ABSTRACT: Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness.
Project description:<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.
Project description:The replication of simian foamy virus from macaques can be inhibited by the nucleoside reverse transcriptase inhibitor azidothymidine (AZT, zidovudine). Four substitutions in the protease-reverse transcriptase (PR-RT) protein (K211I, I224T, S345T, E350K) are necessary to obtain highly AZT resistant and fully replication competent virus. AZT resistance is based on the excision of the incorporated AZTMP in the presence of ATP. I224T is a polymorphism which is not essential for AZT resistance per se, but is important for regaining efficient replication of the resistant virus.We constructed PR-RT enzymes harboring one to four amino acid substitutions to analyze them biochemically and to determine their ability to remove the incorporated AZTMP. S345T is the only single substitution variant exhibiting significant AZTMP excision activity. Although K211I alone showed no AZTMP excision activity, excision efficiency doubled when K211I was present in combination with S345T and E350K. K211I also decreased nucleotide binding affinity and increased fidelity. NMR titration experiments revealed that a truncated version of the highly AZT resistant mt4 variant, comprising only the fingers-palm subdomains was able to bind ATP with a KD-value of ca. 7.6 mM, whereas no ATP binding could be detected in the corresponding wild type protein. We could show by NMR spectroscopy that S345T is responsible for ATP binding, probably by making a tryptophan residue accessible.Although AZT resistance in SFVmac is based on excision of the incorporated AZTMP like in HIV-1, the functions of the resistance substitutions in SFVmac PR-RT appear to be different. No mutation resulting in an aromatic residue like F/Y215 in HIV, which is responsible for ?-?-stacking interactions with ATP, is present in SFVmac. Instead, S345T is responsible for creating an ATP binding site, probably by making an already existing tryptophan more accessible, which in turn can interact with ATP. This is in contrast to HIV-1 RT, in which an ATP binding site is present in the WT RT but differs from that of the AZT resistant enzyme.
Project description:Foamy viruses (FVs) synthesize the Pol precursor protein from a specific transcript. Thus, in contrast to what was found for orthoretroviruses, e.g., human immunodeficiency virus, no Gag-Pol precursor protein is synthesized. Foamy viral Pol consists of a protease (PR) domain, a reverse transcriptase domain, and an integrase domain and is processed into a mature protease-reverse transcriptase (PR-RT) fusion protein and the integrase. Protease activity has to be strictly regulated in order to avoid premature Gag and Pol processing before virus assembly. We have demonstrated recently that FV protease is an inactive monomer with a very weak dimerization tendency and postulated protease activation through dimerization. Here, we identify a specific protease-activating RNA motif (PARM) located in the pol region of viral RNA which stimulates PR activity in vitro and in vivo, revealing a novel and unique mechanism of retroviral protease activation. This mechanism is strikingly different to that of orthoretroviruses, where the protease can be activated even in the absence of viral RNA during the assembly of virus-like particles. Although it has been shown that the integrase domain is important for Pol uptake, activation of the foamy virus protease is integrase independent. We show that at least two foamy virus PR-RT molecules bind to the PARM and only RNAs containing the PARM result in significant activation of the protease. DNA harboring the PARM is not capable of protease activation. Structure determination of the PARM by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) revealed a distinct RNA folding, important for protease activation and thus virus maturation.
Project description:Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76:10069-10073, 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5' long terminal repeat and one at the 3' end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.
Project description:Foamy viruses (FVs) differ from all other genera of retroviruses (orthoretroviruses) in many aspects of viral replication. In this review, we discuss FV assembly, with special emphasis on Pol incorporation. FV assembly takes place intracellularly, near the pericentriolar region, at a site similar to that used by betaretroviruses. The regions of Gag, Pol and genomic RNA required for viral assembly are described. In contrast to orthoretroviral Pol, which is synthesized as a Gag-Pol fusion protein and packaged through Gag-Gag interactions, FV Pol is synthesized from a spliced mRNA lacking all Gag sequences. Thus, encapsidation of FV Pol requires a different mechanism. We detail how WT Pol lacking Gag sequences is incorporated into virus particles. In addition, a mutant in which Pol is expressed as an orthoretroviral-like Gag-Pol fusion protein is discussed. We also discuss temporal regulation of the protease, reverse transcriptase and integrase activities of WT FV Pol.
Project description:Foamy viruses (FVs) are ancient retrovirus that infect most nonhuman primates and several animals, but are rarely reported in tree shrew Tupaia belangeri. In the present study, foamy virus was detected in tree shrew. Phylogenetic analysis indicated that FVtup shared the highest homology with SFVmac (99.3%) in China. The discovery of FVtup indicated that the tree shrew is a new host of foamy virus. FVtup is highly prevalent in Tupaia in China and there is the possibility of cross-species transmission from nonhuman primate to Tupaia.
Project description:Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.
Project description:Foamy virus (FV) replication is resistant to most nucleoside analog reverse transcriptase (RT) inhibitors. In an attempt to create a 2',3'-dideoxy-3'-thiacytidine (3TC)-sensitive virus, the second residue in the highly conserved YXDD motif of simian foamy virus-chimpanzee (human isolate) [SFVcpz(hu)] RT was changed from Val (V) to Met (M). Unexpectedly, the resultant virus, SFVcpz(hu) RT-V313M, replicated poorly, and Met rapidly reverted to Val. Despite the presence of approximately 50% of wild-type RT activity in RT-V313M virions, full-length DNA products were not detected in transfected cells. Using purified recombinant enzymes, we found that the wild-type FV RT is significantly more processive than human immunodeficiency virus type 1 RT. However, the V313M mutant has about 40% of the wild-type level of FV RT activity and has a lower processivity than the wild-type FV enzyme. The V313M mutant RT is also relatively resistant to 3TC. These results suggest that the decrease in RT activity and processivity of FV RT-V313M prevents completion of reverse transcription and greatly diminishes viral replication.
Project description:Foamy viruses (FVs) are complex retroviruses that establish lifelong persistent infection without evident pathology. However, the roles of cellular factors in FV latency are poorly understood. This study revealed that N-Myc interactor (Nmi) could inhibit the replication of prototype foamy virus (PFV). Overexpression of Nmi reduced PFV replication, whereas its depletion by small interfering RNA increased PFV replication. The Nmi-mediated impairment of PFV replication resulted from the diminished transactivation by PFV Tas of the viral long terminal repeat (LTR) and an internal promoter (IP). Nmi was determined to interact with Tas and abrogate its function by sequestration in the cytoplasm. In addition, human and bovine Nmi proteins were found to inhibit the replication of bovine foamy virus (BFV) and PFV. Together, these results indicate that Nmi inhibits both human and bovine FVs by interfering with the transactivation function of Tas and may have a role in the host defense against FV infection.From this study, we report that the N-Myc interactor (Nmi), an interferon-induced protein, can interact with the regulatory protein Tas of the prototype foamy virus and sequester it in the cytoplasm. The results of this study suggest that Nmi plays an important role in maintaining foamy virus latency and may reveal a new pathway in the interferon-mediated antiviral barrier against viruses. These findings are important for understanding virus-host relationships not only with FVs but potentially for other retroviruses as well.
Project description:Foamy virus (FV) vector systems have recently demonstrated their power as efficient gene transfer tools for different target tissues. Unfortunately, FVs cannot be naturally pseudotyped by heterologous viral glycoproteins due to an unusual particle morphogenesis involving a FV Env-dependent particle release process. Therefore, current FV vector systems are constrained to the broad host cell range provided by the cognate viral glycoprotein. We evaluated different approaches for pseudotyping of FV vectors, in which the specific FV Gag-Env interaction, essential for particle egress, is substituted by a small-molecule controlled heterodimerization (HD) system. In one system developed, one HD-domain (HDD) is fused to a membrane-targeting domain (MTD), such as the human immunodeficiency virus (HIV) Gag matrix (MA) subunit, with a second fused to the FV capsid protein. Coexpression of both components with different heterologous viral glycoproteins allowed an efficient, dimerizer-dependent pseudotyping of FV capsids. With this system FV vesicular stomatitis virus glycoprotein (VSV-G) pseudotype titers greater than 1 × 10(6) IU/ml were obtained, at levels comparable to authentic FV vector particles. As a proof-of-principle we demonstrate that Pac2 cells, naturally resistant to FV vectors, become permissive to FV VSV-G pseudotypes. Similar to other retroviral vectors, this FV pseudotyping system now enables adaptation of cell-specific targeting approaches for FVs.