Poxviruses have evolved elaborate mechanisms for cell entry, assembly, and exocytosis. Recently, four vaccinia virus membrane proteins, namely A21, A28, H2 and L5, were reported to be necessary for cell entry and virus-induced cell-cell fusion but not for virion morphogenesis or attachment of virus particles to cells. Using immunoaffinity purification followed by mass spectrometry, we now show that these four proteins as well as four additional previously uncharacterized putative membrane proteins (A16, G3, G9, and J5) form a stable complex. These proteins fall into two groups: A21, A28, G3, H2, and L5 have an N-terminal transmembrane domain, 0 -2 intramolecular disulfide bonds, and no sequence similarity, whereas A16, G9, and J5 have a C-terminal transmembrane domain and 4 -10 predicted disulfide bonds and are homologous. Studies with conditional-lethal null mutants indicated that the viral membrane was crucial for assembly of the complex and that the absence of individual polypeptide components profoundly decreased complex formation or stability, suggesting a complicated interaction network. Analysis of purified virions, however, demonstrated that the polypeptides of the complex trafficked independently to the viral membrane even under conditions in which the complex itself could not be isolated. All eight proteins comprising the entry-fusion complex are conserved in all poxviruses, suggesting that they have nonredundant functions and that the basic entry mechanism evolved before the division between vertebrate and invertebrate poxvirus species. mass spectrometry ͉ transmembrane proteins ͉ vaccinia virus ͉ viral envelope proteins ͉ virus entry

Understanding human immunodeficiency virus type 1 (HIV-1) transmission is central to developing effective prevention strategies, including a vaccine. We compared phenotypic and genetic variation in HIV-1 env genes from subjects in acute/early infection and subjects with chronic infections in the context of subtype C heterosexual transmission. We found that the transmitted viruses all used CCR5 and required high levels of CD4 to infect target cells, suggesting selection for replication in T cells and not macrophages after transmission. In addition, the transmitted viruses were more likely to use a maraviroc-sensitive conformation of CCR5, perhaps identifying a feature of the target T cell. We confirmed an earlier observation that the transmitted viruses were, on average, modestly underglycosylated relative to the viruses from chronically infected subjects. This difference was most pronounced in comparing the viruses in acutely infected men to those in chronically infected women. These features of the transmitted virus point to selective pressures during the transmission event. We did not observe a consistent difference either in heterologous neutralization sensitivity or in sensitivity to soluble CD4 between the two groups, suggesting similar conformations between viruses from acute and chronic infection. However, the presence or absence of glycosylation sites had differential effects on neutralization sensitivity for different antibodies. We suggest that the occasional absence of glycosylation sites encoded in the conserved regions of env, further reduced in transmitted viruses, could expose specific surface structures on the protein as antibody targets.

The vaccinia virus A16L open reading frame encodes a 378-amino-acid protein with a predicted C-terminal transmembrane domain and 20 invariant cysteine residues that is conserved in all sequenced members of the poxvirus family. The A16 protein was expressed late in infection and incorporated into intracellular virus particles with the N-terminal segment of the protein exposed on the surface. The cysteine residues were disulfide bonded via the poxvirus cytoplasmic redox system. Unsuccessful attempts to isolate a mutant virus with the A16L gene deleted suggested that the protein is essential for replication. To study the role of the A16 protein, we made a recombinant vaccinia virus that has the Escherichia coli lac operator system regulating transcription of the A16L gene. In the absence of inducer, A16 synthesis was repressed and plaque size and virus yield were greatly reduced. Nevertheless, virus morphogenesis occurred and normal-looking intracellular and extracellular virus particles formed. Purified virions made in the presence and absence of inducer were indistinguishable, though the latter had 60-to 100-fold-lower specific infectivity. A16-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, A16-deficient virions were unable to induce low-pH-triggered syncytium formation. The phenotype of the inducible A16L mutant was similar to those of mutants in which synthesis of the A21, A28, H2, or L5 membrane protein was repressed, indicating that at least five conserved viral proteins are required for entry of poxviruses into cells as well as for cell-cell fusion.Vaccinia virus (VACV) is a member of the poxvirus family of large, double-stranded DNA viruses that replicate entirely in the cytoplasm (17). VACV strain Western Reserve (WR), the prototype member of the orthopoxvirus genus, contains approximately 200 genes of which 44 have a putative transmembrane domain (Poxvirus Bioinformatics Resource Center [www.poxvirus.org]). VACV membrane proteins have diverse essential and nonessential roles in virus-host interactions, virion assembly, intracellular movement, and cell-to-cell spread. A novel class of poxvirus membrane proteins, required for cell entry but not for virion assembly, was recently identified (20-22, 27, 28). These four VACV proteins (A21, A28, H2, and L5) are conserved in all sequenced poxviruses, have common features including N-terminal or near N-terminal transmembrane domains and cysteines that form two to four intramolecular disulfide bonds, and are located on the surfaces of infectious intracellular mature virions (IMVs). Each of the four proteins is also required for cell-cell fusion, implying that cell entry involves a related or identical fusion mechanism. Fusion of IMVs with the plasma membrane was previously suggested on the basis of biochemical and microscopic studies (4,7,8,11,15). Cellular receptor proteins that participate in cell entry, however, remain to be identified.Following virus morphogenesis, IMVs are completely enveloped by an additio...

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.The mechanisms by which enveloped DNA viruses enter cells are poorly understood compared to those for many enveloped RNA viruses (17). Entry of the latter is typically mediated by one or two viral glycoproteins and involves virus attachment to the cell, activation of a fusion protein, and ultimately, merging of the viral and cellular membranes to allow entry of the genome and associated proteins. In contrast, three to four glycoproteins are required for entry of herpesviruses (35) and even more are needed for entry of vaccinia virus (VACV), the prototype poxvirus (26). Studies of VACV entry have been complicated by the existence of two infectious forms: the mature virion (MV), which contains a nucleoprotein core surrounded by a membrane containing more than 20 nonglycosylated proteins, and the extracellular virion (EV), which is essentially an MV surrounded by an additional membrane containing five proteins that are glycosylated and one that is not (9,26,34). The MV, which is extremely stable and can be liberated by cell lysis, is thought to mediate transmission between host animals, whereas the EV mediates cell-to-cell spread. There is evidence that the MV and EV bind differently to cells (39), consistent with their different outer membrane proteins. Binding of the MV to some cells appears to be due at least in part to three membrane proteins that can bind heparan or chondroitin sulfate (7,19,20,23,40), although individ...