Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelopemediated infectivity of murine leukemia virus pseudotypes.Signal peptides (SP) are key determinants for targeting and membrane insertion of secretory and membrane proteins (reviewed in reference 25). They can be removed co-or posttranslationally by the cellular membrane-bound signal peptidase or may, if not cleaved, serve as membrane anchors for proteins with distinct membrane orientations. In general, SP are composed of three domains, of which a central 6-to 15-amino-acid (aa)-long hydrophobic domain (h-domain) is the most essential. An N-terminal polar domain (n-domain) usually of net positive charge shows high variability in overall length, ranging from 15 to more than 50 aa. The composition and structure of the n-domain influences protein orientation in the membrane. The polar C-terminal domain (c-domain) often contains helixbreaking as well as small uncharged residues in positions -3 and -1 which determine the site of SP cleavage. In most cases, SP cleavage is thought to occur cotranslationally; however, for some proteins, e.g., the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp160, SP cleavage occurs inefficiently and very late after translocation (21). A basic amino acid stretch in the n-domain of gp160 is responsible for this phenomenon and believed to influence folding and exit of HIV-1 Env from the endoplasmic reticulum (ER) (21). Recent studies revealed that SP bear specific information accounting for distinct functions in targeting and membrane insertion or even for defined metabolic pathways after their cleavage from the parent protein (reviewed in reference 25). The HIV-1 SP Env , for example, is further processed by the signal peptidase, leading to the release of an SP fragment into the cytosol, where it binds to calmodulin (26). The function of this process in viral replication is not known.Foamy viruses (FV), as studied with the prototype member human foamy virus (HFV), follow a replication cycle which is charact...

Supplemental Digital Content is available in the text.

Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus replication, but their functions have remained unknown. In the present study, we deleted the essential US22 family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells, which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its target protein, the eukaryotic translation initiation factor 2␣, suggesting that m142 and m143 are necessary to block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both required for inhibition of the PKR-mediated host antiviral response.Cytomegaloviruses (CMVs) are prototypes of the ␤ subfamily of the Herpesviridae. Their genomes span 230 kb and are the largest among the herpesviruses. Of the approximately 170 genes on a CMV genome, about 46 are conserved among the herpesviruses (42). An additional 34 genes are characteristic of the ␤-herpesviruses (12,21,44,58), and the remaining genes are unique to a particular virus.A typical property of ␤-herpesvirus genomes is the presence of gene families, which probably arose by duplication from ancestral genes. One of the largest, the US22 gene family, was first identified in human cytomegalovirus (HCMV) and was later also found in other ␤-herpesviruses (12,21,44,51,58). It is characterized by four conserved sequence motifs consisting of hydrophobic residues interspersed with charged amino acids. HCMV and murine cytomegalovirus (MCMV) both include 12 members of the US22 gene family on their genomes (12, 51). The rat CMV and human herpesviruses 6 and 7, two other human ␤-herpesviruses, also possess up to 11 US22 family genes (20,21,44,58).Little is known about the functions of US22 gene products. However, 5 of the 12 US22 gene products of MCMV affect the virus' ability to replicate in macrophages: M36, M43, m139, m140, and m141 (28,29,32,41). The molecular mechanism of action for only one of these has been elucidated. The M36 gene encodes an antiapoptotic protein that binds to procaspase 8 and inhibits death receptor-mediated induction of apoptosis (41). The positional and sequence homolog of M36 in HCMV, UL36, was originally proposed to function as a transcriptional transactivator (16...

Apoptosis of infected cells can limit virus replication and serves as an innate defense mechanism against viral infections. Consequently, viruses delay apoptosis by expressing antiapoptotic proteins, many of which structurally resemble the cellular antiapoptotic protein Bcl-2. Like Bcl-2, the viral analogs inhibit apoptosis by preventing activation and/or oligomerization of the proapoptotic mitochondrial proteins Bax and Bak. Here we show that cytomegaloviruses (CMVs) have adopted a different strategy. They encode two separate mitochondrial proteins that lack obvious sequence similarities to Bcl-2-family proteins and specifically counteract either Bax or Bak. We identified a small mitochondrion-localized protein encoded by the murine CMV open reading frame (ORF) m41.1, which functions as a viral inhibitor of Bak oligomerization (vIBO). It blocks Bak-mediated cytochrome c release and Bak-dependent induction of apoptosis. It protects cells from cell death-inducing stimuli together with the previously identified Bax-specific inhibitor viral mitochondria-localized inhibitor of apoptosis (vMIA) (encoded by ORF m38.5). Similar vIBO proteins are encoded by CMVs of rats, and possibly by other CMVs as well. These results suggest a non-redundant function of Bax and Bak during viral infection, and a benefit for CMVs derived from the ability to inhibit Bak and Bax separately with two viral proteins.

In general, enveloped viruses use two different entry strategies and are classified accordingly into pHdependent 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.