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1.  Vaccinia virus F16 protein, a predicted catalytically inactive member of the prokaryotic serine recombinase superfamily, is targeted to nucleoli 
Virology  2011;417(2):334-342.
The F16L gene of vaccinia virus (VACV) is conserved in all chordopoxviruses except avipoxviruses. The crocodile poxvirus F16 protein ortholog has highly significant similarity to prokaryotic serine recombinases and contains all amino acids that comprise the catalytic site. In contrast, F16 orthologs encoded by other poxviruses show only marginally significant similarity to serine recombinases, lack essential amino acids of the active site and are most likely inactive derivatives of serine recombinases. Nevertheless, the conservation of F16L in non-avian poxviruses suggested an important function. However, a VACV mutant with the F16L gene knocked out replicated normally in dividing and quiescent cells. The F16 protein was synthesized early after infection and detected in virus cores. When expressed in infected or uninfected cells, F16 accumulated in nucleoli depending on the level of expression and confluency of cells. Evidence was obtained that F16 forms multimers, which might regulate concentration-dependent intracellular localization.
PMCID: PMC3163719  PMID: 21752417
poxvirus orthologs; poxvirus prokaryotic homologs; vaccinia virus replication; poxvirus recombinase
2.  Cellular DNA Ligase I Is Recruited to Cytoplasmic Vaccinia Virus Factories and Masks the Role of the Vaccinia Ligase in Viral DNA Replication 
Cell host & microbe  2009;6(6):563-569.
Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. Knock-down of human DNA ligase I but not other ligases with siRNA or a specific inhibitor severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding of a ligase might allow VACV to “jump-start” DNA synthesis.
PMCID: PMC2846536  PMID: 20006844
3.  Vaccinia Virus A26 and A27 Proteins Form a Stable Complex Tethered to Mature Virions by Association with the A17 Transmembrane Protein ▿  
Journal of Virology  2008;82(24):12384-12391.
During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.
PMCID: PMC2593336  PMID: 18842719
4.  A conserved poxvirus NLPC/P60 superfamily protein contributes to vaccinia virus virulence in mice but not to replication in cell culture 
Virology  2008;374(2):506-514.
Of the vaccinia virus genes that are conserved in all sequenced poxviruses, each one except for VACWR084 (G6R) has been at least partially characterized. The poxvirus protein encoded by G6R belongs to the NLPC/P60 superfamily, which consists of proteins with a papain-like fold and known or predicted protease, amidase or acyltransferase activity. The G6 protein was synthesized late in infection and localized to the interior of virions, primarily between the membrane and core. Unlike other conserved poxvirus genes, G6R was not required for virus propagation and spread in a variety of cells. Nevertheless, G6R null mutants caused less severe disease in mice than the parent or revertant virus. Moreover, mutation of the predicted catalytic cysteine led to the same level of attenuation as a null mutant, suggesting that the G6 protein has enzymatic activity that is important in vivo. Conservation of G6R amongst poxviruses and the disparity between its role in vitro and in vivo imply that the protein is involved in an aspect of the virus-host interaction that is common to vertebrates and insects.
PMCID: PMC2412903  PMID: 18281072
5.  Vaccinia Virus F9 Virion Membrane Protein Is Required for Entry but Not Virus Assembly, in Contrast to the Related L1 Protein 
Journal of Virology  2006;80(19):9455-9464.
All sequenced poxviruses encode orthologs of the vaccinia virus L1 and F9 proteins, which are structurally similar and share about 20% amino acid identity. We found that F9 further resembles L1 as both proteins are membrane components of the mature virion with similar topologies and induce neutralizing antibodies. In addition, a recombinant vaccinia virus that inducibly expresses F9, like a previously described L1 mutant, had a conditional-lethal phenotype: plaque formation and replication of infectious virus were dependent on added inducer. However, only immature virus particles are made when L1 is repressed, whereas normal-looking intracellular and extracellular virions formed in the absence of F9. Except for the lack of F9, the polypeptide components of such virions were indistinguishable from those of wild-type virus. These F9-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, cells infected with F9-negative virions did not fuse after a brief low-pH treatment, as did cells infected with virus made in the presence of inducer. In these respects, the phenotype associated with F9 deficiency was identical to that produced by the lack of individual components of a previously described poxvirus entry/fusion complex. Moreover, F9 interacted with proteins of that complex, supporting a related role. Thus, despite the structural relationships of L1 and F9, the two proteins have distinct functions in assembly and entry, respectively.
PMCID: PMC1617236  PMID: 16973551
6.  The ancient Virus World and evolution of cells 
Biology Direct  2006;1:29.
Recent advances in genomics of viruses and cellular life forms have greatly stimulated interest in the origins and evolution of viruses and, for the first time, offer an opportunity for a data-driven exploration of the deepest roots of viruses. Here we briefly review the current views of virus evolution and propose a new, coherent scenario that appears to be best compatible with comparative-genomic data and is naturally linked to models of cellular evolution that, from independent considerations, seem to be the most parsimonious among the existing ones.
Several genes coding for key proteins involved in viral replication and morphogenesis as well as the major capsid protein of icosahedral virions are shared by many groups of RNA and DNA viruses but are missing in cellular life forms. On the basis of this key observation and the data on extensive genetic exchange between diverse viruses, we propose the concept of the ancient virus world. The virus world is construed as a distinct contingent of viral genes that continuously retained its identity throughout the entire history of life. Under this concept, the principal lineages of viruses and related selfish agents emerged from the primordial pool of primitive genetic elements, the ancestors of both cellular and viral genes. Thus, notwithstanding the numerous gene exchanges and acquisitions attributed to later stages of evolution, most, if not all, modern viruses and other selfish agents are inferred to descend from elements that belonged to the primordial genetic pool. In this pool, RNA viruses would evolve first, followed by retroid elements, and DNA viruses. The Virus World concept is predicated on a model of early evolution whereby emergence of substantial genetic diversity antedates the advent of full-fledged cells, allowing for extensive gene mixing at this early stage of evolution. We outline a scenario of the origin of the main classes of viruses in conjunction with a specific model of precellular evolution under which the primordial gene pool dwelled in a network of inorganic compartments. Somewhat paradoxically, under this scenario, we surmise that selfish genetic elements ancestral to viruses evolved prior to typical cells, to become intracellular parasites once bacteria and archaea arrived at the scene. Selection against excessively aggressive parasites that would kill off the host ensembles of genetic elements would lead to early evolution of temperate virus-like agents and primitive defense mechanisms, possibly, based on the RNA interference principle. The emergence of the eukaryotic cell is construed as the second melting pot of virus evolution from which the major groups of eukaryotic viruses originated as a result of extensive recombination of genes from various bacteriophages, archaeal viruses, plasmids, and the evolving eukaryotic genomes. Again, this vision is predicated on a specific model of the emergence of eukaryotic cell under which archaeo-bacterial symbiosis was the starting point of eukaryogenesis, a scenario that appears to be best compatible with the data.
The existence of several genes that are central to virus replication and structure, are shared by a broad variety of viruses but are missing from cellular genomes (virus hallmark genes) suggests the model of an ancient virus world, a flow of virus-specific genes that went uninterrupted from the precellular stage of life's evolution to this day. This concept is tightly linked to two key conjectures on evolution of cells: existence of a complex, precellular, compartmentalized but extensively mixing and recombining pool of genes, and origin of the eukaryotic cell by archaeo-bacterial fusion. The virus world concept and these models of major transitions in the evolution of cells provide complementary pieces of an emerging coherent picture of life's history.
W. Ford Doolittle, J. Peter Gogarten, and Arcady Mushegian.
PMCID: PMC1594570  PMID: 16984643
7.  Entry of Vaccinia Virus and Cell-Cell Fusion Require a Highly Conserved Cysteine-Rich Membrane Protein Encoded by the A16L Gene 
Journal of Virology  2006;80(1):51-61.
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.
PMCID: PMC1317547  PMID: 16352530
8.  The Product of the Vaccinia Virus L5R Gene Is a Fourth Membrane Protein Encoded by All Poxviruses That Is Required for Cell Entry and Cell-Cell Fusion 
Journal of Virology  2005;79(17):10988-10998.
The L5R gene of vaccinia virus is conserved among all sequenced members of the Poxviridae but has no predicted function or recognized nonpoxvirus homolog. Here we provide the initial characterization of the L5 protein. L5 is expressed following DNA replication with kinetics typical of a viral late protein, contains a single intramolecular disulfide bond formed by the virus-encoded cytoplasmic redox pathway, and is incorporated into intracellular mature virus particles, where it is exposed on the membrane surface. To determine whether L5 is essential for virus replication, we constructed a mutant that synthesizes L5 only in the presence of an inducer. The mutant exhibited a conditional-lethal phenotype, as cell-to-cell virus spread and formation of infectious progeny were dependent on the inducer. Nevertheless, all stages of replication occurred in the absence of inducer and intracellular and extracellular progeny virions appeared morphologically normal. Noninfectious virions lacking L5 could bind to cells, but the cores did not enter the cytoplasm. In addition, virions lacking L5 were unable to mediate low-pH-triggered cell-cell fusion from within or without. The phenotype of the L5R conditional lethal mutant is identical to that of recently described mutants in which expression of the A21, A28, and H2 genes is repressed. Thus, L5 is the fourth component of the poxvirus cell entry/fusion apparatus that is required for entry of both the intracellular and extracellular infectious forms of vaccinia virus.
PMCID: PMC1193616  PMID: 16103150
9.  Vaccinia Virus A21 Virion Membrane Protein Is Required for Cell Entry and Fusion 
Journal of Virology  2005;79(15):9458-9469.
We provide the initial characterization of the product of the vaccinia virus A21L (VACWR140) gene and demonstrate that it is required for cell entry and low pH-triggered membrane fusion. The A21L open reading frame, which is conserved in all sequenced members of the poxvirus family, encodes a protein of 117 amino acids with an N-terminal hydrophobic domain and four invariant cysteines. Expression of the A21 protein occurred at late times of infection and was dependent on viral DNA replication. The A21 protein contained two intramolecular disulfide bonds, the formation of which required the vaccinia virus-encoded cytoplasmic redox pathway, and was localized on the surface of the lipoprotein membrane of intracellular mature virions. A conditional lethal mutant, in which A21L gene expression was regulated by isopropyl-β-d-thiogalactopyranoside, was constructed. In the absence of inducer, cell-to-cell spread of virus did not occur, despite the formation of morphologically normal intracellular virions and extracellular virions with actin tails. Purified virions lacking A21 were able to bind to cells, but cores did not penetrate into the cytoplasm and synthesize viral RNA. In addition, virions lacking A21 were unable to mediate low pH-triggered cell-cell fusion. The A21 protein, like the A28 and H2 proteins, is an essential component of the poxvirus entry/fusion apparatus for both intracellular and extracellular virus particles.
PMCID: PMC1181583  PMID: 16014909
10.  Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion 
Journal of Virology  2005;79(8):4744-4754.
The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.
PMCID: PMC1069540  PMID: 15795260
11.  Vaccinia Virus Entry into Cells Is Dependent on a Virion Surface Protein Encoded by the A28L Gene 
Journal of Virology  2004;78(5):2357-2366.
The A28L gene of vaccinia virus is conserved in all poxviruses and encodes a protein that is anchored to the surface of infectious intracellular mature virions (IMV) and consequently lies beneath the additional envelope of extracellular virions. A conditional lethal recombinant vaccinia virus, vA28-HAi, with an inducible A28L gene, undergoes a single round of replication in the absence of inducer, producing IMV, as well as extracellular virions with actin tails, but fails to infect neighboring cells. We show here that purified A28-deficient IMV appeared to be indistinguishable from wild-type IMV and were competent to synthesize RNA in vitro. Nevertheless, A28-deficient virions did not induce cytopathic effects, express early genes, or initiate a productive infection. Although A28-deficient IMV bound to the surface of cells, their cores did not penetrate into the cytoplasm. An associated defect in membrane fusion was demonstrated by the failure of low pH to trigger syncytium formation when cells were infected with vA28-HAi in the absence of inducer (fusion from within) or when cells were incubated with a high multiplicity of A28-deficient virions (fusion from without). The correlation between the entry block and the inability of A28-deficient virions to mediate fusion provided compelling evidence for a relationship between these events. Because repression of A28 inhibited cell-to-cell spread, which is mediated by extracellular virions, all forms of vaccinia virus regardless of their outer coat must use a common A28-dependent mechanism of cell penetration. Furthermore, since A28 is conserved, all poxviruses are likely to penetrate cells in a similar way.
PMCID: PMC369249  PMID: 14963132
12.  Vaccinia Virus A28L Gene Encodes an Essential Protein Component of the Virion Membrane with Intramolecular Disulfide Bonds Formed by the Viral Cytoplasmic Redox Pathway 
Journal of Virology  2004;78(5):2348-2356.
We report the initial characterization of the product of the vaccinia virus A28L gene, which is highly conserved in all sequenced poxviruses. Our studies showed that the A28 protein is expressed at late times during the virus replication cycle and is a membrane component of the intracellular mature virion. An N-terminal hydrophobic sequence, present in all poxvirus A28 orthologs, anchors the protein in the virion surface membrane so that most of it is exposed to the cytoplasm. The cytoplasmic domain contains four conserved cysteines, which form two intramolecular disulfide bonds. Disulfide bond formation depended on the expression of three viral proteins, E10, A2.5, and G4, which together comprise a conserved cytoplasmic redox pathway. A28 is the third identified substrate of this pathway; the others are the L1 and F9 proteins. We constructed a conditional-lethal recombinant vaccinia virus with an inducible A28L gene. The recombinant virus was propagated in the presence of inducer but was unable to replicate and spread in its absence. During a single round of an abortive infection in the absence of inducer, the synthesis and processing of viral proteins, assembly of intra- and extracellular virions, and formation of actin tails occurred normally. In another paper (T. Senkevich, B. M. Ward, and B. Moss, J. Virol. 78:2357-2366, 2004), we have demonstrated that virions assembled without A28 cannot carry out a second round of infection because they are defective in cell penetration.
PMCID: PMC369248  PMID: 14963131
13.  Vaccinia Virus G4L Glutaredoxin Is an Essential Intermediate of a Cytoplasmic Disulfide Bond Pathway Required for Virion Assembly 
Journal of Virology  2002;76(2):467-472.
Our previous studies provided evidence that E10R, a vaccinia virus protein belonging to the ERV1/ALR family, has a redox function and is required for virion assembly. Repression of E10R prevented the formation of intramolecular disulfide bonds of the G4L glutaredoxin, the L1R membrane protein, and the structurally related F9L protein. Here, we demonstrate an oxidation pathway (E10RSS → G4LSS → L1RSS, F9LSS) in which G4L occupies an intermediate position. By reacting free thiols with 4-acetamido-4′-malemideylstilbene-2,2′-disulfonic acid, alkylated and nonalkylated disulfide-bonded forms of G4L could be resolved from each other by polyacrylamide gel electrophoresis. The cysteines of intracellular G4L were in both disulfide and reduced forms, whereas those of E10R, L1R, and F9L and virion-associated G4L were mostly disulfide bonded. Repression of G4L expression prevented the formation of disulfide bonds in both L1R and F9L but not E10R. Both cysteines of G4L were required for L1R and F9L disulfide bond formation or for trans-complementation of virus infectivity when G4L expression was repressed. No role in the E10R-G4L redox pathway was found for O2L, a nonessential glutaredoxin encoded by vaccinia virus. We suggest that cytoplasmic G4L is a redox shuttle between membrane-associated E10R and L1R or F9L.
PMCID: PMC136847  PMID: 11752136

Results 1-13 (13)