Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.

Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol proteins are expressed independently of one another, and both proteins undergo single cleavage events. Thus, the mature FV Gag protein does not consist of the matrix, capsid, and nucleocapsid (NC) proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol protein.The process of retroviral assembly is largely driven by the Gag protein. For all Orthoretrovirinae, such as human immunodeficiency virus (HIV), specific domains within the Gag precursor polyprotein regulate the major steps of assembly (reviewed in reference 32). To properly assemble an infectious virus particle, the genomic RNA must be selectively packaged into the virion. This is no easy task given that the viral RNA makes up much less than 1% of the total mRNA pool existing in the cytoplasm of the cell. The regions of viral RNA responsible for orthoretroviral RNA packaging are referred to as the packaging signal () and are located near the 5Ј end of the RNA genome. The packaging signal is believed to comprise both specific sequences and higher-order structural features (3,4,28). Viral proteins, in addition to cis-acting sequences, play an important role in viral genomic RNA packaging. The virion nucleocapsid protein (NC) has been shown to be specifically involved in viral genomic RNA dimerization and packaging (12)(13)(14)23). In addition, mutagenesis studies have shown that the basic residues surrounding the Cys-His boxes within NC are critical for RNA packaging for many orthoretroviruses (6,11,19,27,31).In contrast, foamy viruses (FV) have a fundamentally different replication strategy. FV are classified as complex retroviruses based largely on their genomic organization and their reverse transcription pathway. A major difference between FV and other retroviruses is the mode of Pol expression. Unlike orthoretroviruses, which express Pol as part of a Gag-Pol fusion protein, FV e...

BackgroundFoamy viruses (FV) are ancient complex retroviruses that differ from orthoretroviruses such as human immunodeficiency virus (HIV) and murine leukemia virus (MLV) and comprise a distinct subfamily of retroviruses, the Spumaretrovirinae. FV are ubiquitous in their natural hosts, which include cows, cats, and nonhuman primates (NHP). FV are transmitted mainly through saliva and appear nonpathogenic by themselves, but they may increase morbidity of other pathogens in coinfections.ConclusionsThis review summarizes and discusses what is known about FV infection of natural hosts. It also emphasizes what is known about FV zoonotic infections A large number of studies have revealed that the FV of NHP, simian foamy viruses (SFV), are transmitted to humans who interact with infected NHP. SFV from a variety of NHP establish persistent infection in humans, while bovine foamy virus and feline foamy virus rarely or never do. The possibility of FV recombination and mutation leading to pathogenesis is considered. Since humans can be infected by SFV, a seemingly nonpathogenic virus, there is interest in using SFV vectors for human gene therapy. In this regard, detailed understanding of zoonotic SFV infection is highly relevant.

e Foamy viruses (FV) are complex retroviruses that naturally infect all nonhuman primates (NHP) studied to date. Zoonotic transmission of Old World NHP simian foamy viruses (SFV) has been documented, leading to nonpathogenic persistent infections. To date, there have been no reports concerning zoonotic transmission of New World monkey (NWM) SFV to humans and resulting infection. In this study, we developed a Western blot assay to detect antibodies to NWM SFV, a nested PCR assay to detect NWM SFV DNA, and a ␤-galactosidase-containing indicator cell line to assay replication of NWM SFV. Using these tools, we analyzed the plasma and blood of 116 primatologists, of whom 69 had reported exposures to NWM. While 8 of the primatologists tested were seropositive for SFV from a NWM, the spider monkey, none had detectable levels of viral DNA in their blood. We found that SFV isolated from three different species of NWM replicated in some, but not all, human cell lines. From our data, we conclude that while humans exposed to NWM SFV produce antibodies, there is no evidence for long-term viral persistence. Foamy viruses (FV) are unusual complex retroviruses that infect cattle, horses, cats, and all species of nonhuman primates (NHP) examined to date (reviewed in reference 1). Simian foamy viruses (SFV) can cause life-long infections in natural hosts without any apparent pathogenicity (2). In cell culture models, SFV can establish latent infections in some cell types and lytic infections in others, resulting in cytopathic effects (CPE) that include syncytium formation (reviewed in reference 3). In infected macaques, FV DNA is found in almost all tissues, while FV RNA and replicating virus are limited to the superficial epithelial cells of the oral mucosa in immunocompetent animals (4, 5). Consistent with the site of viral replication detected in vivo, it is thought that FV are transmitted via saliva, through grooming, biting, and other behaviors. Studies of natural transmission suggest that infant and young NHP are resistant to infection, presumably because of passive immunity from maternal antibodies, but typically (for macaques) become infected by 3 years of age (6). Epidemiological studies indicate that SFV seroprevalence can reach up to 100% in adults (reviewed in reference 7).Although FV do not naturally infect humans, SFV have been found to be zoonotically transmitted, most likely through contact with saliva from an infected NHP. Several studies have documented the transmission of SFV to humans who interact directly with Old World (OW) NHP, including Cercopithicus species, baboons, macaques, mandrills, gorillas, and chimpanzees (reviewed in reference 7). SFV antibody-positive humans have been found in a variety of natural settings, including people in Asia who live in areas with free-ranging macaques, villagers in Gabon with known exposure to NHP, and a population of hunters in Cameroon with bites from Old World NHP (6,(8)(9)(10)(11). SFV antibody-positive humans have also been documented in various laboratory, veterina...

Foamy virus (FV) replication, while related to that of orthoretroviruses, differs at a number of steps. Several of these differences involve the reverse transcriptase (RT). There appear to be fewer RTs present in FV than in orthoretroviruses; we previously proposed that the polymerase of FV RT was more active than orthoretroviral RTs to compensate for the numerical difference. Here we present further characterization of the RT of FV. The polymerase activity of FV RT was greater than that of human immunodeficiency virus type 1 RT in a variety of assays. We also examined the RNase H activity of FV RT, and we propose that FV RT has a basic loop in the RNase H domain. Although the sequence of the basic loop of FV RT is different from the basic loop of either Moloney leukemia virus RNase H or Escherichia coli RNase H, the FV RT basic loop appears to have a similar function.Foamy viruses (FVs) are retroviruses (subfamily Spumaretrovirinae) but are in some ways distinct from typical retroviruses. The organization of the gag, pol, and env genes in the FV genome is the same as in orthoretroviruses and, like orthoretroviruses, FVs convert their single-stranded RNA genomes into integrated double-stranded DNA proviruses flanked by two long terminal repeats (LTRs), using the enzymes reverse transcriptase (RT) and integrase (IN). However, FVs have distinct features that set them apart from orthoretroviruses. Several of these differences involve either reverse transcription or the RT enzyme itself. In orthoretroviruses, such as human immunodeficiency virus type 1 (HIV-1), RT converts the single-stranded RNA genome into double-stranded DNA after the virion enters the target cell (8). In contrast, a significant portion of FV particles contain double-stranded DNA (39,41). Inhibitor studies make it clear that it is the virions with DNA genomes that infect the target cells, indicating that FV RT must be active in the cells producing the viral particles (27,41).Orthoretroviruses use a single full-length RNA transcript as the mRNA for both the gag and pol genes. Translation of the full-length RNA produces both the Gag and Gag-Pol polyproteins (8). Approximately 20 Gag proteins are produced for every Gag-Pol fusion protein (8). Gag self-assembles to form viral particles; during virus assembly, the Gag portion of the Gag-Pol fusion protein interacts with the Gag proteins. This is how the Pol proteins are incorporated into virions; the resulting virions have approximately 50 to 100 Gag-Pol polyproteins per particle (14,18,28,38). However, in an FV-infected cell, the Pol polyprotein is made independently from Gag; Pol is translated from a spliced message (13,19,39). The FV Pol polyprotein is proteolytically processed; however, it undergoes only limited processing. There is a single protease cleavage between RT and IN which releases IN and a protease-RT fusion protein (30). Protease is not cleaved from RT. The mechanism for packaging FV Pol protein into the virion appears to involve interactions of FV Pol with specific sequences in the RN...

We compared the in vitro fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and the prototype foamy virus (PFV) RT. Both enzymes had similar error rates for single nucleotide substitutions; however, PFV RT did not appear to make errors at specific hotspots, like HIV-1 RT. In addition, PFV RT made more deletions and insertions than HIV-1 RT. Although the majority of the missense errors made by HIV-1 RT and PFV RT are different, relatively few of the mutations caused by either enzyme can be explained by a misalignment/slippage mechanism. We suggest that the higher polymerase activity of PFV RT could contribute to the ability of the enzyme to jump to the same or a different template.

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.

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