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The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.

Viruses in the family Retroviridae are found in a wide variety of vertebrate hosts. Enveloped virions are 80–100 nm in diameter with an inner core containing the viral genome and replicative enzymes. Core morphology is often characteristic for viruses within the same genus. Replication involves reverse transcription and integration into host cell DNA, resulting in a provirus. Integration into germline cells can result in a heritable provirus known as an endogenous retrovirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Retroviridae, which is available at ictv.global/report/retroviridae.

Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.

The Eleventh International Foamy Virus Conference took place on 9–10 June 2016 at the Institut Pasteur, Paris, France. The meeting reviewed progress on foamy virus (FV) research, as well as related current topics in retrovirology. FVs are complex retroviruses that are widespread in several animal species. Several research topics on these viruses are relevant to human health: cross-species transmission and viral emergence, vectors for gene therapy, development of antiretroviral drugs, retroviral evolution and its influence on the human genome. In this article, we review the conference presentations on these viruses and highlight the major questions to be answered.

Here we report a possible mechanistic link between coronavirus disease 2019 (COVID-19) and diabetes. In addition to its known role on the respiratory system, the human coronavirus SARS-CoV-2 has been shown to affect the endocrine system including the pancreas 1-4. It has been suggested that the virus can induce type 1 diabetes 5-8. Therefore, we isolated human pancreatic islets and examined the expression of angiotensin-converting enzyme 2 (ACE2) and the protease TMPRSS2, known to be important for SARS-CoV-2 entry 9. Furthermore, we investigated the expression of an alternative entry receptor, dipeptidyl peptidase-4 (DPP4 also known as CD26) 10. We found all three proteins expressed in pancreatic beta-cells and confirmed that beta-cells are permissive to infection with SARS-CoV-2 pseudoviruses. Additionally, we performed a comprehensive analysis of ACE2, TMPRSS2 and DPP4 expression in pancreata of 10 patients who died of COVID-19. We report significant variation between the samples and detected the highest levels of ACE2 and DPP4 expression in patients exhibiting SARS-CoV-2 infiltration shown by confocal microscopy, RNAscope and electron microscopy. Furthermore, necroptotic cell death was observed in beta-cells of the COVID-19 patients. Taken together, these data suggest that SARS-CoV-2 viral infection of pancreatic beta-cells may trigger necroptosis and islet impairment.

The family of human APOBEC3 (A3) restriction factors is formed by seven different proteins, A3A-D and A3F-H. Among these A3s, A3B harbors strong restriction activity against several retroviruses, such as SIV, and MLV. How lentiviruses and other retroviruses, prevalent in many primate species, counteract A3B is poorly understood. In this study, we found that A3B strongly inhibited SIVmac and HIV-2 infectivity, which was antagonized by their Vif proteins. Both SIVmac and HIV-2 Vifs diminished the protein level of A3B in viral producer cells, and hindered A3B incorporation into viral particles. We observed that HIV-2 Vif binds A3B and induces its degradation by assembly of an A3-Vif-CUL5-ElonginB/C E3-ligase complex. A3B and HIV-2 Vif localize and interact in the nucleus. In addition, we also found that the accessory protein Bet of prototype foamy virus (PFV) significantly antagonized the anti-SIVmac activity of A3B. Like Vif, Bet prevented the incorporation of A3B into viral particles. However, in contrast to Vif Bet did not induce the degradation of A3B. Rather, Bet binds A3B to block formation of high molecular weight A3B complexes and induces A3B cytoplasmic trapping. In summary, these findings indicate that A3B is recognized by diverse retroviruses and counteracted by virus-specific pathways that could be targeted to inhibit A3B mutating activity in cancers.

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders. Keywords: foamy virus, spumavirus, viral vector, gene therapy, vector tropism, bioimaging, hippocampus

Spumaretroviruses, commonly referred to as foamy viruses, are complex retroviruses belonging to the subfamily Spumaretrovirinae, family Retroviridae, which naturally infect a variety of animals including nonhuman primates (NHPs). Additionally, cross-species transmissions of simian foamy viruses (SFVs) to humans have occurred following exposure to tissues of infected NHPs. Recent research has led to the identification of previously unknown exogenous foamy viruses, and to the discovery of endogenous spumaretrovirus sequences in a variety of host genomes. Here, we describe an updated spumaretrovirus taxonomy that has been recently accepted by the International Committee on Taxonomy of Viruses (ICTV) Executive Committee, and describe a virus nomenclature that is generally consistent with that used for other retroviruses, such as lentiviruses and deltaretroviruses. This taxonomy can be applied to distinguish different, but closely related, primate (e.g., human, ape, simian) foamy viruses as well as those from other hosts. This proposal accounts for host-virus co-speciation and cross-species transmission.

The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A–H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFVmac) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards.

Human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs), provide access to hard-to-obtain cells for studies under physiological and disease conditions. For the study of neurodegenerative diseases, especially sporadic cases where the “disease condition” might be restricted towards the neuroectodermal lineage, obtaining the affected neurons is important to help unravel the underlying molecular mechanism leading to the diseases. Although differentiation of iPSCs to neural lineage allows acquisition of cell types of interest, the technology suffers from low efficiency leading to low yield of neurons. Here, we investigated the potential of adult neuroprogenitor cells (aNPCs) for iPSC derivation and possible confounders such as cell density of infected NPCs on their subsequent neuronal differentiation potential from reprogrammed cells under isogenic conditions. Characterized hiPSCs of defined cell densities generated from aNPCs were subjected to neuronal differentiation on PA6 stromal cells. The results showed that hiPSC clones obtained from low seeding density (iPSC-aNPCLow) differentiated less efficiently compared to those from higher density (iPSC-aNPCHigh). Our findings might help to further improve the yield and quality of neurons for in vitro modelling of neurodegenerative diseases.

International audience; Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae, and fungi (1, 2). This broad distribution suggests the ancient origin(s) of these viruses, possibly [...]

Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1,2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

NK cells are emerging as new effectors for immunotherapy of cancer. In particular, the genetic engraftment of chimeric Ag receptors (CARs) in NK cells is a promising strategy to redirect NK cells to otherwise NK cell–resistant tumor cells. On the basis of DNAX-activation protein 12 (DAP12), a signaling adaptor molecule involved in signal transduction of activating NK cell receptors, we generated a new type of CAR targeting the prostate stem cell Ag (PSCA). We demonstrate in this article that this CAR, designated anti–PSCA-DAP12, consisting of DAP12 fused to the anti-PSCA single-chain Ab fragment scFv(AM1) confers improved cytotoxicity to the NK cell line YTS against PSCA-positive tumor cells when compared with a CAR containing the CD3ζ signaling chain. Further analyses revealed phosphorylation of the DAP12-associated ZAP-70 kinase and IFN-γ release of CAR-engineered cells after contact with PSCA-positive target cells. YTS cells modified with DAP12 alone or with a CAR bearing a phosphorylation-defective ITAM were not activated. Notably, infused YTS cells armed with anti–PSCA-DAP12 caused delayed tumor xenograft growth and resulted in complete tumor eradication in a significant fraction of treated mice. The feasibility of the DAP12-based CAR was further tested in human primary NK cells and confers specific cytotoxicity against KIR/HLA-matched PSCA-positive tumor cells, which was further enhanced by KIR-HLA mismatches. We conclude that NK cells engineered with DAP12-based CARs are a promising tool for adoptive tumor immunotherapy.

Foamy viruses are non-pathogenic retroviruses and represent a tool for vector development. For gene therapy applications and for analyses of viral protein composition infectious particles need to be purified, which has been difficult for foamy viruses in the past. Here, we describe a novel, simple, and fast purification method for prototype foamy viruses with high purity using size exclusion and affinity chromatography. More than 99,9% of the contaminating proteins were removed. The purified viruses were used to determine the amount of the incorporated Pol protein relative to Gag. The determined Gag to Pol PR-RT ratio of 30:1 confirmed previous studies suggesting FV virions encapsidate fewer number of Pol molecules than orthoretroviruses.

The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1β production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti–RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold., Author Summary Foamyviruses (FVs) or Spuma-retroviruses derive their name from the cytopathic effects they cause in cell culture. However, infection in humans is benign and FVs have entered the human population through zoonosis from apes resulting in the emergence of Prototype FV (PFV). Like all retroviruses, FVs contain gag, pol and env structural genes and replicate through reverse-transcription and host genome integration. Gag, the major structural protein, is required for genome packaging, virion assembly, trafficking and egress. However, although functionally equivalent, FV and orthoretroviral Gag share little sequence homology and it is unclear how they perform the same function. Therefore, to understand more about relationship between FV and orthoretroviral replication we have carried out structural studies of PFV-Gag. Here we present the structure of CA domains from a central region PFV-Gag and show that despite little sequence similarity they share the same fold as the CA domains of orthoretroviral Gag. These data provide the first information relating the Spuma and Orthoretrovirinae Gag proteins. We discuss our findings in terms of evolutionary divergence of spuma and orthoretroviral lineages.

Human diseases of zoonotic origin are a major public health problem. Simian foamy viruses (SFVs) are complex retroviruses which are currently spilling over to humans. Replication-competent SFVs persist over the lifetime of their human hosts, without spreading to secondary hosts, suggesting the presence of efficient immune control. Accordingly, we aimed to perform an in-depth characterization of neutralizing antibodies raised by humans infected with a zoonotic SFV. We quantified the neutralizing capacity of plasma samples from 58 SFV-infected hunters against primary zoonotic gorilla and chimpanzee SFV strains, and laboratory-adapted chimpanzee SFV. The genotype of the strain infecting each hunter was identified by direct sequencing of the env gene amplified from the buffy coat with genotype-specific primers. Foamy virus vector particles (FVV) enveloped by wild-type and chimeric gorilla SFV were used to map the envelope region targeted by antibodies. Here, we showed high titers of neutralizing antibodies in the plasma of most SFV-infected individuals. Neutralizing antibodies target the dimorphic portion of the envelope protein surface domain. Epitopes recognized by neutralizing antibodies have been conserved during the cospeciation of SFV with their nonhuman primate host. Greater neutralization breadth in plasma samples of SFV-infected humans was statistically associated with smaller SFV-related hematological changes. The neutralization patterns provide evidence for persistent expression of viral proteins and a high prevalence of coinfection. In conclusion, neutralizing antibodies raised against zoonotic SFV target immunodominant and conserved epitopes located in the receptor binding domain. These properties support their potential role in restricting the spread of SFV in the human population., Author summary Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. Simian foamy viruses (SFVs) can be transmitted to humans, in whom they establish persistent infection, as have the simian lenti- and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 (HIV-1) and human T lymphotropic virus type 1 (HTLV-1). Such cross-species transmission of SFV is ongoing in many parts of the world where humans have contact with nonhuman primates. We present the first comprehensive study of neutralizing antibodies in SFV-infected humans. We showed high titers of neutralizing antibodies in the plasma of most SFV-infected individuals. Neutralizing antibodies target the dimorphic portion of the envelope protein surface domain that overlap with the receptor binding domain. SFV-specific antibodies target epitopes conserved over 8 million years of co-speciation with their nonhuman primate host. Greater neutralization potency in infected individuals was statistically associated with smaller SFV-related hematological changes. In conclusion, our results suggest the protective action of neutralizing antibodies against SFV infection and spread in the human population.

SUMMARY Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3′ exonuclease and deoxy-nucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments.

Summary Foamy viruses (FVs), a unique type of retroviruses, are characterized by several unusual features in their replication strategy. FVs, common to all non-human primates and several other species, display an extremely broad tropism in vitro. Basically, all mammalian cells and species examined, but also cells of amphibian or bird origin, are permissive to FV glycoprotein (Env)-mediated capsid release into the cytoplasm. The nature of the broadly expressed, and potentially evolutionary conserved, FV entry receptor molecule(s) is poorly characterized. Although recent data indicate that proteoglycans serve as an important factor for FV Env-mediated target cell attachment, additional uncharacterized molecules appear to be essential for the pH-dependent fusion of viral and cellular lipid membranes after endocytic uptake of virions. Furthermore, FVs show a very special assembly strategy. Unlike other retroviruses, the FV capsid precursor protein (Gag) undergoes only very limited proteolytic processing during assembly. This results in an immature morphology of capsids found in released FV virions. In addition, the FV Gag protein appears to lack a functional membrane-targeting signal. As a consequence, FVs utilize a specific interaction between capsid and cognate viral glycoprotein for initiation of thebudding process. Genetic fusion of heterologous targeting domains for plasma but not endosomal membranes to FV Gag enables glycoprotein-independent particle egress. However, this is at the expense of normal capsid morphogenesis and infectivity. The low-level Gag precursor processing and the requirement for a reversible, artificial Gag membrane association for effective pseudotyping of FV capsids by heterologous glycoproteins strongly suggest that FVs require a transient interaction of capsids with cellular membranes for viral replication. Under natural condition, this appears to be achieved by the lack of a membrane-targeting function of the FV Gag protein and the accomplishment of capsid membrane attachment through an unusual specific interaction with the cognate glycoprotein.

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells., Author Summary Viruses are masters at exploiting host cell machineries for their replication. For human immunodeficiency virus type 1 (HIV-1), the best-studied representative of the Orthoretrovirinae subfamily from the genus lentiviruses, numerous important virus-host interactions have been described. In contrast, only a few cellular proteins are known to influence the replication of foamy viruses (FVs, also known as spumaviruses), an intriguing type of complex retrovirus of the Spumaretrovirinae subfamily that combines features of both retroviruses and hepadnaviruses in its replication strategy. Given the increasing interest in FVs as gene transfer tools and their unique status within the retrovirus family, this discrepancy urged the identification of novel host cell interaction partners of FV structural components. This study focused on prototype FV (PFV), the best-characterized member of FVs, and its capsid protein, Gag, as the central player of viral replication. Members of the mitosis-regulatory, polo-like kinase (PLK) family were identified as novel Gag binding partners. The Gag interaction with PLK1 (and possibly also PLK2) facilitated efficient PFV genome integration into host chromatin, ensuring successful replication and viral spread in infected target cell cultures. Collectively, our results elucidate the first link between cell cycle regulatory networks and the mitosis-dependent PFV integration process.

Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae., Author Summary Foamy viruses (FVs), which belong to the retroviral genus Spumavirus, are endemic to non-human primates and can be transmitted to humans. They are considered as potential vectors for gene therapy due to their broad cell tropism and their apparent apathogenicity in natural hosts and humans. In order to gain more insight into the ultrastructure of the prototype FV (PFV) we performed (cryo-)electron tomography and microscopy of infected cells and of isolated virions. We find that PFV contains a nucleocapsid of constant dimensions at its center, an intermediate shell of protein positioned between the core capsid and the viral membrane and glycoprotein that arranges into regular hexagonal lattices on the virus membrane. Structural analysis of the glycoprotein was performed in situ to a resolution of 9Å, which shows regular helical features such as a trimeric coiled coil of the fusion protein subunit, a hallmark of class I fusion proteins, spacer arms between the glycoprotein trimers and the arrangement of six transmembrane helices, a characteristic feature of the PFV Env glycoprotein. We discuss our results in light of the evolutionary relationship of PFV with other retroviruses as well as the role of the unique glycoprotein architecture on the virus life cycle.

The simultaneous overexpression of several transcription factors has emerged as a successful strategy to convert fibroblasts into other cell types including pluripotent cells, neurons, and cardiomyocytes. The selection and screening of factors are critical, and have often involved testing a large pool of transcription factors, followed by successive removal of single factors. Here, to identify a cardiac transcription factor combination facilitating mouse fibroblast reprogramming into cardiomyocytes, we directly screened all triplet combinations of 10 candidate factors combined with a Q-PCR assay reporting induction of multiple cardiac-specific genes. Through this screening method the combination of Tbx5, Mef2c, and Myocd was identified to upregulate a broader spectrum of cardiac genes compared to the combination of Tbx5, Mef2c, and Gata4 that was recently shown to induce reprogramming of fibroblasts into cardiomyocytes. Cells cotransduced with Tbx5, Mef2c, Myocd expressed cardiac contractile proteins, had cardiac-like potassium and sodium currents and action potentials could be elicited. In summary the alternative screening approach that is presented here avoided the elimination of transcription factors whose potency is masked in complex transcription factor mixes. Furthermore, our results point to the importance of verifying multiple lineage specific genes when assessing reprogramming.