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Foamy viruses (FV), the oldest known genus of Retroviridae, are unique among the retroviruses in having no disease association. It is not known why FV are non-pathogenic while infection by their closest relatives can be deadly. This may be related to the estimated 60 million years of coevolution of FV and their primate hosts. We review the current state of knowledge of FV infection, including information about the sites of viral replication and host immune responses, and discuss the role these may play in establishing persistent yet non-pathogenic infections. Whether FV has pathologic consequences in immunosuppressed hosts has not been thoroughly investigated. As most primates in HIV/SIV research are coinfected with FV, investigation into possible interactions between these viruses is of interest. The use of FV as a vector for gene therapy is also discussed.

Foamy viruses (FV) are the oldest known genus of retroviruses and have persisted in nonhuman primates for over 60 million years. FV are efficiently transmitted, leading to a lifelong nonpathogenic infection. Transmission is thought to occur through saliva, but the detailed mechanism is unknown. Interestingly, this persistent infection contrasts with the rapid cytopathicity caused by FV in vitro, suggesting a host defense against FV. To better understand the tissue specificity of FV replication and host immunologic defense against FV cytopathicity, we quantified FV in tissues of healthy rhesus macaques (RM) and those severely immunosuppressed by simian immunodeficiency virus (SIV). Contrary to earlier findings, we find that all immunocompetent animals consistently have high levels of viral RNA in oral tissues but not in other tissues examined, including the small intestine. Strikingly, abundant viral transcripts were detected in the small intestine of all of the SIV-infected RM, which has been shown to be a major site of SIV (and human immunodeficiency virus)-induced CD4+ T-cell depletion. In contrast, there was a trend to lower viral RNA levels in oropharyngeal tissues of SIV-infected animals. The expansion of FV replication to the small intestine but not to other CD4+ T-cell-depleted tissues suggests that factors other than T-cell depletion, such as dysregulation of the jejunal microenvironment after SIV infection, likely account for the expanded tissue tropism of FV replication.

Foamy virus (FV) replication is distinct from that of all other retroviruses in many respects, including viral assembly. In fact, the viral assembly pathway is rather similar to that of hepadnaviruses such as hepatitis B virus. Foamy virus Gag does not contain landmark retroviral assembly domains such as the major homology region, Cys-His boxes, or a defined M domain. Like hepadnaviruses, the FV Gag protein is not cleaved and contains arginine-rich regions at the carboxyl terminus. In addition, egress of FV particles requires presence of the envelope glycoproteins. Finally, the cis-acting sequences in the FV genome required for genome incorporation, although poorly defined, differ in location from other retroviruses.

In contrast to all retroviruses but similar to the hepatitis B virus, foamy viruses (FV) require expression of the envelope protein for budding of intracellular capsids from the cell, suggesting a specific interaction between the Gag and Env proteins. Capsid assembly occurs in the cytoplasm of infected cells in a manner similar to that for the B- and D-type viruses; however, in contrast to these retroviruses, FV Gag lacks an N-terminal myristylation signal and capsids are not targeted to the plasma membrane (PM). We have found that mutation of an absolutely conserved arginine (Arg) residue at position 50 to alanine (R50A) of the simian foamy virus SFV cpz(hu) inhibits proper capsid assembly and abolishes viral budding even in the presence of the envelope (Env) glycoproteins. Particle assembly and extracellular release of virus can be restored to this mutant with the addition of an N-terminal Src myristylation signal (Myr-R50A), presumably by providing an alternate site for assembly to occur at the PM. In addition, the strict requirement of Env expression for capsid budding can be bypassed by addition of a PM-targeting signal to Gag. These results suggest that intracellular capsid assembly may be mediated by a signal akin to the cytoplasmic targeting and retention signal CTRS found in Mason-Pfizer monkey virus and that FV Gag has the inherent ability to assemble capsids at multiple sites like conventional retroviruses. The necessity of Env expression for particle egress is most probably due to the lack of a membrane-targeting signal within FV Gag to direct capsids to the PM for release and indicates that Gag-Env interactions are essential to drive particle budding.

The foamy virus (FV) genome contains two promoters, the canonical long terminal repeat (LTR) promoter, containing three consensus AP-1 binding sites, and an internal promoter (IP) within the env gene. We investigated the regulation of the two promoters in lytic and persistent infections and found that in the presence of a constitutive source of the viral transactivator protein Tas, transactivation of the LTR promoter and that of the IP differ. In lytic infections, both the LTR promoter and the IP are efficiently transactivated by Tas, while in persistent infections, the IP is efficiently transactivated by Tas, but the LTR promoter is not. Analysis of proteins expressed from the LTR promoter and the IP during infection indicated that IP transcription is more robust than that of the LTR promoter in persistently infected cells, while the opposite is true for lytically infected cells. Coculture experiments also showed that LTR promoter transcription is greatest in cells which support lytic replication. Replacement of much of the LTR promoter with the IP leads to increased viral replication in persistent but not lytic infections. We also found that the induction of persistently infected cells with phorbol 12-myristate 13-acetate (PMA) greatly enhanced viral replication and transcription from the SFVcpz(hu) (new name for human FV) LTR promoter. However, mutation of three consensus AP-1 binding sites in the FV LTR promoter did not affect viral replication in lytically or persistently infected cells, nor did the same mutations affect LTR promoter transactivation by Tas in PMA-treated cells. Our data indicate that differential regulation of transcription is important in the outcome of FV infection but is unlikely to depend on AP-1.

Foamy viruses (FV) are complex retroviruses which are widespread in many species. Despite being discovered over 40 years ago, FV are among the least well characterized retroviruses. The replication of these viruses is different in many interesting respects from that of all other retroviruses. Infection of natural hosts by FV leads to a lifelong persistent infection, without any evidence of pathology. A large number of studies have looked at the prevalence of primate foamy viruses in the human population. Many of these studies have suggested that FV infections are prevalent in some human populations and are associated with specific diseases. More recent data, using more rigorous criteria for the presence of viruses, have not confirmed these studies. Thus, while FV are ubiquitous in all nonhuman primates, they are only acquired as rare zoonotic infections in humans. In this communication, we briefly discuss the current status of FV research and review the history of FV epidemiology, as well as the lack of pathogenicity in natural, experimental, and zoonotic infections.

Foamy viruses (FV) comprise one of the seven genera of retroviruses. These viruses infect most non-human primates as well as cats, cows and horses. Infections are persistent and life-long, but have no pathogenic consequences. Viral replication in vivo is at a very low level, but virus can be recovered years after infection. Humans can acquire FV as zoonotic infections which are also life-long. Although FV are highly cytopathic to fibroblast cultures, some cell lines can be infected with FV without any syncytium formation or cell death. Thus, material obtained from infected animals could contain FV proviruses without any obvious cytopathicity. The consequences of FV in biological material is not known. Little is known about how FV package their genomes, or whether they are able to package cellular genes which could have consequences for recipient cells. In addition, possible recombination between FV and other retroviruses has not been studied. Future studies of this virus should focus on the packaging ability of FV and whether they can recombine with other retroviruses.

Foamy viruses are complex retroviruses that lead to either highly cytopathic or persistent infections in vitro, but to non-pathogenic lifelong infections in naturally or accidentally infected hosts. Factors that could contribute to these benign persistent infections include regulated transcription from the two viral promoters, the functions of the Bet accessory protein and the host immune response.

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete tas gene, which encodes the viral transactivator, and are not derived from Deltatas cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.

Human foamy virus (HFV) is the prototype member of the spumaviruses. While similar in genomic organization to other complex retroviruses, foamy viruses share several features with their more distant relatives, the hepadnaviruses such as human hepatitis B virus (HBV). Both HFV and HBV express their Pol proteins independently from the structural proteins. However unlike HBV, Pol is not required for assembly of HFV core particles or for packaging of viral RNA. These results suggest that the assembly of Pol into HFV particles must occur by a mechanism different from those used by retroviruses and hepadnaviruses. We have examined possible mechanisms for HFV Pol incorporation, including the role of proteolysis in assembly of Pol and the role of initiation of reverse transcription. We have found that proteolytic activity is not required for Pol incorporation. p4 Gag and the residues immediately upstream of the cleavage site in Gag are also not important. Deletion of the primer binding site had no effect on assembly, ruling out early steps of reverse transcription in the process of Pol incorporation.

The genomes of the spumaviruses, of which human foamy virus (HFV) is the prototype, are very similar to those of other complex retroviruses. However, in some aspects of the viral replicative cycle, HFV more closely resembles pararetroviruses such as hepatitis B virus. Previous work indicated that HFV extracellular particles contain apparently full-length double-stranded DNA, as well as RNA. We have further characterized the amount of DNA in particles and the role that this DNA has in viral replication. Experiments with the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) suggest that reverse transcription is largely complete before extracellular virus infects new cells. In addition, we have been able to show that DNA extracted from virions can lead to production of virus after transfection. Taken together, these data suggest that complete, or nearly complete, proviral-length DNA is present in viral particles and that this DNA is sufficient for new rounds of viral replication.

Human foamy virus (HFV) is the prototype of the Spumavirus genus of retroviruses. These viruses have a genomic organization close to that of other complex retroviruses but have similarities to hepadnaviruses such as human hepatitis B virus (HBV). Both HFV and HBV express their Pol protein independently of their structural proteins. Retroviruses and hepadnaviruses differ in their requirements for particle assembly and genome packaging. Assembly of retroviral particles containing RNA genomes requires only the Gag structural protein. The Pol protein is not required for capsid assembly, and the Env surface glycoprotein is not required for release of virions from the cell. In contrast, assembly of extracellular HBV particles containing DNA requires core structural protein and polymerase (P protein) for assembly of nucleocapsids and requires surface glycoproteins for release from the cell. We investigated the requirements for synthesis of extracellular HFV particles by constructing mutants with either the pol or env gene deleted. We found that the Pol protein is dispensable for production of extracellular particles containing viral nucleic acid. In the absence of Env, intracellular particles are synthesized but few or no extracellular particles could be detected. Thus, foamy virus assembly is distinct from that of other reverse transcriptase-encoding mammalian viruses.

The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes). We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties. Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays. Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication. A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells. Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture. This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection. Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag protein.

Human foamy virus (HFV) is the prototype of the Spumavirus genus of Retroviridae. In all other retroviruses, the pol gene products, including reverse transcriptase, are synthesized as Gag-Pol fusion proteins and are cleaved to functional enzymes during viral budding or release. In contrast, the Pol protein of HFV is translated from a spliced messenger RNA and lacks Gag domains. Infectious HFV particles contain double-stranded DNA similar in size to full-length provirus, suggesting that reverse transcription has taken place in viral particles before new rounds of infection, reminiscent of hepadnaviruses. These data suggest that foamy viruses possess a replication pathway containing features of both retroviruses and hepadnaviruses but distinct from both.

Human foamy virus can establish persistent infections in human hematopoietic cell lines, such as H92.1.7 (erythroblastoid cells), Jurkat (CD4+ T cells), and U937 (myeloid-monocytic cells). The infection is characterized by constant production of infectious viruses (for > 2 1/2 years) with no cytopathic effects on the host cells. Electron microscopy of the infected cells showed a viral morphology similar to that observed for particles produced after acute infection. We have detected, in addition to the full-length form of bel1, a previously described deletion in the bel1 gene of the proviral DNA in these cells. RNA containing this 301-bp deletion, which mapped to the splice donor and acceptor sites of the intron of the bet gene, was also found in encapsidated virion RNA. However, the presence of this defective provirus harboring the deletion in bel1 does not prevent productive persistence in these chronically infected cells, since the virus titer does not decrease during cultivation.

The human foamy or spumaretrovirus (HFV) is a complex retrovirus that codes for the three retroviral genes gag, pol, and env and the regulatory and accessory bel genes. A particular feature of HFV gene expression was recently described: not only does the HFV provirus contain the classical retroviral long terminal repeat promoter, a second functionally active promoter is present in the env gene upstream of the bel genes (M. Löchelt, W. Muranyi, and R. M. Flügel, 1993, Proc. Natl. Acad. Sci. USA 90, 7317-7321). Both, the HFV long terminal repeat promoter I and internal promoter II depend upon the HFV transcriptional transactivator Bel 1 for efficient gene expression. The internal promoter directs the synthesis of functionally active Bel 1 transactivator and Bet proteins that are expressed early after HFV infection. In this report, it is shown that mutation of the promoter II TATA box resulted in HFV proviral clones with a reduction in infectivity by a factor of approximately 100. Gene expression by promoter II TATA box mutant HFV proviruses was reduced. HFV proviruses with the mutated promoter II TATA box used cryptic start sites of transcription upstream of the original promoter II TATA box, resulting in an inefficient and less accurate transcriptional initiation. The reduced HFV structural gene expression by the mutated HFV proviruses was relieved by providing Bel 1 protein in trans. This demonstrates that HFV promoter II-directed Bel 1 expression is important for producing the high levels of Bel 1 that increases virus replication.

We have constructed a BHK-21-derived indicator cell line containing a single integrated copy of the beta-galactosidase (beta-Gal) gene under control of the human foamy virus (HFV) long terminal repeat promoter (from -533 to +20). These foamy virus-activated beta-Gal expression (FAB) cells can be used in a quantitative assay to measure the infectious titer of HFV. Our results show that the FAB assay is 50 times more sensitive than determination of the virus titer by the end-point dilution method. Using the FAB assay, we have found that HFV can productively replicate in several erythroblastoid cell lines as well as in the Jurkat T-cell line. We have also examined the roles of bel2, bet, and bel3 in viral replication by constructing proviral HFV clones in which the reading frame of Bel2, Bet, or Bel3 is disrupted by placement of translation stop codons. Analysis of these mutants reveals that while the bel3 gene is not required for viral replication in vitro, mutations in the bel2 or bet gene decrease cell-free viral transmission approximately 10-fold.