animals. Here, we report that species-adapted nucleofection procedures combined with optimized cultivation conditions render proliferating primary T cells, B cells, and natural killer cells from widely used rat and mouse strains susceptible to high-level gene delivery. As a result, transgene expression levels were enhanced approximately 10- to 370-fold over established protocols. The effectiveness of the nucleofection approach for functional analyses was demonstrated by specific down-regulation of CD4 cell surface molecules by either transient expression of the endocytosis-inducing Nef protein from human immunodeficiency virus or by specific gene silencing mediated by small interfering RNA. In conclusion, this species-adapted procedure for nonviral gene delivery renders primary rodent lymphocytes accessible to rapid functional ex vivo studies, which until now have not been feasible. Furthermore, nucleofection may aid the advancement of therapeutic nonviral gene delivery approaches.

Background In response to viral infections, interferons induce the transcription of several hundred genes in mammalian cells. Specific antiviral functions, however, have only been attributed to a few of them. 90K/LGALS3BP has been reported to be an interferon-stimulated gene that is upregulated in individuals with cancer or HIV-1 infection. Results Here, we show that 90K expression dose-dependently decreased the particle infectivity of HIV-1 progeny. The lower infectivity of released particles correlated with reduced virion incorporation of mature envelope glycoproteins gp120 and gp41. Further, proteolytic processing of the gp160 precursor and surface expression of gp120 in the producer cell were impaired in the presence of 90K expression. In contrast, expression of Gag, Nef and Vpu, and virus release were not grossly affected by 90K expression. 90K-imposed restriction occurred in the absence of direct interaction of 90K with HIV-1 Env or entrapment of Env in the ER. The cell-associated, but not the secreted species of 90K, mediated the antiviral effect. A truncated version of human 90K, solely consisting of the two intermediate domains, displayed a similar antiviral activity as the full-length wildtype 90K, indicating that the N-terminal SRCR-like domain and the C-terminal domain are dispensable for 90K’s antiviral activity. The murine homolog of 90K, CypCAP (Cyclophilin C-associated protein), neither modulated particle infectivity of HIV-1 nor lowered the virion incorporation of mature gp120, suggesting a species-specific mode of action. 90K was expressed at basal levels in TZM-bl cells and in primary macrophages, and at low levels in CD4+ T-cells and PBMCs. 90K’s susceptibility to IFN-mediated stimulation of expression was cell type-specific. siRNA-mediated knockdown of 90K in TZM-bl cells and primary macrophages enhanced the incorporation of Env glycoproteins into progeny virions, boosted the particle infectivity of released HIV-1, and accelerated HIV-1 spread. Conversely, treatment of HIV-1 infected macrophages with IFN-α induced 90K expression and lowered the particle infectivity of HIV-1. Conclusions Thus, 90K constitutes a novel antiviral factor that reduces the particle infectivity of HIV-1, involving interference with the maturation and incorporation of HIV-1 Env molecules into virions.

SummaryThe interferon-induced host restriction factor tetherin poses a barrier for SIV transmission from primates to humans. After cross-species transmission, the chimpanzee precursor of pandemic HIV-1 switched from the accessory protein Nef to Vpu to effectively counteract human tetherin. As we report here, the experimental reintroduction of HIV-1 into its original chimpanzee host resulted in a virus that can use both Vpu and Nef to antagonize chimpanzee tetherin. Functional analyses demonstrated that alterations in and near the highly conserved ExxxLL motif in the C-terminal loop of Nef were critical for the reacquisition of antitetherin activity. Strikingly, just two amino acid changes allowed HIV-1 Nef to counteract chimpanzee tetherin and promote virus release. Our data demonstrate that primate lentiviruses can reacquire lost accessory gene functions during a single in vivo passage and suggest that other functional constraints keep Nef ready to regain antitetherin activity.

Interferon-stimulated genes fulfill innate antiviral effector functions. Among them, tetherin (THN) blocks the release of many enveloped viruses from infected cells. Vaccinia virus (VACV) encodes immune modulators interfering with antiviral host responses. Therefore, it was tempting to study a potential VACV-THN interaction. Remarkably, THN expression did not inhibit VACV release and replication. VACV infection did not diminish THN surface levels or impair its function on retroviral release. This suggests that THN is unable to restrict VACV replication.

Vpu antagonizes human immunodeficiency virus type 1 (HIV-1) particle release inhibition by CD317/BST-2/Tetherin. Whether this Vpu activity strictly requires cellular depletion of the restriction factor is unclear. Here, we characterized CD317 variants with mutations in putative sorting or ubiquitination motifs. All mutants still potently impaired release of Vpu-defective HIV-1 and remained sensitive to Vpu-mediated release enhancement. Importantly, this virological antagonism correlated with surface downregulation of CD317 mutants by Vpu, while intracellular pools of these mutants, which were consistently depleted of the wild-type protein, were highly variable or even enhanced. Thus, Vpu can efficiently antagonize virion tethering in the absence of CD317 degradation.

Mammals encode proteins that inhibit viral replication at the cellular level. In turn, certain viruses have evolved genes that can functionally counteract these intrinsic restrictions. Human CD317 (BST-2/tetherin) was recently identified as a restriction factor that blocks release of HIV-1 from the cell surface and can be overcome by HIV-1 Vpu. Here, we show that mouse and rat CD317 potently inhibit HIV-1, but are resistant to Vpu. To promote virus release, Vpu depletes cellular pools of human CD317, but not of the rodent orthologs, by accelerating its degradation via the 20S proteasome. Importantly, HIV-1 downregulates surface-exposed CD317 and depletes endogenous pools of the restriction factor in infected human T-cells in a Vpu-dependent manner. Distinct mutants of CD317 indicate that also degradation-independent abilities of Vpu contribute to its ability to promote virus release. Interspecies-chimeras of CD317 reveal that the rodent-specific resistance and human-specific sensitivity to Vpu antagonism have a complex genetic basis involving all three major structural domains of the restriction factor. In rodent cells, knock-down of endogenous CD317 enhances HIV release and accelerates MLV spread, suggesting that CD317 contributes to the control of retroviral pathogenesis. Thus, HIV-1 Vpu suppresses the expression of the CD317 antiviral factor in infected human cells, and the species-specific resistance to Vpu antagonism may guide the advancement of small animal models of HIV infection.

Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.

Background Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1). Results Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env) that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. Conclusion Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

Background Aqueous extracts from leaves of well known species of the Lamiaceae family were examined for their potency to inhibit infection by human immunodeficiency virus type 1 (HIV-1). Results Extracts from lemon balm (Melissa officinalis L.), peppermint (Mentha × piperita L.), and sage (Salvia officinalis L.) exhibited a high and concentration-dependent activity against the infection of HIV-1 in T-cell lines, primary macrophages, and in ex vivo tonsil histocultures with 50% inhibitory concentrations as low as 0.004%. The aqueous Lamiaceae extracts did not or only at very high concentrations interfere with cell viability. Mechanistically, extract exposure of free virions potently and rapidly inhibited infection, while exposure of surface-bound virions or target cells alone had virtually no antiviral effect. In line with this observation, a virion-fusion assay demonstrated that HIV-1 entry was drastically impaired following treatment of particles with Lamiaceae extracts, and the magnitude of this effect at the early stage of infection correlated with the inhibitory potency on HIV-1 replication. Extracts were active against virions carrying diverse envelopes (X4 and R5 HIV-1, vesicular stomatitis virus, ecotropic murine leukemia virus), but not against a non-enveloped adenovirus. Following exposure to Lamiaceae extracts, the stability of virions as well as virion-associated levels of envelope glycoprotein and processed Gag protein were unaffected, while, surprisingly, sucrose-density equilibrium gradient analyses disclosed a marked increase of virion density. Conclusion Aqueous extracts from Lamiaceae can drastically and rapidly reduce the infectivity of HIV-1 virions at non-cytotoxic concentrations. An extract-induced enhancement of the virion's density prior to its surface engagement appears to be the most likely mode of action. By harbouring also a strong activity against herpes simplex virus type 2, these extracts may provide a basis for the development of novel virucidal topical microbicides.

Background In vivo studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are non-permissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1YU-2 infection, demonstrating HIV-1 susceptibility in vivo. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread ex vivo. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors. Results Levels of virus entry, HIV-1 cDNA synthesis, nuclear import, and integration into the host genome were shown to be remarkably similar in cell lines and, where technically accessible, in primary T-cells from both species. In contrast, a profound impairment at the level of early HIV gene expression was disclosed at the single-cell level in primary rat T-cells and most other rat-derived cells. Macrophages were a notable exception, possibly reflecting the unique transcriptional milieu in this evolutionarily conserved target cell of all lentiviruses. Importantly, transient trans-complementation by ex vivo nucleofection with the Tat-interacting protein Cyclin T1 of human origin markedly elevated HIV gene expression in primary rat T-cells. Conclusion This is the first study that has quantitatively determined the efficiency of consecutive steps in the HIV-1 replication cycle in infected primary HIV target cells from a candidate transgenic small animal and compared it to human cells. Unlike cells derived from mice or rabbits, rat cells complete all of the early steps in the HIV-1 replication cycle, including provirus integration in vivo, with high efficiency. A deficiency in gene expression was disclosed at the single cell level and could be counteracted by the human pTEFb transcription complex factor Cyclin T1. Collectively, these results provide the basis for the advancement of this transgenic rat model through strategies aimed at boosting HIV-1 gene expression in primary rat CD4 T-cells, including human Cyclin T1 transgenesis.

Background Human CD317 (BST-2/HM1.24/tetherin) restricts the release of multiple viruses including HIV-1, XMRV, Lassa virus and KSHV from infected cells in culture. Its relevance for infection control in humans is however unclear, in part due to its poorly defined in vivo-expression pattern. Materials and methods To provide a framework for studies into the biological functions, regulation and therapeutic potential of CD317, we performed a tissue microarray-based expression profiling in 468 samples of 25 healthy organs from over 250 patients, not suffering from clinically apparent infections. Results We report that CD317 protein was expressed to varying degrees in all organs tested and detected in a number of specialized cell types including hepatocytes, pneumocytes in the lung, ducts of major salivary glands, pancreas and kidney, Paneth cells in the small intestine, epithelia of multiple organs, Leydig cells in the testis, plasma cells, bone marrow stromal cells, monocytes, and vascular endothelium. Remarkably, many of these CD317-positive cell types are in vivo-targets for pathogenic viruses, only for some of which restriction by CD317 or virus-encoded antagonists have thus far been investigated. Of note, major HIV target cells in tonsil and gut-associated lymphoid tissue did not express CD317. Limited, cell type-dependent co-expression of CD317 with the interferon biomarker MxA in vivo and an unresponsiveness to cytokine stimulation in lymphoid organ explants suggest that type I interferons may only in part regulate CD317. Conclusions

Background The development of an immunocompetent, genetically modified mouse model to study HIV-1 pathogenesis and to test antiviral strategies has been hampered by the fact that cells from native mice do not or only inefficiently support several steps of the HIV-1 replication cycle. Upon HIV-1 infection, mouse T-cell lines fail to express viral proteins, but the underlying replication barrier has thus far not been unambiguously identified. Here, we performed a kinetic and quantitative assessment of consecutive steps in the early phase of the HIV-1 replication cycle in T-cells from mice and humans. Results Both T-cell lines and primary T-cells from mice harbor a severe post-entry defect that is independent of potential species-specTR transactivation. Reverse transcription occurred efficiently following VSV-G-mediated entry of virions into mouse T-cells, and abundant levels of 2-LTR circles indicated successful nuclear import of the pre-integration complex. To probe the next step in the retroviral replication cycle, i.e. the integration of HIV-1 into the host cell genome, we established and validated a nested real-time PCR to specifically quantify HIV-1 integrants exploiting highly repetitive mouse B1 elements. Importantly, we demonstrate that the frequency of integrant formation is diminished 18- to > 305-fold in mouse T-cell lines compared to a human counterpart, resulting in a largely abortive infection. Moreover, differences in transgene expression from residual vector integrants, the transcription off which is cyclin T1-independent, provided evidence for an additional, peri-integrational deficit in certain mouse T-cell lines. Conclusion In contrast to earlier reports, we find that mouse T-cells efficiently support early replication steps up to and including nuclear import, but restrict HIV-1 at the level of chromosomal integration.

Table of contents Oral presentations Session 1: Entry & uncoating O1 Host cell polo-like kinases (PLKs) promote early prototype foamy virus (PFV) replication Irena Zurnic, Sylvia Hütter, Ute Lehmann, Nicole Stanke, Juliane Reh, Tobias Kern, Fabian Lindel, Gesche Gerresheim, Martin Hamann, Erik Müllers, Paul Lesbats, Peter Cherepanov, Erik Serrao, Alan Engelman, Dirk Lindemann O2 A novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized HIV-1 infection Claire Da Silva Santos, Kevin Tartour, Andrea Cimarelli O3 Dynamics of nuclear envelope association and nuclear import of HIV-1 complexes Rya Burdick, Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay Pathak O4 Human papillomavirus protein E4 potently enhances the susceptibility to HIV infection Oliver T. Keppler Session 2: Reverse transcription & integration O5 Structure and function of HIV-1 integrase post translational modifications Karine Pradeau, Sylvia Eiler, Nicolas Levy, Sarah Lennon, Sarah Cianferani, Stéphane Emiliani, Marc Ruff O6 Regulation of retroviral integration by RNA polymerase II associated factors and chromatin structure Vincent Parissi Session 3: Transcription and latency O7 A novel single-cell analysis pipeline to identify specific biomarkers of HIV permissiveness Sylvie Rato, Antonio Rausell, Miguel Munoz, Amalio Telenti, Angela Ciuffi O8 A capsid-dependent integration program linking T cell activation to HIV-1 gene expression Alexander Zhyvoloup, Anat Melamed, Ian Anderson, Delphine Planas, Janos Kriston-Vizi, Robin Ketteler, Chen-Hsuin Lee, Andy Merritt, Petronela Ancuta, Charles Bangham, Ariberto Fassati O9 Characterisation of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome Anthony Rodari, Benoit Van Driessche, Mathilde Galais, Nadége Delacourt, Sylvain Fauquenoy, Caroline Vanhulle, Anna Kula, Arsène Burny, Olivier Rohr, Carine Van Lint O10 Tissue-specific dendritic cells differentially modulate latent HIV-1 reservoirs Thijs van Montfort, Renee van der Sluis, Dave Speijer, Ben Berkhout Session 4: RNA trafficking & packaging O11 A novel cis-acting element affecting HIV replication Bo Meng, Andrzej Rutkowski, Neil Berry, Lars Dölken, Andrew Lever O12 Tolerance of HIV’s late gene expression towards stepwise codon adaptation Thomas Schuster, Benedikt Asbach, Ralf Wagner Session 5: Assembly & release O13 Importance of the tax-inducible actin-bundling protein fascin for transmission of human T cell leukemia virus Type 1 (HTLV-1) Christine Gross, Veit Wiesmann, Martina Kalmer, Thomas Wittenberg, Jan Gettemans, Andrea K. Thoma-Kress O14 Lentiviral nef proteins antagonize TIM-mediated inhibition of viral release Minghua Li, Eric O. Freed, Shan-Lu Liu Session 6: Pathogenesis & evolution O15 SEVI and semen prolong the half-life of HIV-1 Janis Müller, Jan Münch O16 CD169+ macrophages mediate retrovirus trans-infection of permissive lymphocytes to establish infection in vivo Xaver Sewald, Pradeep Uchil, Mark Ladinsky, Jagadish Beloor, Ruoxi Pi, Christin Herrmann, Nasim Motamedi, Thomas Murooka, Michael Brehm, Dale Greiner, Thorsten Mempel, Pamela Bjorkman, Priti Kumar, Walther Mothes O17 Efficient replication of a vpu containing SIVagm construct in African Green Monkeys requires an HIV-1 nef gene Simone Joas, Erica Parrish, Clement Wesley Gnanadurai, Edina Lump, Christina M. Stürzel, Nicholas F. Parrish, Ulrike Sauermann, Katharina Töpfer, Tina Schultheiss, Steven Bosinger, Guido Silvestri, Cristian Apetrei, Nicholas Huot, Michaela Müller-Trutwin, Daniel Sauter, Beatrice H. Hahn, Christiane Stahl-Hennig, Frank Kirchhoff O18 Reprogramming initiates mobilization of endogenous mutagenic LINE-1, Alu and SVA retrotransposons in human induced pluripotent stem cells with consequences for host gene expression Gerald Schumann, Sabine Jung-Klawitter, Nina V. Fuchs, Kyle R. Upton, Martin Muñoz-Lopez, Ruchi Shukla, Jichang Wang, Marta Garcia-Canadas, Cesar Lopez-Ruiz, Daniel J. Gerhardt, Attila Sebe, Ivana Grabundzija, Patricia Gerdes, Sylvia Merkert, Andres Pulgarin, Anja Bock, Ulrike Held, Anett Witthuhn, Alexandra Haase, Ernst J. Wolvetang, Ulrich Martin, Zoltán Ivics, Zsuzsanna Izsvák, J. Garcia-Perez, Geoffrey J. Faulkner O19 NF-κB activation induces expression of human endogenous retrovirus and particle production Tara Hurst, Aris Katzourakis, Gkikas Magiorkinis Session 7a and b: Innate sensing & intrinsic immunity O20 Identification of the phosphatase acting on T592 in SAMHD1 during M/G1 transition Kerstin Schott, Rita Derua, Janna Seifried, Andreas Reuter, Heike Schmitz, Christiane Tondera, Alberto Brandariz-Nuñez, Felipe Diaz-Griffero, Veerle Janssens, Renate König O21 Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells Hanna-Mari Baldauf, Lena Stegmann, Sarah-Marie Schwarz, Maud Trotard, Margarethe Martin, Gina Lenzi, Manja Burggraf, Xiaoyu Pan, Oliver I. Fregoso, Efrem S. Lim, Libin Abraham, Elina Erikson, Laura Nguyen, Ina Ambiel, Frank Rutsch, Renate König, Baek Kim, Michael Emerman, Oliver T. Fackler, Oliver T. Keppler O22 The role of SAMHD1 in antiviral restriction and immune sensing in the mouse Sabine Wittmann, Rayk Behrendt, Bianca Volkmann, Kristin Eissmann, Thomas Gramberg O23 T cells expressing reduced restriction factors are preferentially infected in therapy naïve HIV-1 patients Sebastian Bolduan, Herwig Koppensteiner, Stefanie Regensburg, Ruth Brack-Werner, Rika Draenert, Michael Schindler O24 cGAS-mediated innate immunity spreads through HIV-1 env-induced membrane fusion sites from infected to uninfected primary HIV-1 target cells Aurélie Ducroux, Shuting Xu, Aparna Ponnurangam, Sergej Franz, Angelina Malassa, Ellen Ewald, Christine Goffinet O25 Perturbation of innate RNA and DNA sensing by human T cell leukemia virus type 1 oncoproteins Sin-Yee Fung, Ching-Ping Chan, Chun-Kit Yuen, Kin-Hang Kok, Chin-Ping Chan, Dong-Yan Jin O26 Induction and anti-viral activity of Interferon α subtypes in HIV-1 infection Ulf Dittmer O27 Vpu-mediated counteraction of tetherin is a major determinant of HIV-1 interferon resistance Dorota Kmiec, Shilpa Iyer, Christina Stürzel, Daniel Sauter, Beatrice Hahn, Frank Kirchhoff O28 DNA repair protein Rad18 restricts HIV-1 and LINE-1 life cycle Yasuo Ariumi, Mariko Yasuda-Inoue, Koudai Kawano, Satoshi Tateishi, Priscilla Turelli O29 Natural mutations in IFITM3 allow escape from post-translational regulation and toggle antiviral specificity Alex Compton, Nicolas Roy, Françoise Porrot, Anne Billet, Nicoletta Casartelli, Jacob Yount, Chen Liang, Oliver Schwartz Session 8: Adaptive immunity & immune evasion O30 Observing evolution in HIV-1 infection: phylogenetics and mutant selection windows to infer the influence of the autologous antibody response on the viral quasispecies Carsten Magnus, Lucia Reh, Penny Moore, Therese Uhr, Jacqueline Weber, Lynn Morris, Alexandra Trkola O31 Dose and subtype specific analyses of the anti-HIV effects of IFN-alpha family members Rashel V. Grindberg, Erika Schlaepfer, Gideon Schreiber, Viviana Simon, Roberto F. Speck Session 9: Novel antiviral strategies O32 LEDGIN-mediated inhibition of the integrase-LEDGF/p75 interaction reduces reactivation of residual latent HIV Zeger Debyser, Lenard Vranckx, Jonas Demeulemeester, Suha Saleh, Eric Verdin, Anna Cereseto, Frauke Christ, Rik Gijsbers O33 NKG2D-mediated clearance of reactivated viral reservoirs by natural killer cells O34 Inhibition of HIV reactivation in brain cells by AAV-mediated delivery of CRISPR/Cas9 O35 CRISPR-Cas9 as antiviral: potent HIV-1 inhibition, but rapid virus escape and the subsequent design of escape-proof antiviral strategies Ben Berkhout, Gang Wang, Na Zhao, Atze T. Das Session 10: Recent advances in HIV vaccine development O36 Priming with a potent HIV-1 DNA vaccine frames the quality of T cell and antibody responses prior to a poxvirus and protein boost Benedikt Asbach, Josef Köstler, Beatriz Perdiguero, Mariano Esteban, Bertram L. Jacobs, David C. Montefiori, Celia C. LaBranche, Nicole L. Yates, Georgia D. Tomaras, Guido Ferrari, Kathryn E. Foulds, Mario Roederer, Gary Landucci, Donald N. Forthal, Michael S. Seaman, Natalie Hawkins, Steven G. Self, Sanjay Phogat, James Tartaglia, Susan W. Barnett, Brian Burke, Anthony D. Cristillo, Song Ding, Jonathan L. Heeney, Giuseppe Pantaleo, Ralf Wagner O37 Passive immunisation with a neutralising antibody against HIV-1 Env prevents infection of the first cells in a mucosal challenge rhesus monkey model Christiane Stahl-Hennig, Viktoria Stab, Armin Ensser, Ulrike Sauermann, Bettina Tippler, Dennis Burton, Matthias Tenbusch, Klaus Überla O38 HIV antibody Fc-glycoforms drive B cell affinity maturation Galit Alter, Giuseppe Lofano, Anne-Sophie Dugast, Viraj Kulkarni, Todd Suscovich Poster presentations Topic 1: Entry & uncoating P1 Dynein light chain is required for murine leukemia virus infection Tatiana Opazo, Felipe Barraza, Diego Herrera, Andrea Garces, Tomas Schwenke, Diego Tapia, Jorge Cancino, Gloria Arriagada P2 Peptide paratope mimics of the broadly neutralising HIV-1 antibody b12 Christina Haußner, Dominik Damm, Anette Rohrhofer, Barbara Schmidt, Jutta Eichler P3 Investigating cellular pathways involved in the transmission of HIV-1 between dendritic cells and T cells using RNAi screening techniques Rebecca Midgley, James Wheeldon, Vincent Piguet P4 Co-receptor tropism in HIV-1, HIV-2 monotypic and dual infections Priyanka Khopkar, Megha Rohamare, Smita Kulkarni P5 Characterisation of the role of CIB1 and CIB2 as HIV-1 helper factors Ana Godinho-Santos, Allan Hance, Joao Goncalves, Fabrizio Mammano P6 Buffering deleterious polymorphisms in the highly constrained C2 region of HIV-1 envelope by the flexible V3 domain Romain Gasser, Meriem Hamoudi, Martina Pellicciotta, Zhicheng Zhou, Clara Visdeloup, Philippe Colin, Martine Braibant, Bernard Lagane, Matteo Negroni P7 Entry inhibition of HERV-K(HML-2) by an Env-IgG fusion protein Jula Wamara, Norbert Bannert Topic 2: Reverse transcription & integration P8 The R263K/H51Y resistance substitutions in HIV integrase decreases levels of integrated HIV DNA over time Thibault Mesplede, Nathan Osman, Kaitlin Anstett, Jiaming Calvin Liang, Hanh Thi Pham, Mark Wainberg P9 The Retrovirus Integration Database (RID) Wei Shao, Jigui Shan, Mary Kearney, Xiaolin Wu, Frank Maldarelli, John Mellors, Brian Luke, John Coffin, Stephen Hughes P10 The small molecule 3G11 inhibits HIV-1 reverse transcription Thomas Fricke, Silvana Opp, Caitlin Shepard, Dmitri Ivanov, Baek Kim, Jose Valle-Casuso, Felipe Diaz-Griffero P11 Dual and opposite regulation of HIV-1 integration by hRAD51: impact on therapeutical approaches using homologous DNA repair modulators Vincent Parissi P12 A flexible motif essential for integration by HIV-1 integrase Marine Kanja, Pierre Cappy, Matteo Negroni, Daniela Lener P13 Interaction between HIV-1 integrase and the host protein Ku70: identification of the binding site and study of the influence on integrase-proteasome interplay Ekaterina Knyazhanskaya, Andrey Anisenko, Timofey Zatsepin, Marina Gottikh P14 Normalisation based method for deep sequencing of somatic retroelement integrations in human genome Alexander Komkov, Anastasia Minervina, Gaiaz Nugmanov, Vadim Nazarov, Konstantin Khodosevich, Ilgar Mamedov, Yuri Lebedev Topic 3: Transcription and latency P15 BCA2/RABRING7 restricts HIV-1 transcription by preventing the nuclear translocation of NF-κB Marta Colomer-Lluch, Ruth Serra-Moreno P16 MATR3 post-transcriptional regulation of HIV-1 transcription during latency Ambra Sarracino, Anna Kula, Lavina Gharu, Alexander Pasternak, Carine Van Lint, Alessandro Marcello P17 HIV-1 tat intersects the SUMO pathway to regulate HIV-1 promoter activity Ann Marie McCartin, Anurag Kulkarni, Valentin Le Douce, Virginie Gautier P18 Conservation in HIV-1 Vpr guides tertiary gRNA folding and alternative splicing Ann Baeyens, Evelien Naessens, Anouk Van Nuffel, Karin Weening, Anne-Marie Reilly, Eva Claeys, Wim Trypsteen, Linos Vandekerckhove, Sven Eyckerman, Kris Gevaert, Bruno Verhasselt P19 The majority of reactivatable latent HIV are genetically distinct Hoi Ping Mok, Nicholas Norton, Axel Fun, Jack Hirst, Mark Wills, Andrew Lever P20 Do mutations in the tat exonic splice enhancer contribute to HIV-1 latency? Nicholas Norton, Hoi Ping Mok, Jack Hirst, Andrew Lever P21 Culture-to-Ct: A fast and direct RT-qPCR HIV gene reactivation screening method using primary T cell culture Valentin Le Douce, Ann Marie McCartin, Virginie Gautier P22 A novel approach to define populations of early silenced proviruses Dalibor Miklik, Filip Senigl, Jiri Hejnar Topic 4: RNA trafficking & packaging P23 Functional analysis of the structure and conformation of HIV-1 genome RNA DIS Jun-ichi Sakuragi, Sayuri Sakuragi, Masaru Yokoyama, Tatsuo Shioda, Hironori Sato P24 Regulation of foamy viral env splicing controls gag and pol expression Jochen Bodem, Rebecca Moschall, Sarah Denk, Steffen Erkelenz, Christian Schenk, Heiner Schaal Topic 5: Assembly & release P25 Transfer of HTLV-1 p8 to target T cells depends on VASP: a novel interaction partner of p8 Norbert Donhauser, Ellen Socher, Sebastian Millen, Heinrich Sticht, Andrea K. Thoma-Kress P26 COL4A1 and COL4A2 are novel HTLV-1 tax targets with a putative role in virus transmission Christine Gross, Sebastian Millen, Melanie Mann, Klaus Überla, Andrea K. Thoma-Kress P27 The C terminus of foamy virus gag protein is required for particle formation, and virus budding: starting assembly at the C terminus? Guochao Wei, Matthew J. Betts, Yang Liu, Timo Kehl, Robert B. Russell, Martin Löchelt P28 Generation of an antigen-capture ELISA and analysis of Rec and Staufen-1 effects on HERV-K(HML-2) virus particle production Oliver Hohn, Saeed Mostafa, Kirsten Hanke, Stephen Norley, Norbert Bannert P29 Antagonism of BST-2/tetherin is a conserved function of primary HIV-2 Env glycoproteins Chia-Yen Chen, Masashi Shingai, Pedro Borrego, Nuno Taveira, Klaus Strebel P30 Mutations in the packaging signal region of the HIV-1 genome cause a late domain mutant phenotype Chris Hellmund, Bo Meng, Andrew Lever P31 p6 regulates membrane association of HIV-1 gag Melanie Friedrich, Friedrich Hahn, Christian Setz, Pia Rauch, Kirsten Fraedrich, Alina Matthaei, Petra Henklein, Maximilian Traxdorf, Torgils Fossen, Ulrich Schubert Topic 6: Pathogenesis & evolution P32 Molecular and structural basis of protein evolution during viral adaptation Aya Khwaja, Meytal Galilee, Akram Alian P33 HIV-1 enhancement and neutralisation by soluble gp120 and its role for the selection of the R5-tropic “best fit” Birco Schwalbe, Heiko Hauser, Michael Schreiber P34 An insertion of seven amino acids in the Env cytoplasmic tail of Human Immunodeficiency Virus type 2 (HIV-2) selected during disease progression enhances viral replication François Dufrasne, Mara Lucchetti, Patrick Goubau, Jean Ruelle P35 Cell-associated HIV-1 unspliced to multiply spliced RNA ratio at 12 weeks ART correlates with markers of immune activation and apoptosis and predicts the CD4 T-cell count at 96 weeks ART Mirte Scherpenisse, Ben Berkhout, Alexander Pasternak P36 Faster progression in non-B subtype HIV-1-infected patients than Korean subclade of subtype B is accompanied by higher variation and no induction of gross deletion in non-B nef gene by Korean red ginseng treatment Young-Keol Cho, Jungeun Kim, Daeun Jeong P37 Aberrant expression of ERVWE1 endogenous retrovirus and overexpression of TET dioxygenases are characteristic features of seminoma Katerina Trejbalova, Martina Benesova, Dana Kucerova, Zdenka Vernerova, Rachel Amouroux, Petra Hajkova, Jiri Hejnar P38 Life history of the oldest lentivirus: characterisation of ELVgv integrations and the TRIM5 selection pattern in dermoptera Daniel Elleder, Tomas Hron, Helena Farkasova, Abinash Padhi, Jan Paces P39 Characterisation of a highly divergent endogenous retrovirus in the equine germ line Henan Zhu, Robert Gifford, Pablo Murcia P40 The emergence of pandemic retroviral infection in small ruminants Maria Luisa Carrozza, Anna-Maria Niewiadomska, Maurizio Mazzei, Mounir Abi-Said, Joseph Hughes, Stéphane Hué, Robert Gifford P41 Near full-length genome (NFLG) Characterisation of HIV-1 subtype B identified in South Africa Adetayo Obasa, Graeme Jacobs, Susan Engelbrecht P42 Acquisition of Vpu-mediated tetherin antagonism by an HIV-1 group O strain Katharina Mack, Kathrin Starz, Daniel Sauter, Matthias Geyer, Frederic Bibollet-Ruche, Christina Stürzel, Marie Leoz, Jean Christophe Plantier, Beatrice H. Hahn, Frank Kirchhoff P43 The human endogenous retrovirus type K is involved in cancer stem cell markers expression and in human melanoma malignancy Ayele Argaw-Denboba, Emanuela Balestrieri, Annalucia Serafino, Ilaria Bucci, Chiara Cipriani, Corrado Spadafora, Paolo Sinibaldi-Vallebona, Claudia Matteucci P44 Natural infection of Indian non-human primates by unique lentiviruses S. Nandi Jayashree, Ujjwal Neogi, Anil K. Chhangani, Shravan Sing Rathore, Bajrang R. J. Mathur P45 Free cervical cancer screening among HIV-positive women receiving antiretroviral treatment in Nigeria Adeyemi Abati P46 Molecular evolutionary status of feline immunodeficiency virus in Turkey B. Taylan Koç, Tuba Çiğdem Oğuzoğlu Topic 7: Innate sensing & intrinsic immunity P47 Cell-to-cell contact with HTLV-1-infected T cells reduces dendritic cell immune functions and contributes to infection in trans. Takatoshi Shimauchi, Stephan Caucheteux, Jocelyn Turpin, Katja Finsterbusch, Charles Bangham, Yoshiki Tokura, Vincent Piguet P48 Deciphering the mechanisms of HIV-1 exacerbation induced by Mycobacterium tuberculosis in monocytes/macrophages Shanti Souriant, Luciana Balboa, Karine Pingris, Denise Kviatcowsky, Brigitte Raynaud-Messina, Céline Cougoule, Ingrid Mercier, Marcelo Kuroda, Pablo González-Montaner, Sandra Inwentarz, Eduardo Jose Moraña, Maria del Carmen Sasiain, Olivier Neyrolles, Isabelle Maridonneau-Parini, Geanncarlo Lugo-Villarino, Christel Vérollet P49 The SAMHD1-mediated inhibition of LINE-1 retroelements is regulated by phosphorylation Alexandra Herrmann, Sabine Wittmann, Caitlin Shepard, Dominique Thomas, Nerea Ferreirós Bouzas, Baek Kim, Thomas Gramberg P50 Activities of nuclear envelope protein SUN2 in HIV infection Xavier Lahaye, Anvita Bhargava, Takeshi Satoh, Matteo Gentili, Silvia Cerboni, Aymeric Silvin, Cécile Conrad, Hakim Ahmed-Belkacem, Elisa C. Rodriguez, Jean-François Guichou, Nathalie Bosquet, Matthieu Piel, Roger Le Grand, Megan King, Jean-Michel Pawlotsky, Nicolas Manel P51 Activation of TLR7/8 with a small molecule agonist induces a novel restriction to HIV-1 infection of monocytes Henning Hofmann, Benedicte Vanwalscappel, Nicolin Bloch, Nathaniel Landau P52 Steady state between the DNA polymerase and Rnase H domain activities of reverse transcriptases determines the sensitivity of retroviruses to inhibition by APOBEC3 proteins Stanislav Indik, Benedikt Hagen P53 HIV restriction in mature dendritic cells is related to p21 induction and p21-mediated control of the dNTP pool and SAMHD1 activity. José Carlos Valle-Casuso, Awatef Allouch, Annie David, Françoise Barré-Sinoussi, Michaela Müller-Trutwin, Monsef Benkirane, Gianfranco Pancino, Asier Saez-Cirion P54 IFITM protens restrict HIV-1 protein synthesis Wing-Yiu Lee, Chen Liang, Richard Sloan P55 Characterisation and functional analysis of the novel restriction factor Serinc5 Bianca Schulte, Silvana Opp, Felipe Diaz-Griffero P56 piRNA sequences are common in Human Endogenous Retroviral Sequences (HERVs): An antiretroviral restriction mechanism? Jonas Blomberg, Luana Vargiu, Patricia Rodriguez-Tomé, Enzo Tramontano, Göran Sperber P57 Ferroportin restricts HIV-1 infection in sickle cell disease Namita Kumari, Tatiana Ammosova, Sharmeen Diaz, Patricia Oneal, Sergei Nekhai P58 APOBEC3G modulates the response to antiretroviral drugs in humanized mice Audrey Fahrny, Gustavo Gers-Huber, Annette Audigé, Roberto F. Speck, Anitha Jayaprakash, Ravi Sachidanandam, Matt Hernandez, Marsha Dillon-White, Viviana Simon P59 High-throughput epigenetic analysis of evolutionarily young endogenous retrovirus presents in the mule deer (Odocoileus hemionus) genome Tomas Hron, Helena Farkasova, Daniel Elleder P60 Characterisation of the expression of novel endogenous retroviruses and immune interactions in a macaque model Neil Berry, Emmanuel Maze, Claire Ham, Neil Almond, Greg Towers, Robert Belshaw P61 HIV-1 restriction by orthologs of SERINC3 and SERINC5 Patrícia de Sousa-Pereira, Joana Abrantes, Massimo Pizzato, Pedro J. Esteves, Oliver T. Fackler, Oliver T. Keppler, Hanna-Mari Baldauf P62 TRIM19/PML restricts HIV infection in a cell type-dependent manner Bianca Volkmann, Tanja Kahle, Kristin Eissmann, Alexandra Herrmann, Sven Schmitt, Sabine Wittmann, Laura Merkel, Nina Reuter, Thomas Stamminger, Thomas Gramberg P63 Recent invasion of the mule deer genome by a retrovirus Helena Farkasova, Tomas Hron, Daniel Elleder P64 Does the antiviral protein SAMHD1 influence mitochondrial function? Ilaria Dalla Rosa, Kate Bishop, Antonella Spinazzola, Harriet Groom P65 cGAMP transfers intercellularly via HIV-1 Env-mediated cell–cell fusion sites and triggers an innate immune response in primary target cells Shuting Xu, Aurélie Ducroux, Aparna Ponnurangam, Sergej Franz, Gabrielle Vieyres, Mathias Müsken, Thomas Zillinger, Angelina Malassa, Ellen Ewald, Veit Hornung, Winfried Barchet, Susanne Häussler, Thomas Pietschmann, Christine Goffinet P66 Pre-infection transcript levels of FAM26F in PBMCS inform about overall plasma viral load in acute and postacute phase after SIV-infection Ulrike Sauermann, Aneela Javed, Nicole Leuchte, Gabriela Salinas, Lennart Opitz, Christiane Stahl-Hennig, Sieghart Sopper P67 Sequence-function analysis of three T cell receptors targeting the HIV-1 p17 epitope SLYNTVATL Christiane Mummert, Christian Hofmann, Angela G. Hückelhoven, Silke Bergmann, Sandra M. Müller-Schmucker, Ellen G. Harrer, Jan Dörrie, Niels Schaft, Thomas Harrer P68 An immunodominant region of the envelope glycoprotein of small ruminant lentiviruses may function as decoy antigen Laure Cardinaux, M.-L. Zahno, H.-R. Vogt, R. Zanoni, G. Bertoni P69 Impact of immune activation, immune exhaustion, broadly neutralising antibodies and viral reservoirs on disease progression in HIV-infected children Maximilian Muenchhoff, Philip Goulder, Oliver Keppler Topic 9: Novel antiviral strategies P70 Identification of natural compounds as new antiviral products by bioassay-guided fractionation Alexandra Herrmann, Stephanie Rebensburg, Markus Helfer, Michael Schindler, Ruth Brack-Werner P71 The PPARG antagonism disconnects the HIV replication and effector functions in Th17 cells Yuwei Zhang, Huicheng Chen, Delphine Planas, Annie Bernier, Annie Gosselin, Jean-Pierre Routy, Petronela Ancuta P72 Characterisation of a multiresistant subtype AG reverse transcriptase: AZT resistance, sensitivity to RNase H inhibitors and inhibitor binding Birgitta Wöhrl, Anna Schneider, Angela Corona, Imke Spöring, Mareike Jordan, Bernd Buchholz, Elias Maccioni, Roberto Di Santo, Jochen Bodem, Enzo Tramontano, Kristian Schweimer P73 Insigths into the acetylation pattern of HDAC inhibitors and their potential role in HIV therapy Christian Schölz, Brian Weinert, Sebastian Wagner, Petra Beli, Yasuyuki Miyake, Jun Qi, Lars Jensen, Werner Streicher, Anna McCarthy, Nicholas Westwood, Sonia Lain, Jürgen Cox, Patrick Matthias, Matthias Mann, James Bradner, Chunaram Choudhary P74 HPV-derived and seminal amyloid peptides enhance HIV-1 infection and impair the efficacy of broadly neutralising antibodies and antiretroviral drugs Marcel Stern, Oliver T. Keppler P75 D(−)lentiginosine inhibits both proliferation and virus expression in cells infected by HTLV-1 in vitro Elena Valletta, Caterina Frezza, Claudia Matteucci, Francesca Marino-Merlo, Sandro Grelli, Anna Lucia Serafino, Antonio Mastino, Beatrice Macchi P76 HIV-1 resistance analyses of the Cape Winelands districts, South Africa Sello Mikasi, Graeme Jacobs, Susan Engelbrecht Topic 10: Recent advances in HIV vaccine development P77 Induction of complex retrovirus antigen-specific immune responses by adenovirus-based vectors depends on the order of vector administration Meike Kaulfuß, Sonja Windmann, Wibke Bayer P78 Direct impact of structural properties of HIV-1 Env on the regulation of the humoral immune response Rebecca Heß, Michael Storcksdieck gen. Bonsmann, Viktoria Stab, Carsten Kirschning, Bernd Lepenies, Matthias Tenbusch, Klaus Überla P79 Lentiviral virus-like particles mediate gerenration of T-follicular helper cells in vitro Anne Kolenbrander, Klaus Überla, Vladimir Temchura P80 Recruitment of HIV-1 Vpr to DNA damage sites and protection of proviral DNA from nuclease activity Kenta Iijima, Junya Kobayashi, Yukihito Ishizaka

COVID-19-induced ‘acute respiratory distress syndrome’ (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyzed pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single cell genomics, immunohistology and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not Influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS., SARS-CoV-2 infection, but not influenza A, triggers immunological and pathological changes in the lung that are hallmarks of pulmonary fibrosis. A subset of CD163+ macrophages are found to drive this fibroproliferative acute respiratory distress.