Inhibition of spumavirus gene expression by PHF11.

The foamy viruses (FV) or spumaviruses are an ancient subfamily of retroviruses that infect a variety of vertebrates. FVs are endemic, but apparently apathogenic, in modern non-human primates. Like other retroviruses, FV replication is inhibited by type-I interferon (IFN). In a previously described screen of IFN-stimulated genes (ISGs), we identified the macaque PHD finger domain protein-11 (PHF11) as an inhibitor of prototype foamy virus (PFV) replication. Here, we show that human and macaque PHF11 inhibit the replication of multiple spumaviruses, but are inactive against several orthoretroviruses. Analysis of other mammalian PHF11 proteins revealed that antiviral activity is host species dependent. Using multiple reporter viruses and cell lines, we determined that PHF11 specifically inhibits a step in the replication cycle that is unique to FVs, namely basal transcription from the FV internal promoter (IP). In so doing, PHF11 prevents expression of the viral transactivator Tas and subsequent activation of the viral LTR promoter. These studies reveal a previously unreported inhibitory mechanism in mammalian cells, that targets a family of ancient viruses and may promote viral latency.

The Late Domain of Prototype Foamy Virus Gag Facilitates Autophagic Clearance of Stress Granules by Promoting Amphisome Formation.

Prototype foamy virus (PFV), a complex retrovirus belonging to Spumaretrovirinae, maintains lifelong latent infection. The maintenance of lifelong latent infection by viruses relies on the repression of the type I interferon (IFN) response. However, the mechanism involving PFV latency, especially regarding the suppression of the IFN response, is poorly understood. Our previous study showed that PFV promotes autophagic flux. However, the underlying mechanism and the role of PFV-induced autophagy in latent infection have not been clarified. Here, we report that the PFV viral structural protein Gag induced amphisome formation and triggered autophagic clearance of stress granules (SGs) to attenuate type I IFN production. Moreover, the late domain (L-domain) of Gag played a central role in Alix recruitment, which promoted endosomal sorting complex required for transport I (ESCRT-I) formation and amphisome accumulation by facilitating late endosome formation. Our data suggest that PFV Gag represses the host IFN response through autophagic clearance of SGs by activating the endosome-autophagy pathway. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.IMPORTANCE Maintenance of lifelong latent infection for viruses relies on repression of the type I IFN response. Autophagy plays a double-edged sword in antiviral immunity. However, the role of autophagy in the regulation of the type I IFN response and the mechanism involving virus-promoted autophagy have not been fully elucidated. SGs are an immune complex associated with the antiviral immune response and are critical for type I IFN production. Autophagic clearance of SGs is one means of degradation of SGs and is associated with regulation of immunity, but the detailed mechanism remains unclear. In this article, we demonstrate that PFV Gag recruits ESCRT-I to facilitate amphisome formation. Our data also suggest that amphisome formation is a critical event for autophagic clearance of SGs and repression of the type I IFN response. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.

Structural basis for spumavirus GAG tethering to chromatin.

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.

Single residue mutation in integrase catalytic core domain affects feline foamy viral DNA integration.

DD(35)E motif in catalytic core domain (CCD) of integrase (IN) is extremely involved in retroviral integration step. Here, nine single residue mutants of feline foamy virus (FFV) IN were generated to study their effects on IN activities and on viral replication. As expected, mutations in the highly conserved D107, D164, and E200 residues abolished all IN catalytic activities (3'-end processing, strand transfer, and disintegration) as well as viral infectivity by blocking viral DNA integration into cellular DNA. However, Q165, Y191, and S195 mutants, which are located closely to DDE motif were observed to have diverse levels of enzymatic activities, compared to those of the wild type IN. Their mutant viruses produced by one-cycle transfection showed different infectivity on their natural host cells. Therefore, it is likely that effects of single residue mutation at DDE motif is critical on viral replication depending on the position of the residues.

The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag.

BACKGROUND: Nuclear localization of Gag is a property shared by many retroviruses and retrotransposons. The importance of this stage for retroviral replication is still unknown, but studies on the Rous Sarcoma virus indicate that Gag might select the viral RNA genome for packaging in the nucleus. In the case of Foamy viruses, genome encapsidation is mediated by Gag C-terminal domain (CTD), which harbors three clusters of glycine and arginine residues named GR boxes (GRI-III). In this study we investigated how PFV Gag subnuclear distribution might be regulated. RESULTS: We show that the isolated GRI and GRIII boxes act as nucleolar localization signals. In contrast, both the entire Gag CTD and the isolated GRII box, which contains the chromatin-binding motif, target the nucleolus exclusively upon mutation of the evolutionary conserved arginine residue at position 540 (R540), which is a key determinant of FV Gag chromatin tethering. We also provide evidence that Gag localizes in the nucleolus during FV replication and uncovered that the viral protein interacts with and is methylated by Protein Arginine Methyltransferase 1 (PRMT1) in a manner that depends on the R540 residue. Finally, we show that PRMT1 depletion by RNA interference induces the concentration of Gag C-terminus in nucleoli. CONCLUSION: Altogether, our findings suggest that methylation by PRMT1 might finely tune the subnuclear distribution of Gag depending on the stage of the FV replication cycle. The role of this step for viral replication remains an open question.

Prototype foamy virus elicits complete autophagy involving the ER stress-related UPR pathway.

BACKGROUND: Prototype foamy virus (PFV) is a member of the Spumaretrovirinae subfamily of retroviruses, which maintains lifelong latent infection while being nonpathogenic to their natural hosts. Autophagy is a cell-programmed mechanism that plays a pivotal role in controlling homeostasis and defense against exotic pathogens. However, whether autophagy is the mechanism for host defense in PFV infection has not been investigated. FINDINGS: Our results revealed that PFV infection induced the accumulation of autophagosomes and triggered complete autophagic flux in BHK-21 cells. PFV infection also altered endoplasmic reticulum (ER) homeostasis. The PERK, IRE1 and ATF6 pathways, all of which are components of the ER stress-related unfolded protein response (UPR), were activated in PFV-infected cells. In addition, accelerating autophagy suppressed PFV replication, and inhibition of autophagy promoted viral replication. CONCLUSIONS: Our data indicate that PFV infection can induce complete autophagy through activating the ER stress-related UPR pathway in BHK-21 cells. In turn, autophagy negatively regulates PFV replication.

A comparative analysis of the foamy and ortho virus capsid structures reveals an ancient domain duplication.

BACKGROUND: The Spumaretrovirinae (foamy viruses) and the Orthoretrovirinae (e.g. HIV) share many similarities both in genome structure and the sequences of the core viral encoded proteins, such as the aspartyl protease and reverse transcriptase. Similarity in the gag region of the genome is less obvious at the sequence level but has been illuminated by the recent solution of the foamy virus capsid (CA) structure. This revealed a clear structural similarity to the orthoretrovirus capsids but with marked differences that left uncertainty in the relationship between the two domains that comprise the structure. METHODS: We have applied protein structure comparison methods in order to try and resolve this ambiguous relationship. These included both the DALI method and the SAP method, with rigorous statistical tests applied to the results of both methods. For this, we employed collections of artificial fold 'decoys' (generated from the pair of native structures being compared) to provide a customised background distribution for each comparison, thus allowing significance levels to be estimated. RESULTS: We have shown that the relationship of the two domains conforms to a simple linear correspondence rather than a domain transposition. These similarities suggest that the origin of both viral capsids was a common ancestor with a double domain structure. In addition, we show that there is also a significant structural similarity between the amino and carboxy domains in both the foamy and ortho viruses. CONCLUSIONS: These results indicate that, as well as the duplication of the double domain capsid, there may have been an even more ancient gene-duplication that preceded the double domain structure. In addition, our structure comparison methodology demonstrates a general approach to problems where the components have a high intrinsic level of similarity.

The mechanism of retroviral integration from X-ray structures of its key intermediates.

To establish productive infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA. This process is operated by the intasome, a nucleoprotein complex composed of an integrase tetramer (IN) assembled on the viral DNA ends. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) recombinase superfamily have been reported, the mechanics of target DNA capture and strand transfer by these enzymes remained unclear. Here we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results resolve the structural basis for retroviral DNA integration and provide a framework for the design of INs with altered target sequences.

Key determinants of target DNA recognition by retroviral intasomes.

BACKGROUND: Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joining and generates a short (4-6 bp) duplication of host DNA flanking the provirus. We previously determined two key parameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiency virus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integration sites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase. Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings with respect to length of target site duplication (TSD). RESULTS: Integration datasets covering six viral genera and the three lengths of TSD were accessed from the literature or generated in this work. All viruses exhibited significant enrichments of flexible YR and/or selection against rigid RY dinucleotide steps at the centers of integration sites, and the magnitude of this enrichment inversely correlated with TSD length. The DNA sequence environments of in vivo-generated HIV-1 and PFV sites were consistent with integration into nucleosomes, however, the local sequence preferences were largely independent of target DNA chromatinization. Integration sites derived from cells infected with the gammaretrovirus reticuloendotheliosis virus strain A (Rev-A), which yields a 5 bp TSD, revealed the targeting of global chromatin features most similar to those of Moloney murine leukemia virus, which yields a 4 bp duplication. In vitro assays revealed that Rev-A integrase interacts with and is catalytically stimulated by cellular bromodomain containing 4 protein. CONCLUSIONS: Retroviral integrases have likely evolved to bend target DNA to fit scissile phosphodiester bonds into two active sites for integration, and viruses that cut target DNA with a 6 bp stagger may not need to bend DNA as sharply as viruses that cleave with 4 bp or 5 bp staggers. For PFV and HIV-1, the selection of signature bases and central flexibility at sites of integration is largely independent of chromatin structure. Furthermore, global Rev-A integration is likely directed to chromatin features by bromodomain and extraterminal domain proteins.

Knockdown of the host cellular protein transportin 3 attenuates prototype foamy virus infection.

Transportin 3 (TNPO3) is a member of the importin-ß superfamily proteins. Despite numerous studies, the exact molecular mechanism of TNPO3 in retroviral infection is still controversial. Here, we provide evidence for the role and mechanism of TNPO3 in the replication of prototype foamy virus (PFV). Our findings revealed that PFV infection was reduced 2-fold by knockdown (KD) of TNPO3. However, late stage of viral replication including transcription, translation, viral assembly, and release was not influenced. The differential cellular localization of PFV integrase (IN) in KD cells pinpointed a remarkable reduction of viral replication at the nuclear import step. We also found that TNPO3 interacted with PFV IN but not with Gag, suggesting that IN-TNPO3 interaction is important for nuclear import of PFV pre-integration complex. Our report enlightens the mechanism of PFV interaction with TNPO3 and support ongoing research on PFV as a promising safe vector for gene therapy.

The cooperative function of arginine residues in the Prototype Foamy Virus Gag C-terminus mediates viral and cellular RNA encapsidation.

BACKGROUND: One unique feature of the foamy virus (FV) capsid protein Gag is the absence of Cys-His motifs, which in orthoretroviruses are irreplaceable for multitude functions including viral RNA genome recognition and packaging. Instead, FV Gag contains glycine-arginine-rich (GR) sequences at its C-terminus. In case of prototype FV (PFV) these are historically grouped in three boxes, which have been shown to play essential functions in genome reverse transcription, virion infectivity and particle morphogenesis. Additional functions for RNA packaging and Pol encapsidation were suggested, but have not been conclusively addressed. RESULTS: Here we show that released wild type PFV particles, like orthoretroviruses, contain various cellular RNAs in addition to viral genome. Unlike orthoretroviruses, the content of selected cellular RNAs in capsids of PFV vector particles was not altered by viral genome encapsidation. Deletion of individual GR boxes had only minor negative effects (2 to 4-fold) on viral and cellular RNA encapsidation over a wide range of cellular Gag to viral genome ratios examined. Only the concurrent deletion of all three PFV Gag GR boxes, or the substitution of multiple arginine residues residing in the C-terminal GR box region by alanine, abolished both viral and cellular RNA encapsidation (>50 to >3,000-fold reduced), independent of the viral production system used. Consequently, those mutants also lacked detectable amounts of encapsidated Pol and were non-infectious. In contrast, particle release was reduced to a much lower extent (3 to 20-fold). CONCLUSIONS: Taken together, our data provides the first identification of a full-length PFV Gag mutant devoid in genome packaging and the first report of cellular RNA encapsidation into PFV particles. Our results suggest that the cooperative action of C-terminal clustered positively charged residues, present in all FV Gag proteins, is the main viral protein determinant for viral and cellular RNA encapsidation. The viral genome independent efficiency of cellular RNA encapsidation suggests differential packaging mechanisms for both types of RNAs. Finally, this study indicates that analogous to orthoretroviruses, Gag - nucleic acid interactions are required for FV capsid assembly and efficient particle release.

Prototype foamy virus protease activity is essential for intraparticle reverse transcription initiation but not absolutely required for uncoating upon host cell entry.

Foamy viruses (FVs) are unique among retroviruses in performing genome reverse transcription (RTr) late in replication, resulting in an infectious DNA genome, and also in their unusual Pol biosynthesis and encapsidation strategy. In addition, FVs display only very limited Gag and Pol processing by the viral protease (PR) during particle morphogenesis and disassembly, both thought to be crucial for viral infectivity. Here, we report the generation of functional prototype FV (PFV) particles from mature or partially processed viral capsid and enzymatic proteins with infectivity levels of up to 20% of the wild type. Analysis of protein and nucleic acid composition, as well as infectivity, of virions generated from different Gag and Pol combinations (including both expression-optimized and authentic PFV open reading frames [ORFs]) revealed that precursor processing of Gag, but not Pol, during particle assembly is essential for production of infectious virions. Surprisingly, when processed Gag (instead of Gag precursor) was provided together with PR-deficient Pol precursor during virus production, infectious, viral DNA-containing particles were obtained, even when different vector or proviral expression systems were used. Although virion infectivity was reduced to 0.5 to 2% relative to that of the respective parental constructs, this finding overturns the current dogma in the FV literature that viral PR activity is absolutely essential at some point during target cell entry. Furthermore, it demonstrates that viral PR-mediated Gag precursor processing during particle assembly initiates intraparticle RTr. Finally, it shows that reverse transcriptase (RT) and integrase are enzymatically active in the Pol precursor within the viral capsid, thus enabling productive host cell infection.

Expression of prototype foamy virus pol as a Gag-Pol fusion protein does not change the timing of reverse transcription.

Foamy viruses are retroviruses whose Pol protein is synthesized without Gag from a spliced mRNA. Unlike orthoretroviruses, reverse transcription occurs during viral assembly, leading to DNA-containing virions. When prototype foamy virus Pol is expressed as an orthoretroviral-like Gag-Pol fusion protein, reverse transcription also occurs late in viral replication, as measured by the timing of reverse transcriptase sensitivity to the inhibitor 3'-azido-3'deoxythymidine (AZT). Thus, timing of reverse transcription is intrinsic to Pol itself.

Identification and functional characterization of Bet protein as a negative regulator of BFV3026 replication.

Foamy virus (FV) establishes persistent infection in the host without causing apparent disease. Besides the transactivator Tas protein, another auxiliary protein--Bet--has been reported in prototype foamy virus, equine foamy virus, and feline foamy virus. Here, we found the putative bbet gene in clone C74 from a cDNA library of bovine foamy virus strain 3026 (BFV3026) by comparison of gene localization, composition, and splicing features with other known bet genes. Subsequently, BBet protein was detected in BFV3026-infected cells by Western blot and immunofluorescence analyses. Analysis of the BBet mutant infectious clone (pBS-BFVdelBBet) revealed that BBet could inhibit BFV3026 replication. Consistent with this result, overexpression of BBet in Cf2Th cells reduced BFV replication by approximately threefold. Furthermore, virus replication levels similarly were reduced by approximately threefold in pBS-BFV-transfected and BFV3026-infected Cf2Th cells stably expressing BBet compared with control cells. After three passages, BFV3026 replicated more slowly in BBet-expressing cells. This study implicates BBet as a negative regulator of BFV replication and provides a resource for future studies on the function of this protein in the virus lifecycle.

Identification of the feline foamy virus Bet domain essential for APOBEC3 counteraction.

BACKGROUND: APOBEC3 (A3) proteins restrict viral replication by cytidine deamination of viral DNA genomes and impairing reverse transcription and integration. To escape this restriction, lentiviruses have evolved the viral infectivity factor (Vif), which binds A3 proteins and targets them for proteolytic degradation. In contrast, foamy viruses (FVs) encode Bet proteins that allow replication in the presence of A3, apparently by A3 binding and/or sequestration, thus preventing A3 packaging into virions and subsequent restriction. Due to a long-lasting FV-host coevolution, Bet proteins mainly counteract restriction by A3s from their cognate or highly related host species. RESULTS: Through bioinformatics, we identified conserved motifs in Bet, all localized in the bel2 exon. In line with the localization of these conserved motifs within bel2, this part of feline FV (FFV) Bet has been shown to be essential for feline A3 (feA3) inactivation and feA3 protein binding. To study the function of the Bet motifs in detail, we analyzed the ability of targeted deletion, substitution, and chimeric FFV-PFV (prototype FV) Bet mutants to physically bind and/or inactivate feA3. Binding of Bet to feA3Z2b is sensitive to mutations in the first three conserved motifs and N- and C-terminal deletions and substitutions across almost the complete bel2 coding sequence. In contrast, the Bel1 (also designated Tas) domain of Bet is dispensable for basal feA3Z2b inactivation and binding but mainly increases the steady state level of Bet. Studies with PFV Bel1 and full-length FFV Bel2 chimeras confirmed the importance of Bel2 for A3 inactivation indicating that Bel1 is dispensable for basal feA3Z2b inactivation and binding but increases Bet stability. Moreover, the bel1/tas exon may be required for expression of a fully functional Bet protein from a spliced transcript. CONCLUSIONS: We show that the Bel2 domain of FV Bet is essential for the inactivation of APOBEC3 cytidine deaminase restriction factors. The Bel1/Tas domain increases protein stability and can be exchanged by related sequence. Since feA3 binding and inactivation by Bet are highly correlated, the data support the view that FV Bet prevents A3-mediated restriction of viral replication by creating strong complexes with these proteins.

An N-terminal domain helical motif of Prototype Foamy Virus Gag with dual functions essential for particle egress and viral infectivity.

BACKGROUND: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. RESULTS: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag - Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. CONCLUSIONS: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

U1snRNP-mediated suppression of polyadenylation in conjunction with the RNA structure controls poly (A) site selection in foamy viruses.

BACKGROUND: During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5'LTR and activate polyadenylation in the 3'LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal. RESULTS: Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5'LTR. In contrast, polyadenylation at the 3'LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation. CONCLUSION: Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5'end of their RNA. At the 3'end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression.

Innate sensing of foamy viruses by human hematopoietic cells.

Foamy viruses (FV) are nonpathogenic retroviruses that have cospeciated with primates for millions of years. FV can be transmitted through severe bites from monkeys to humans. Viral loads remain generally low in infected humans, and no secondary transmission has been reported. Very little is known about the ability of FV to trigger an innate immune response in human cells. A few previous reports suggested that FV do not induce type I interferon (IFN) in nonhematopoietic cells. Here, we examined how human hematopoietic cells sense FV particles and FV-infected cells. We show that peripheral blood mononuclear cells (PBMCs), plasmacytoid dendritic cells (pDCs), and the pDC-like cell line Gen2.2 detect FV, produce high levels of type I IFN, and express the IFN-stimulated gene MxA. Fewer than 20 FV-infected cells are sufficient to trigger an IFN response. Both prototypic and primary viruses stimulated IFN release. Donor cells expressing a replication-defective virus, carrying a mutated reverse transcriptase, induced IFN production by target cells as potently as wild-type virus. In contrast, an FV strain with env deleted, which does not produce viral particles, was inactive. IFN production was blocked by an inhibitor of endosomal acidification (bafilomycin A1) and by an endosomal Toll-like receptor (TLR) antagonist (A151). Silencing experiments in Gen2.2 further demonstrated that TLR7 is involved in FV recognition. Therefore, FV are potent inducers of type I IFN by pDCs and by PBMCs. This previously underestimated activation of the innate immune response may be involved in the control of viral replication in humans.

Heparan sulfate is an attachment factor for foamy virus entry.

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

The DEAD-box RNA helicase DDX6 is required for efficient encapsidation of a retroviral genome.

Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging.