Restriction of foamy viruses by primate Trim5alpha

Foamy viruses (FVs) are unconventional retroviruses with a replication strategy that is significantly different from orthoretroviruses and bears some homology to that of hepadnaviruses. Although some cellular proteins, such as APOBEC3, have been reported to block FVs, no restriction by Trim5alpha has been described to date. The sensitivity of three FV isolates of human-chimpanzee or prototypic (PFV), macaque (SFVmac), and feline (FFV) origin to a variety of primate Trim5alphas was therefore tested. PFV and SFVmac were restricted by Trim5alphas from most New World monkeys, but not from other primates, whereas FFV-based vectors were restricted by Trim5alphas from the great apes gorilla and orangutan. Trim5alphas from Old World monkeys did not restrict any FV isolate tested. Capuchin Trim5alpha was unique, as it restricted SFVmac and FFV but not PFV. Trim5alpha specificity for FVs was determined by the B30.2 domain, interestingly involving, in some instances, the same residues of the variable regions previously implicated as major determinants for human immunodeficiency virus type 1 restriction. FVs with chimeric Gags were made to map the viral determinants of sensitivity to restriction. The N-terminal half of the Gag molecule was found to contain the regions that control susceptibility. This region most likely corresponds to the capsid of conventional retroviruses. Due to their unique replication strategy, FVs should provide a valuable new system to examine the mechanism of retroviral restriction by Trim5alpha.     

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.     

Prototype foamy virus gag nuclear localization: a novel pathway among retroviruses

Gag nuclear localization has long been recognized as a hallmark of foamy virus (FV) infection. Two required motifs, a chromatin-binding site (CBS) and a nuclear localization signal (NLS), both located in glycine-arginine-rich box II (GRII), have been described. However, the underlying mechanisms of Gag nuclear translocation are largely unknown. We analyzed prototype FV (PFV) Gag nuclear localization using a novel live-cell fluorescence microscopy assay. Furthermore, we characterized the nuclear localization route of Gag mutants tagged with the simian vacuolating virus 40-NLS (SV40-NLS) and also dissected the respective contributions of the CBS and the NLS. We found that PFV Gag does not translocate to the nucleus of interphase cells by NLS-mediated nuclear import and does not possess a functional NLS. PFV Gag nuclear localization occurred only by tethering to chromatin during mitosis. This mechanism was found for endogenously expressed Gag as well as for Gag delivered by infecting viral particles. Thereby, the CBS was absolutely essential, while the NLS was dispensable. Gag CBS-dependent nuclear localization was neither essential for infectivity nor necessary for Pol encapsidation. Interestingly, Gag localization was independent of the presence of Pol, Env, and viral RNA. The addition of a heterologous SV40-NLS resulted in the nuclear import of PFV Gag in interphase cells, rescued the nuclear localization deficiency but not the infectivity defect of a PFV Gag ΔGRII mutant, and did not enhance FV's ability to infect G(1)/S-phase-arrested cells. Thus, PFV Gag nuclear localization follows a novel pathway among orthoretroviral Gag proteins.     

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles

ABSTRACT: BACKGROUND: It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. RESULTS: N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Monitoring the fraction of viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. CONCLUSIONS: The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques.     

Characterization of the prototype foamy virus envelope glycoprotein receptor-binding domain

The foamy virus (FV) glycoprotein precursor gp130(Env) undergoes a highly unusual biosynthesis, resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM). Little structural and functional information on the extracellular domains of FV Env is available. In this study, we characterized the prototype FV (PFV) Env receptor-binding domain (RBD) by flow cytometric analysis of recombinant PFV Env immunoadhesin binding to target cells. The extracellular domains of the C-terminal TM subunit as well as targeting of the recombinant immunoadhesins by the cognate LP to the secretory pathway were dispensable for target cell binding, suggesting that the PFV Env RBD is contained within the SU subunit. N- and C-terminal deletion analysis of the SU domain revealed a minimal continuous RBD spanning amino acids (aa) 225 to 555; however, internal deletions covering the region from aa 397 to 483, but not aa 262 to 300 or aa 342 to 396, were tolerated without significant influence on host cell binding. Analysis of individual cysteine point mutants in PFV SU revealed that only most of those located in the nonessential region from aa 397 to 483 retained residual binding activity. Interestingly, analysis of various N-glycosylation site mutants suggests an important role of carbohydrate chain attachment to N391, either for direct interaction with the receptor or for correct folding of the PFV Env RBD. Taken together, these results suggest that a bipartite sequence motif spanning aa 225 to 396 and aa 484 to 555 is essential for formation of the PFV Env RBD, with N-glycosylation site at position 391 playing a crucial role for host cell binding.     

Molecular dissection of the prototype foamy virus (PFV) RNA 5′-UTR identifies essential elements of a ribosomal shunt

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs.     

Characterization of prototype foamy virus gag late assembly domain motifs and their role in particle egress and infectivity

Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101.     

Foamy virus envelope glycoprotein-mediated entry involves a pH-dependent fusion process

In general, enveloped viruses use two different entry strategies and are classified accordingly into pH-dependent and pH-independent viruses. Different members of the retrovirus family use one or the other strategy. Little is known about the uptake of foamy viruses (FV), a special group of retroviruses, into the target cells. In this study, we examined the pH dependence of FV entry by analyzing FV envelope glycoprotein (Env)-mediated infection of target cells with murine leukemia virus or FV vector pseudotypes in the presence of various lysosomotropic agents. Similar to vesicular stomatitis virus glycoprotein G (VSV-G)-mediated uptake, FV Env-mediated entry was inhibited by various lysosomotropic agents, suggesting a pH-dependent endocytic pathway. However, in contrast to its effect on VSV-G pseudotypes, chloroquine failed to reduce the infectivity of FV Env pseudotypes, implying that the pathway is different from that of VSV-G. Glycoproteins of various other FV species showed inhibition profiles similar to that of the prototype FV (PFV) Env. Analysis of the pH dependence of the FV Env-mediated fusion process in a cell-to-cell fusion assay revealed an induction of syncytium formation by a short exposure to acidic pH, peaking around pH 5.5. Interestingly, of all FV Env species analyzed, only the PFV Env had a significant fusion activity at neutral pH. Taken together, these data suggest a pH-dependent endocytic pathway for infection of target cells by FV.     

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.     

Replication in a superficial epithelial cell niche explains the lack of pathogenicity of primate foamy virus infections

Foamy viruses (FVs) are ancient retroviruses that are ubiquitous in nonhuman primates (NHPs). While FVs share many features with pathogenic retroviruses, such as human immunodeficiency virus, FV infections of their primate hosts have no apparent pathological consequences. Paradoxically, FV infections of many cell types in vitro are rapidly cytopathic. Previous work has shown that low levels of proviral DNA are found in most tissues of naturally infected rhesus macaques, but these proviruses are primarily latent. In contrast, viral RNA, indicative of viral replication, is restricted to tissues of the oral mucosa, where it is abundant. Here, we perform in situ hybridization on tissues from rhesus macaques naturally infected with simian FV (SFV). We show that superficial differentiated epithelial cells of the oral mucosa, many of which appear to be shedding from the tissue, are the major cell type in which SFV replicates. Thus, the innocuous nature of SFV infection can be explained by replication that is limited to differentiated superficial cells that are short-lived and shed into saliva. This finding can also explain the highly efficient transmission of FVs among NHPs.     

The promyelocytic leukemia protein does not mediate foamy virus latency in vitro

Spumaviruses, commonly called foamy viruses, are complex retroviruses that establish life-long persistent infections in the absence of accompanying pathology. Depending upon cell type, infection of cells in tissue culture cells can result in either lytic replication, persistence, or latency. The cellular factors that mediate foamy virus (FV) latency are poorly understood. In this study we show that the only known inhibitor of FV replication, the promyelocytic leukemia protein (PML), which binds the FV transactivator (Tas), does not play an important role in FV latency in vitro. We found no significant differences in PML levels in cells that supported lytic replication compared to those that were latently infected. Furthermore, endogenous PML levels did not change following exposure to phorbol myristate acetate (PMA), which induces FV replication. We demonstrated that FV replication proceeded in the presence of substantial levels of PML, both in fully permissive cells and during reactivation of latent FV. Endogenous PML did not efficiently colocalize with Tas, even after upregulation by alpha interferon (IFN-alpha) treatment. IFN-alpha did, however, partially suppress the reactivation of latent FV by PMA. Finally, depletion of endogenous PML by small interfering RNA did not promote activation of FV in cells that responded to PMA treatment. Taken together, these data indicate that endogenous PML does not play an important role in mediating FV latency.