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The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. A comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structures than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Purpose of Review: Despite suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and reignite viral replication if therapy is interrupted. Persistence of the viral reservoir in people with HIV-1 (PWH) is the main obstacle to an HIV-1 cure. The reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals. Here, we review the recent progress in the characterization of persistent HIV-1 transcription during ART., Recent Findings: Evidence from several studies indicates that, although cell-associated unspliced (US) HIV-1 RNA is abundantly expressed in ART-treated PWH, intact full-length US transcripts are rare and most US RNA is derived from defective proviruses. The transcription- and translation-competent defective proviruses, previously considered irrelevant, are increasingly being linked to residual HIV-1 pathogenesis under suppressive ART. Recent data suggest a continuous crosstalk between the residual HIV-1 activity under ART and the immune system. Persistent HIV-1 transcription on ART, despite being mostly derived from defective proviruses, predicts viral rebound upon therapy interruption, suggesting its role as an indicator of the strength of the host antiviral immune response that is shaping the viral rebound., Summary: In light of the recent findings, the significance of persistent HIV-1 transcription during ART for the long-term health of PWH and the cure research should be reassessed., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Importance: Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA. It was previously shown that this inactivation was due to small inactivating mutations at the targeted sites in the HIV genome and to excision or inversion of the viral DNA fragment between two target sites. We, here, demonstrate that CRISPR-Cas treatment also causes large unintended deletions, which can include surrounding chromosomal sequences. As the loss of chromosomal sequences may cause oncogenic transformation of the cell, such unintended large deletions form a potential safety risk in clinical application of this antiviral application and possibly all CRISPR-Cas gene-editing approaches., Competing Interests: The authors declare no conflict of interest.

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Christian J Buchholz reports financial support was provided by National Institutes of Health. Christian J Buchholz, Vanessa Riechert has patent pending to Assignee., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates., Methods: Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes., Results: Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype., Conclusions: These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits., (© 2023. The Author(s).)

The development of prophylatic or therapeutic medicines for infectious diseases is one of the priorities for health organizations worldwide. Innovative solutions are required to achieve effective, safe, and accessible treatments for most if not all infectious diseases, particularly those that are chronic in nature or that emerge unexpectedly over time. Genetic technologies offer versatile possibilities to design therapies against pathogens. Recent developments such as mRNA vaccines, CRISPR gene editing, and immunotherapies provide unprecedented hope to achieve significant results in the field of infectious diseases. This review will focus on advances in this domain, showcasing the cross-fertilization with other fields ( e.g., oncology), and addressing some of the logistical and economic concerns important to consider when making these advances accessible to diverse populations around the world.

Background: The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a., Methods: We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells., Findings: We demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such "hypermutated" HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation., Interpretation: These results provide in vitro "proof of concept' for CRISPR-Cas12b mediated HIV-1 inactivation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA + cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir., Competing Interests: Conflict of interest V. R., J. H., K. C., and C. J. B. are listed as inventors on a filed patent describing FcγRIIA-specific DARPins., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an in vivo systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles. As the replication of these virus variants is activated by the human T-lymphotropic virus 1 (HTLV-1) Tax protein, a factor that reverses epigenetic silencing of episomal HIV DNA, these data indicate that the 3'PPT-mutated viruses escape from the integrase inhibitor DTG by switching to an integration-independent replication mechanism. IMPORTANCE The integrase inhibitor DTG is a potent inhibitor of HIV replication and is currently recommended in drug regimens for people living with HIV. Whereas HIV normally escapes from antiviral drugs by the acquisition of specific mutations in the gene that encodes the targeted enzyme, mutational inactivation of the viral 3'PPT sequence, an RNA element that has a crucial role in the viral reverse transcription process, was found to allow HIV replication in the presence of DTG in cell culture experiments. While the integration of the viral DNA into the cellular genome is considered one of the hallmarks of retroviruses, including HIV, 3'PPT inactivation caused integration-independent replication, which can explain the reduced DTG sensitivity. Whether this exotic escape route can also contribute to viral escape in HIV-infected persons remains to be determined, but our results indicate that screening for 3'PPT mutations in patients that fail on DTG therapy should be considered., Competing Interests: The authors declare no conflict of interest.

Despite decades of suppressive antiretroviral therapy, human immunodeficiency virus (HIV) reservoirs in infected individuals persist and fuel viral rebound once therapy is interrupted. The persistence of viral reservoirs is the main obstacle to achieving HIV eradication or a long-term remission. The last decade has seen a profound change in our understanding of the mechanisms behind HIV persistence, which appears to be much more complex than originally assumed. In addition to the persistence of transcriptionally silent proviruses in a stable latent reservoir that is invisible to the immune system, HIV is increasingly recognized to persist by resistance to the immune clearance, which appears to play a surprisingly prominent role in shaping the reservoir. In this review, we discuss some emerging insights into the mechanisms of HIV persistence, as well as their implications for the development of strategies towards an HIV cure., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus -RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals.

Background: Activation of RNA-dependent stress kinase PKR, especially by viral double-stranded RNA, induces eukaryotic initiation factor 2 α-chain (eIF2α) phosphorylation, attenuating thereby translation. We report that this RNA-mediated negative control mechanism, considered a cornerstone of the cell's antiviral response, positively regulates splicing of a viral mRNA., Results: Excision of the large human immunodeficiency virus (HIV) rev/tat intron depends strictly on activation of PKR by the viral RNA and on eIF2α phosphorylation. Rev/tat mRNA splicing was blocked by viral PKR antagonists Vaccinia E3L and Ebola VP35, as well as by a trans-dominant negative mutant of PKR, yet enhanced by overexpressing PKR. Expression of non-phosphorylatable mutant eIF2αS51A, but not of wild type eIF2α, abrogated efficient splicing of rev/tat mRNA. By contrast, expression of eIF2αS51D, a phosphomimetic mutant of eIF2α, left rev/tat mRNA splicing intact. Unlike eIF2αS51A, eIF2αS51D does not inhibit eIF2α phosphorylation by activated PKR. All HIV mRNA species contain terminal trans-activation response (TAR) stem-loop sequences that potentially could activate PKR, yet even upon TAR deletion, HIV mRNA production remained sensitive to inhibitors of PKR activation. Bioinformatic and mutational analyses revealed a compact RNA pseudoknot upstream of 3'-terminal TAR that promotes splicing by activating PKR. Supporting its essential role in control of splicing, this pseudoknot is conserved among diverse HIV and nonhuman primate SIVcpz isolates. The pseudoknot and 3'-terminal TAR collaborate in mediating PKR-regulated splicing of rev/tat intron, the pseudoknot being dominant., Conclusions: Our results on HIV provide the first example of a virus co-opting activation of PKR by its RNA, a cellular antiviral mechanism, to promote splicing. They raise the question whether other viruses may use local activation of host kinase PKR through RNA elements within their genome to achieve efficient splicing of their mRNA. Our experiments reveal an indispensable role for eIF2α phosphorylation in HIV rev/tat mRNA splicing that accounts for the need for PKR activation., (© 2023. The Author(s).)

Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure.

Purpose of Review: To summarize the current status and highlight recent findings on predictive biomarkers for posttreatment HIV control (PTC) and virological remission. While historically, many studies focused on virological markers, there is an increasing tendency to enter immune and metabolic factors into the equation., Recent Findings: On the virological side, several groups reported that cell-associated HIV RNA could predict time to viral rebound. Recent data hints at the possible importance of the genic location and chromatin context of the integrated provirus, although these factors still need to be assessed in relation to PTC and virological remission. Evidence from immunological studies highlighted innate and humoral immunity as important factors for prolonged HIV remission. Interestingly, novel metabolic markers have emerged, which offer additional angles to our understanding of latency and viral rebound., Summary: Facilitating PTC and virological remission remain top priorities for the HIV cure research. We advocate for clear and precise definitions for both phenomena in order to avoid misconceptions and to strengthen the conclusions that can be drawn. As no one-size-fits-all marker has emerged yet, more biomarkers are on the horizon, and viral rebound is a complex and heterogeneous process, it is likely that a combination of various biomarkers in cohesion will be necessary for a more accurate prediction of antiretroviral therapy-free HIV remission., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

There is a strong evolutionary tendency of the human immunodeficiency virus (HIV) to accumulate A nucleotides in its RNA genome, resulting in a mere 40 per cent A count. This A bias is especially dominant for the so-called silent codon positions where any nucleotide can be present without changing the encoded protein. However, particular silent codon positions in HIV RNA refrain from becoming A, which became apparent upon genome analysis of many virus isolates. We analyzed these 'noA' genome positions to reveal the underlying reason for their inability to facilitate the A nucleotide. We propose that local RNA structure requirements can explain the absence of A at these sites. Thus, noA sites may be prominently involved in the correct folding of the viral RNA. Turning things around, the presence of multiple clustered noA sites may reveal the presence of important sequence and/or structural elements in the HIV RNA genome., (© The Author(s) 2022. Published by Oxford University Press.)

Background: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC)., Methods: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4 + T-cell models and ex vivo cultures of PBMCs from HIV + individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24 Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA., Findings: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations., Interpretation: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV + patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies., Funding: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and harbors many mutations in the spike protein, but the origin of this virus variant remains unclear. We address the role of unusual virus evolution mechanisms such as hypermutation, out-of-frame reading, and recombination. Rather, regular Darwinian evolution, that is, the repeated selection of beneficial spike mutations, seems to have led to the appearance of the grossly altered spike protein of the Omicron variant.

Set-point viral load (SPVL), a common measure of human immunodeficiency virus (HIV)-1 virulence, is partially determined by viral genotype. Epidemiological evidence suggests that this viral property has been under stabilising selection, with a typical optimum for the virus between 10 4 and 10 5 copies of viral RNA per ml. Here we aimed to detect transmission fitness differences between viruses from individuals with different SPVLs directly from phylogenetic trees inferred from whole-genome sequences. We used the local branching index (LBI) as a proxy for transmission fitness. We found that LBI is more sensitive to differences in infectiousness than to differences in the duration of the infectious state. By analysing subtype-B samples from the Bridging the Evolution and Epidemiology of HIV in Europe project, we inferred a significant positive relationship between SPVL and LBI up to approximately 10 5 copies/ml, with some evidence for a peak around this value of SPVL. This is evidence of selection against low values of SPVL in HIV-1 subtype-B strains, likely related to lower infectiousness, and perhaps a peak in the transmission fitness in the expected range of SPVL. The less prominent signatures of selection against higher SPVL could be explained by an inherent limit of the method or the deployment of antiretroviral therapy., (© The Author(s) 2022. Published by Oxford University Press.)

CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack. Here, we tested the antiviral activity of seven dual crRNA combinations and analyzed the HIV proviral genomes for mutations at the target sites. We demonstrated that dual crRNA combinations exhibit more robust antiviral activity than a single crRNA attack and, more important, that the dual-crRNA therapy can prevent virus escape in long-term cultures. We confirmed the absence of any replication-competent virus in these apparently cured cultures. Surprisingly, we did not detect excision of the HIV sequences located between two Cas12a cleavage sites. Instead, we observed almost exclusively HIV inactivation by "hypermutation," that is, the introduction of indel mutations at both target sites due to the error-prone cellular DNA repair machinery., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics.

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log 10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence.

Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the stable expression of CRISPR-Cas9 or Cas12a systems targeting HIV DNA. Because lentiviral transduction and long-term CRISPR-Cas activity are less suitable for in vivo application of this antiviral strategy, we investigated whether HIV can also be completely inactivated by transient CRISPR-Cas activity. Latently infected SupT1 T-cells were repeatedly transfected with different Cas9 and Cas12a mRNA/protein sources in combination with dual gRNAs/crRNAs targeting highly conserved viral sequences. Upon repeated Cas9 protein treatment, viral replication could no longer be reactivated. We demonstrate that this was due to complete mutational inactivation of the proviral DNA, mostly through mutations at the target sites, but also through excision or inversion of the viral DNA fragment between the two target sites. These results demonstrate that repeated transient CRISPR-Cas treatment of a latently infected T-cell culture can lead to the permanent inactivation of HIV replication, indicating that transient CRISPR-Cas delivery methods can be considered for in vivo application.

Zhang et al. (Proc Natl Acad Sci 118:e2105968118, 2021) recently reported that SARS-CoV-2 RNA can be retrotranscribed and integrated into the DNA of human cells by the L1 retrotransposon machinery. This phenomenon could cause persistence of viral sequences in patients and may explain the prolonged PCR-positivity of SARS-CoV-2 infected patients, even long after the phase of active virus replication has ended. This commentary does critically review the available data on this topic and discusses them in the context of findings made for other exogenous viruses and ancestral endogenous retroviral elements., (© 2021. The Author(s).)

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of CD4 + T-cells associated with HTLV-1 infection. In this study, we used the model of immunodeficient NSG mice reconstituted with a functional human immune system (HIS) to investigate early events in HTLV-1 pathogenesis. Upon infection, human T-cells rapidly increased in the blood and lymphoid tissues, particularly CD4 + CD25 + T-cells. Proliferation of CD4 + T-cells in the spleen and mesenteric lymph nodes (MLN) correlated with HTLV-1 proviral load and CD25 expression. In addition, splenomegaly, a common feature of ATLL in humans, was also observed. CD4 + and CD8 + T-cells predominantly displayed an effector memory phenotype (CD45RA - CCR7 - ) and expressed CXCR3 and CCR5 chemokine receptors, suggesting the polarization into a Th1 phenotype. Activated CD8 + T-cells expressed granzyme B and perforin; however, the interferon-γ response by these cells was limited, possibly due to elevated PD-1 expression and increased frequency of CD4 + FoxP3 + regulatory T-cells in MLN. Thus, HTLV-1-infected HIS-NSG mice reproduced several characteristics of infection in humans, and it may be helpful to investigate ATLL-related events and to perform preclinical studies. Moreover, aspects of chronic infection were already present at early stages in this experimental model. Collectively, we suggest that HTLV-1 infection modulates host immune responses to favor viral persistence.

Antiretroviral therapy (ART) suppresses HIV-1 replication but does not eradicate the virus. Persistence of HIV-1 latent reservoirs in ART-treated individuals is considered the main obstacle to achieving an HIV-1 cure. However, these HIV-1 reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals. HIV-1 latency is regulated at the transcriptional and at multiple post-transcriptional levels. Here, we review recent insights into the possible contribution of viral RNA processing to the persistence of HIV-1 reservoirs, and discuss the clinical implications of persistence of viral RNA species in ART-treated individuals.

Background: It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress human immunodeficiency virus (HIV) replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI)., Methods: CA HIV unspliced RNA and total HIV DNA were quantified in two cohorts (n = 100, n = 124) of individuals treated with triple ART regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either an NNRTI or a PI. To compare CA HIV RNA and DNA levels between the regimens, we built multivariable models adjusting for age, gender, current and nadir CD4 + count, plasma viral load zenith, duration of virological suppression, NRTI backbone composition, low-level plasma HIV RNA detectability, and electronically measured adherence to ART., Results: In both cohorts, levels of CA HIV RNA and DNA strongly correlated (rho = 0.70 and rho = 0.54) and both markers were lower in NNRTI-treated than in PI-treated individuals. In the multivariable analysis, CA RNA in both cohorts remained significantly reduced in NNRTI-treated individuals (p adj = 0.02 in both cohorts), with a similar but weaker association between the ART regimen and total HIV DNA (p adj = 0.048 and p adj = 0.10). No differences in CA HIV RNA or DNA levels were observed between individual NNRTIs or individual PIs, but CA HIV RNA was lower in individuals treated with either nevirapine or efavirenz, compared to PI-treated individuals., Conclusions: All current classes of antiretroviral drugs only prevent infection of new cells but do not inhibit HIV RNA transcription in long-lived reservoir cells. Therefore, these differences in CA HIV RNA and DNA levels by treatment regimen suggest that NNRTIs are more potent in suppressing HIV residual replication than PIs, which may result in a smaller viral reservoir size., Funding: This work was supported by ZonMw (09120011910035) and FP7 Health (305522)., Competing Interests: AP, JV, NK, FW, Md, DD, MB, CS, AW, JP, PR, BB No competing interests declared, (© 2021, Pasternak et al.)

The highly conserved trans-acting response element (TAR) present in the RNA genome of human immunodeficiency virus 1 (HIV-1) is a stably folded hairpin structure involved in viral replication. However, TAR is also sensed by viral sensors, leading to antiviral immunity. While high variation in the TAR RNA structure renders the virus replication-incompetent, effects on viral sensing remain unclear. Here, we investigated the role of TAR RNA structure and stability on viral sensing. TAR mutants with deletions in the TAR hairpin that enhanced thermodynamic stability increased antiviral responses. Strikingly, TAR mutants with lower stability due to destabilization of the TAR hairpin also increased antiviral responses without affecting pro-inflammatory responses. Moreover, mutations that affected the TAR RNA sequence also enhanced specific antiviral responses. Our data suggest that mutations in TAR of replication-incompetent viruses can still induce immune responses via viral sensors, hereby underscoring the robustness of HIV-1 RNA sensing mechanisms.

Purpose of Review: Despite decades of suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and fuel viral rebound if therapy is interrupted. The persistence of viral reservoirs in infected individuals is the main obstacle to achieving HIV-1 eradication or a long-term remission. Accurate assessment of the viral reservoir size is necessary for monitoring the effectiveness of the curative interventions. Here, we review the recent progress in the development of assays to measure HIV-1 persistence, highlighting their key advantages and limitations., Recent Findings: To estimate the viral reservoir size, a number of assays have been developed that assess different aspects of HIV-1 persistence in ART-treated individuals. These include viral outgrowth assays to measure proviral replication competence, sequencing-based assays to measure genetic intactness of HIV-1 proviruses, and diverse techniques that measure the ability of proviruses to produce viral RNA and/or proteins (transcription and translation competence), with or without ex vivo stimulation. Recent years have seen the development of next-generation reservoir assays that, in addition to measuring viral persistence markers, assess the proviral integration sites and characterize the HIV-1 reservoir cells on the single-cell level., Summary: Although no assay yet can measure the HIV-1 reservoir with 100% accuracy, recent technical advances allow reliable estimation of its size and composition., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Incomplete restoration of CD4 + T-cell counts on antiretroviral therapy (ART) is a major predictor of HIV-related morbidity and mortality. To understand the possible mechanisms behind this poor immunological response despite viral suppression, we longitudinally measured more than 50 virological and immunological biomarkers in a cohort of HIV-infected individuals at several time points during the first 96 weeks of virologically suppressive ART. No baseline virological or immunological marker was predictive of the degree of immune reconstitution. However, the cell-associated HIV-1 unspliced-to-multiply-spliced (US/MS) RNA ratio at 12 weeks of ART positively correlated with markers of CD4 + T-cell activation and apoptosis and negatively predicted both the absolute and relative CD4 + T-cell counts at 48 and 96 weeks. A higher US/MS RNA ratio may reflect the higher frequency of productively infected cells that could exert pressure on the immune system, contributing to persistent immune activation and apoptosis and subsequently to a poor immunological response to ART. IMPORTANCE Human immunodeficiency virus (HIV) infection is currently managed by antiretroviral drugs, which block virus replication and promote immune restoration. However, the latter effect is not universal, with a proportion of infected individuals failing to sufficiently reconstitute their immune function despite a successful virological response to antiretroviral therapy (ART). No reliable predictive markers of immunological failure have been identified, and there is still no efficient therapeutic strategy, apart from ART itself, to facilitate immune reconstitution. Here, we measured more than 50 viral and host biomarkers at five time points during the first 2 years of ART and identified the cell-associated HIV-1 unspliced-to-multiply-spliced RNA ratio at 12 weeks of ART as a predictive factor for the immunological response to therapy. Moreover, the same marker positively correlated with markers of CD4 + T-cell activation and apoptosis. The fact that a virological biomarker performed better than any immunological biomarker in predicting an immunological outcome highlights the importance of considering the residual HIV activity on ART as a correlate and a possible cause of the residual immune dysfunction that frequently occurs despite virologically suppressive ART., (Copyright © 2021 Scherpenisse et al.)

Combination antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) replication and improves immune function. However, due to the persistence of long-lived HIV reservoirs, therapy interruption almost inevitably leads to a fast viral rebound. A small percentage of individuals who are able to control HIV replication for extended periods after therapy interruption are of particular interest because they may represent a model of long-term HIV remission without ART. These individuals are characterized by a limited viral reservoir and low reservoir measures can predict post-treatment HIV remission. However, most individuals with a low reservoir still experience fast viral rebound. In this Perspective, we discuss the possible reasons behind this and propose to develop an integral profile, composed of viral and host biomarkers, that could allow the accurate prediction of post-treatment HIV remission. We also propose to incorporate information on the chromatin context of the proviral integration sites into the characterization of the HIV reservoir, as this likely influences the reactivation capacity of latent proviruses and, together with the actual number of intact proviruses, contributes to the replication competence of the reservoir., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pasternak, Psomas and Berkhout.)

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18 base pairs (bp) stem and a 3-5 nucleotides (nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5' and 3'-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5' terminus, but the Pol III enzyme generates small RNAs with 3' U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both)., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis -acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions. IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution., (Copyright © 2020 Chen et al.)

Studies of vertebrate genomes have indicated that all species contain in their chromosomes stretches of DNA with sequence similarity to viral genomes. How such 'endogenous' viral elements (EVEs) ended up in host genomes is usually explained in general terms such as 'they entered the germ line at some point during evolution'. This seems a correct statement, but is also rather imprecise. The vast number of endogenous viral sequences suggest that common routes to the 'germ line' may exist, as relying on chance alone may not easily explain the abundance of EVEs in modern mammalian genomes. An increasing number of virus types have been detected in human semen and a growing number of studies have reported on viral infections that cause male infertility or subfertility and on viral infections that threaten in vitro fertilisation practices. Thus, it is timely to survey the pathway(s) that viruses can use to gain access to the human germ line. Embryo transfer and semen quality studies in livestock form another source of relevant information because virus infection during reproduction is clearly unwanted, as is the case for the human situation. In this review, studies on viruses in the male and female reproductive tract and in the early embryo will be discussed to propose a plausible viral route to the mammalian germ line., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tools based on RNA interference (RNAi) and the recently developed clustered regularly short palindromic repeats (CRISPR) system enable the selective modification of gene expression, which also makes them attractive therapeutic reagents for combating HIV infection and other infectious diseases. Several parallels can be drawn between the RNAi and CRISPR-Cas9 platforms. An ideal RNAi or CRISPR-Cas9 therapeutic strategy for treating infectious or genetic diseases should exhibit potency, high specificity and safety. However, therapeutic applications of RNAi and CRISPR-Cas9 have been challenged by several major limitations, some of which can be overcome by optimal design of the therapy or the design of improved reagents. In this review, we will discuss some advantages and limitations of anti-HIV strategies based on RNAi and CRISPR-Cas9 with a focus on the efficiency, specificity, off-target effects and delivery methods., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In adherent individuals, antiretroviral therapy (ART) suppresses HIV replication, restores immune function, and prevents the development of AIDS. However, ART is not curative and has to be followed lifelong. Persistence of viral reservoirs forms the major obstacle to an HIV cure. HIV latent reservoirs persist primarily by cell longevity and proliferation, but replenishment by residual virus replication despite ART has been proposed as another potential mechanism of HIV persistence. It is a matter of debate whether different ART regimens are equally potent in suppressing HIV replication. Here, we summarized the current knowledge on the role of ART regimens in HIV persistence, focusing on differences in residual plasma viremia and other virological markers of the HIV reservoir between infected individuals treated with combination ART composed of different antiretroviral drug classes.

Plasma viral load (VL) and CD4+ T cell count are widely used as biomarkers of HIV type 1 (HIV-1) replication, pathogenesis, and response to antiretroviral therapy (ART). However, the clinical potential of cell-associated (CA) HIV-1 molecular markers is much less understood. Here, we measured CA HIV-1 RNA and DNA in HIV-infected individuals treated with temporary ART initiated during primary HIV-1 infection. We demonstrate substantial predictive value of CA RNA for (a) the virological and immunological response to early ART, (b) the magnitude and time to viral rebound after discontinuation of early ART, and (c) disease progression in the absence of treatment. Remarkably, when adjusted for CA RNA, plasma VL no longer appeared as an independent predictor of any clinical endpoint in this cohort. The potential of CA RNA as an HIV-1 clinical marker, in particular as a predictive biomarker of virological control after stopping ART, should be explored in the context of HIV-1 curative interventions.

Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target sites correlated with permanent virus inactivation. To better understand the mechanism underlying HIV inactivation, we analyzed the proviral DNA upon Cas9 attack with gRNA pairs. We observed that dual-gRNA targeting resulted more frequently in target site mutation than fragment excision, while fragment inversion was rarely observed. The frequencies varied for different gRNA combinations without an obvious relationship with the distance between the target sites, indicating that other gRNA and target DNA characteristics influence the DNA cleavage and repair processes.

The HIV latent reservoir forms the major hurdle to an HIV cure. The discovery of CD32 as marker of this reservoir has aroused much interest, but subsequent reports have challenged this finding. Here, we observe a positive correlation between the percentages of CD32 + cells among CD4 + T cells of aviremic cART-treated, HIV-infected individuals and their HIV DNA loads in peripheral blood. Moreover, optimization of the CD32 + CD4 + T cell purification protocol reveals prominent enrichment for HIV DNA (mean, 292-fold) in these cells. However, no enrichment for HIV RNA is observed in CD32 + CD4 + cells, yielding significantly reduced HIV RNA/DNA ratios. Furthermore, HIV proviruses in CD32 + CD4 + cells can be reactivated ex vivo to produce virus, strongly suggesting that these cells support HIV transcriptional latency. Our results underscore the importance of isolating pure, bona fide CD32 + CD4 + T cells for future studies and indicate that CD32 remains a promising candidate marker of the HIV reservoir., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent studies have shown the potential of broadly neutralizing antibodies (bnAbs) for HIV-1 treatment. One of the candidate antibodies moving into clinical trials is the bnAb PGDM1400. Here, we studied the therapeutic potency and escape pathways of bnAb PGDM1400 during monovalent therapy in human immune system (HIS) mice using the BG505, REJO, MJ4 and AMC008 virus isolates. PGDM1400 administered during chronic infection caused a modest decrease in viral load in the first week of administration in 7 out of 10 animals, which correlated with the in vitro neutralization sensitivity of the viruses to PGDM1400. As expected for monotherapy, viral loads rebounded after about a week and different viral escape pathways were observed, involving the deletion of glycans in the envelope glycoprotein at positions 130 or 160. (Pre)clinical trials should reveal whether PGDM1400 is a useful component of an antibody combination treatment or as part of a tri-specific antibody.

Current antiretroviral drugs can efficiently block HIV replication and prevent transmission, but do not target the HIV provirus residing in cells that constitute the viral reservoir. Because drug therapy interruption will cause viral rebound from this reservoir, HIV-infected individuals face lifelong treatment. Therefore, novel therapeutic strategies are being investigated that aim to permanently inactivate the proviral DNA, which may lead to a cure. Multiple studies showed that CRISPR-Cas9 genome editing can be used to attack HIV DNA. Here, we will focus on not only how this endonuclease attack can trigger HIV provirus inactivation, but also how virus escape occurs and this can be prevented., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Combination antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) replication and improves immune function, but is unable to eradicate the virus. Therefore, development of an HIV cure has become one of the main priorities of the HIV research field. The main obstacle for an HIV cure is the formation of latent viral reservoirs, where the virus is able to "hide" despite decades of therapy, just to reignite active replication once therapy is stopped. Revealing HIV hiding places is thus central to HIV cure research, but the absence of markers of these reservoir cells greatly complicates the search for a cure. Identification of one or several marker(s) of latently infected cells would represent a significant step forward toward a better description of the cell types involved and improved understanding of HIV latency. Moreover, it could provide a "handle" for selective therapeutic targeting of the reservoirs. A number of cellular markers of HIV reservoir have recently been proposed, including immune checkpoint molecules, CD2, and CD30. CD32a is perhaps the most promising of HIV reservoir markers as it is reported to be associated with a very prominent enrichment in HIV DNA, although this finding has been challenged. In this review, we provide an update on the current knowledge about HIV reservoir markers. We specifically highlight studies that characterized markers of persistently infected cells in the lymphoid tissues., (Copyright © 2019 Darcis, Berkhout and Pasternak.)