Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses.

Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.Foamy viruses (FV) are complex retroviruses that comprise one of two subfamilies. The FV replication strategy differs in many ways from that of orthoretroviruses such as human immunodeficiency virus (HIV-1) and is similar in some respects to that of hepadnaviruses such as human hepatitis B viruses (HBV). One of the major differences between FV and orthoretroviruses is the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein. About one Gag-Pol is translated for every 20 Gag proteins, and such translational regulation balances the required levels of structural proteins and enzymatic proteins. The Gag-Pol fusion is packaged into virions by coassembly with self-assembling Gag proteins (reviewed in reference 9). However, FV Pol is synthesized from a separate spliced mRNA independently of Gag. This raises fundamental questions about how Pol expression is regulated and how Pol is selectively incorporated into assembling virus particles without Gag determinants. Recently, we have shown that Pol expression is regulated at the level of transcription by utilization of a suboptimal splicing site (14). The details of FV Pol packaging are not yet known. HBV also expresses its reverse transcriptase (RT) (P protein) independently of the structural proteins. However, HBV P protein is responsible for packaging of genomic RNA and initiating assembly of capsids (18), whereas FV Pol is not required for particle assembly or encapsdation of genomic RNA (1). Thus, the mechanism of FV Pol incorporation is unique, differing from those of orthoretroviruses and hepadnaviruses.

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The Orthoretrovirus Gag interaction (I) domain maps to the nucleocapsid (NC) domain in the Gag polyprotein. We used the yeast two-hybrid system to analyze the role of Alpharetrovirus NC in Gag-Gag interactions and also examined the efficiency of viral assembly and release in vivo. We could delete either or both of the two Cys-His (CH) boxes without abrogating Gag-Gag interactions. We found that as few as eight clustered basic residues, attached to the C terminus of the spacer peptide separating the capsid (CA) and NC domains in the absence of NC, was sufficient for Gag-Gag interactions. Our results support the idea that a sufficient number of basic residues, rather than the CH boxes, play the important role in Gag multimerization. We also examined the requirement for basic residues in Gag for packaging of specific packaging signal (⌿)-containing RNA. Using a yeast three-hybrid RNA-protein interaction assay, second-site suppressors of a packaging-defective Gag mutant were isolated, which restored ⌿ RNA binding. These suppressors mapped to the p10 or CA domains in Gag and resulted in either introduction of a positively charged residue or elimination of a negatively charged one. These results imply that the structural interactions of NC with other domains of Gag are necessary for ⌿ RNA binding. Taken together, our results show that while Gag assembly only requires a certain number of positively charged amino acids, Gag binding to genomic RNA for packaging requires more complex interactions inherent in the protein tertiary structure.The Alpharetrovirus Gag protein, exemplified by Rous sarcoma virus (RSV), is synthesized as a precursor protein consisting of matrix (MA); two small peptides, p2 and p10; capsid (CA), nucleocapsid (NC), and protease (PR). Cleavage of the precursor by PR occurs after, or concomitant with, viral budding from the plasma membrane. Three Gag domains (M [membrane association], I [interaction], and L [late]) are necessary for viral assembly (reviewed in references 18 and 40). The interaction, or I, domain is required for Gag multimerization and virus particle assembly. Deletion of the NC domain has been shown to result in drastic reduction of viral assembly and release, suggesting that I domain function resides in NC. In Orthoretrovirinae, NC has two structural characteristics: one or two copies of the conserved sequence Cys-X 2 -Cys-X 4 -His-X 4 -Cys (Cys-His [CH] box), which coordinates Zn 2ϩ ion binding, and clustered basic residues (BRs) flanking the CH box or boxes. NC plays important roles in essential steps of virus replication, including reverse transcription, integration, RNA packaging, viral assembly, and infectivity (reviewed in reference 15). Some of these functions have been shown to require distinct structural motifs. A previous and extensive yeast twohybrid study (43) found that the minimal domain required for Gag-Gag interactions mapped to human immunodeficiency virus (HIV) NC but did not characterize the specific residues required within the minimal domain. Gag multimerization ...

In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging. We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation. In this study, using both sitedirected mutagenesis and in vivo random screening in the yeast three-hybrid binding assay, we have examined the amino acids in NC required for genomic RNA binding. We found that we could delete either of the two Cys-His boxes without greatly abrogating either RNA binding or packaging, although the two Cys-His boxes are likely to be required for efficient viral assembly and release. In contrast, substitutions for the Zn-coordinating residues within the boxes did prevent RNA binding, suggesting changes in the overall conformation of the protein. In the basic region between the two Cys-His boxes, three positively charged residues, as well as basic residues flanking the two boxes, were necessary for both binding and packaging. Our results suggest that the stretches of positively charged residues within NC that need to be in a proper conformation appear to be responsible for selective recognition and binding to the packaging signal (⌿)-containing RNAs.Retroviral particles which contain two copies of genomic RNA are assembled through Gag-Gag interactions. During the process of RNA encapsidation, sequences in genomic RNA (termed ⌿) are selectively recognized and specifically bound to domains in the Gag polyprotein. In many retroviruses, such as murine leukemia virus (MuLV), human immunodeficiency virus type 1 (HIV-1), and avian sarcoma leukosis virus (ASLV), packaging sequences are located in the 5Ј untranslated leader sequences of the genome. A 160-nucleotide (nt) packaging sequences of ASLV (M⌿) has been identified which confers the efficient packaging of heterologous RNAs into virions, if they are tethered to M⌿ RNA (3).Only the avian retroviral Gag precursor protein is required for viral assembly at the cytoplasmic membrane. Gag is subsequently cleaved by the Gag-encoded protease to yield individual domains; matrix (MA), p2, p10, capsid (CA), nucleocapsid (NC), and protease (PR). Cleavage occurs shortly after or concomitant with viral budding (26). Using deletion analyses and chimeric proteins in which the NC domain of one retrovirus is substituted for the cognate region of another retroviral Gag, the NC domain of the Gag polyprotein has been shown to be solely responsible for specific encapsidation (6, 49). In all Orthoretrovirinae, the NC protein contains one or two copies of the conserved sequence Cys-X 2 -Cys-X 4 -His-X 4 -Cys, which coordinates Zn 2ϩ ion binding (Cys-His zinc finger motif), as well as adjacent basic amino acids flanking the fingers. The zinc finger sequence bears a striking resemblance to those of a variety of eukaryotic transcription factors implicated in recognition of specifi...

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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.