Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 55 records · Page 3Linked to original sources

PubMed2000

Characterization of the vaccinia virus H3L envelope protein: topology and posttranslational membrane insertion via the C-terminal hydrophobic tail.

The vaccinia virus H3L open reading frame encodes a 324-amino-acid immunodominant membrane component of virus particles. Biochemical and microscopic studies demonstrated that the H3L protein was expressed late in infection, accumulated in the cytoplasmic viral factory regions, and associated primarily with amorphous material near immature virions and with intracellular virion membranes. Localization of the H3L protein on the surfaces of viral particles and anchorage via the hydrophobic tail were consistent with its extraction by NP-40 in the absence of reducing agents, its trypsin sensitivity, its reactivity with a membrane-impermeable biotinylation reagent, and its immunogold labeling with an antibody to a peptide comprising amino acids 247 to 259. The H3L protein, synthesized in a coupled in vitro transcription/translation system, was tightly anchored to membranes as determined by resistance to Na(2)CO(3) (pH 11) extraction and cytoplasmically oriented as shown by sensitivity to proteinase K digestion. Further studies demonstrated that membrane insertion of the H3L protein occurred posttranslationally and that the C-terminal hydrophobic domain was necessary and sufficient for this to occur. These data indicated that the H3L protein is a member of the C-terminal anchor family and supported a model in which it is synthesized on free ribosomes and inserts into the membranes of viral particles during their maturation.

Animals↗

PubMed2000

Effects of deletion or stringent repression of the H3L envelope gene on vaccinia virus replication.

The C-terminal membrane anchor protein encoded by the H3L open reading frame of vaccinia virus is located on the surfaces of intracellular mature virions. To investigate the role of the H3L protein, we constructed deletion (vH3Delta) and inducible (vH3i) null mutants. The H3L protein was not detected in lysates of cells infected with vH3Delta or vH3i in the absence of inducer. Under these conditions, plaques were small and round instead of large and comet shaped, indicative of decreased virus replication or cell-to-cell spread. The mutant phenotype was correlated with reduced yields of infectious intra- and extracellular virus in one-step growth experiments. The defect in vH3i replication could not be attributed to a role of the H3L protein in virus binding, internalization, or any event prior to late gene expression. Electron microscopic examination of cells infected with vH3Delta or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumulation of crescent membranes adjacent to granular material and DNA crystalloids. The absence of the H3L protein did not impair the membrane localization of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pH-mediated syncytium formation that could be attributed to decreased virus particle production. The phenotypes of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.

Animals↗

PubMed2000

A glutaredoxin, encoded by the G4L gene of vaccinia virus, is essential for virion morphogenesis.

Vaccinia virus encodes two glutaredoxins, O2L and G4L, both of which exhibit thioltransferase and dehydroascorbate reductase activities in vitro. Although O2L was previously found to be dispensable for virus replication, we now show that G4L is necessary for virion morphogenesis. RNase protection and Western blotting assays indicated that G4L was expressed at late times after infection and was incorporated into mature virus particles. Attempts to isolate a mutant virus with a deleted G4L gene were unsuccessful, suggesting that the protein was required for virus replication. This interpretation was confirmed by the construction and characterization of a conditional lethal recombinant virus with an inducible copy of the G4L gene replacing the original one. Expression of G4L was proportional to the concentration of inducer, and the amount of glutaredoxin could be varied from barely detectable to greater than normal amounts of protein. Immunogold labeling revealed that the induced G4L protein was associated with immature and mature virions and adjacent cytoplasmic depots. In the absence of inducer, the production of infectious virus was severely inhibited, though viral late protein synthesis appeared unaffected except for decreased maturation-dependent proteolytic processing of certain core components. Electron microscopy of cells infected under nonpermissive conditions revealed an accumulation of crescent membranes on the periphery of electron-dense globular masses but few mature particles. We concluded that the two glutaredoxin homologs encoded by vaccinia virus have different functions and that G4L has a role in virion morphogenesis, perhaps by acting as a redox protein.

Gene Expression Regulation, Viral↗

PubMed2000

The vaccinia virus A9L gene encodes a membrane protein required for an early step in virion morphogenesis.

The A9L open reading frame of vaccinia virus was predicted to encode a membrane-associated protein. A transcriptional analysis of the A9L gene indicated that it was expressed at late times in vaccinia virus-infected cells. Late expression, as well as virion membrane association, was demonstrated by the construction and use of a recombinant vaccinia virus encoding an A9L protein with a C-terminal epitope tag. Immunoelectron microscopy revealed that the A9L protein was associated with both immature and mature virus particles and was oriented in the membrane with its C terminus exposed on the virion surface. To determine whether the A9L protein functions in viral assembly or infectivity, we made a conditional-lethal inducible recombinant vaccinia virus. In the absence of inducer, A9L expression and virus replication were undetectable. Under nonpermissive conditions, viral late protein synthesis occurred, but maturational proteolytic processing was inhibited, and there was an accumulation of membrane-coated electron-dense bodies, crescents, and immature virus particles, many of which appeared abnormal. We concluded that the product of the A9L gene is a viral membrane-associated protein and functions at an early stage in virion morphogenesis.

Amino Acid Sequence↗

PubMed2000

Disulfide bonds and membrane topology of the vaccinia virus A17L envelope protein.

The envelope protein encoded by the vaccinia virus A17L open reading frame is essential for virion assembly. Our mutagenesis studies indicated that cysteines 101 and 121 form an intramolecular disulfide bond and that cysteine 178 forms an intermolecular disulfide linking two A17L molecules. This arrangement of disulfide bonds has important implications for the topology of the A17L protein and supports a two-transmembrane model in which cysteines 101 and 121 are intraluminal and cysteine 178 is cytoplasmic. The structure of the A17L protein, however, was not dependent on these disulfide bonds, as a recombinant vaccinia virus with all three cysteine codons mutated to serines retained infectivity.

Blotting, Western↗

PubMed2000

Immunization with a modified vaccinia virus expressing simian immunodeficiency virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge.

The immunogenicity and protective efficacy of a modified vaccinia virus Ankara (MVA) recombinant expressing the simian immunodeficiency virus (SIV) Gag-Pol proteins (MVA-gag-pol) was explored in rhesus monkeys expressing the major histocompatibility complex (MHC) class I allele, MamuA*01. Macaques received four sequential intramuscular immunizations with the MVA-gag-pol recombinant virus or nonrecombinant MVA as a control. Gag-specific cytotoxic T-lymphocyte (CTL) responses were detected in all MVA-gag-pol-immunized macaques by both functional assays and flow cytometric analyses of CD8(+) T cells that bound a specific MHC complex class I-peptide tetramer, with levels peaking after the second immunization. Following challenge with uncloned SIVsmE660, all macaques became infected; however, viral load set points were lower in MVA-gag-pol-immunized macaques than in the MVA-immunized control macaques. MVA-gag-pol-immunized macaques exhibited a rapid and substantial anamnestic CTL response specific for the p11C, C-M Gag epitope. The level at which CTL stabilized after resolution of primary viremia correlated inversely with plasma viral load set point (P = 0.03). Most importantly, the magnitude of reduction in viremia in the vaccinees was predicted by the magnitude of the vaccine-elicited CTL response prior to SIV challenge.

Animals↗

PubMed2000

Comparative efficacy of recombinant modified vaccinia virus Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol and/or Env in macaques challenged with pathogenic SIV.

Prior studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high levels of viremia and AIDS following challenge with a pathogenic strain of SIV (V. M. Hirsch et al., J. Virol. 70:3741-3752, 1996). This MVA-SIV recombinant expressed relatively low levels of the Gag-Pol portion of the vaccine. To optimize protection, second-generation recombinant MVAs that expressed high levels of either Gag-Pol (MVA-gag-pol) or Env (MVA-env), alone or in combination (MVA-gag-pol-env), were generated. A cohort of 24 macaques was immunized with recombinant or nonrecombinant MVA (four groups of six animals) and was challenged with 50 times the dose at which 50% of macaques are infected with uncloned pathogenic SIVsmE660. Although all animals became infected postchallenge, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines as compared with animals that received nonrecombinant MVA (P = 0.0011 by repeated-measures analysis of variance). The differences in the degree of virus suppression achieved by the three MVA-SIV vaccines were not significant. Most importantly, the reduction in levels of viremia resulted in a significant increase in median (P < 0.05 by Student's t test) and cumulative (P = 0.010 by log rank test) survival. These results suggest that recombinant MVA has considerable potential as a vaccine vector for human AIDS.

Animals↗

PubMed2000

Golgi network targeting and plasma membrane internalization signals in vaccinia virus B5R envelope protein.

The vaccinia virus B5R type I integral membrane protein accumulates in the Golgi network, from where it becomes incorporated into the envelope of extracellular virions. Our objective was to determine the domains of B5R responsible for Golgi membrane targeting in the absence of other viral components. Fusion of an enhanced green fluorescent protein to the C terminus of B5R allowed imaging of the chimeric protein without altering intracellular trafficking and Golgi network localization in transfected cells. Deletion or swapping of B5R domains with corresponding regions of the vesicular stomatitis virus G protein, which is targeted to the plasma membrane, indicated that (i) the N-terminal extracellular domain of B5R had no specific role in Golgi apparatus localization, (ii) the transmembrane domain of B5R was sufficient for exiting the endoplasmic reticulum, and (iii) removal of the cytoplasmic tail impaired Golgi network localization and increased the accumulation of B5R in the plasma membrane. Further experiments demonstrated that the cytoplasmic tail mediated internalization of B5R from the plasma membrane, suggesting a retrieval mechanism. Mutagenesis revealed residues required for Golgi membrane localization and efficient plasma membrane retrieval of the B5R protein: a tyrosine at residue 310 and two adjacent leucines at residues 315 and 316.

Amino Acid Sequence↗

PubMed2000

The vaccinia virus A14.5L gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses.

A short sequence, located between the A14L and A15L open reading frames (ORFs) of vaccinia virus, was predicted to encode a hydrophobic protein of 53 amino acids that is conserved in orthopoxviruses, leporipoxviruses, yatapoxiruses, and molluscipoxviruses. We constructed a recombinant vaccinia virus with a 10-codon epitope tag appended to the C terminus of the A14.5L ORF. Synthesis of the tagged protein occurred at late times and was blocked by an inhibitor of DNA replication, consistent with regulation by a predicted late promoter just upstream of the A14.5L ORF. Hydrophobicity of the protein was demonstrated by extraction into the detergent phase of Triton X-114. The protein was associated with purified vaccinia virus particles and with membranes of immature and mature virions that were visualized by electron microscopy of infected cells. Efficient release of the protein from purified virions occurred after treatment with a nonionic detergent and reducing agent. A mutant virus, in which the A14.5L ORF was largely deleted, produced normal-size plaques in several cell lines, and the yields of infectious intra- and extracellular viruses were similar to those of the parent. In contrast, with a mouse model, mutant viruses with the A14.5L ORF largely deleted were attenuated relative to that of the parental virus or a mutant virus with a restored A14.5L gene.

Amino Acid Sequence↗

PubMed2000

Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies.

Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.

Animals↗

PubMed1999

Priming and boosting immunity to respiratory syncytial virus by recombinant replication-defective vaccinia virus MVA.

Intranasal and intramuscular immunizations of mice with the highly attenuated MVA strain of vaccinia virus expressing the respiratory syncytial virus (RSV) F or G glycoprotein induced higher RSV antibody titers than those achieved by infection with RSV and greatly restricted the replication of RS challenge virus in both the upper and lower respiratory tracts. In addition, a recombinant MVA expressing both RSV F and G was stable and was as immunogenic as a combination of two single recombinant viruses. The levels of antibodies to RSV F and G, induced by previous intranasal infection with attenuated RSV, were boosted by intramuscular immunization with recombinant MVA. These data support further development of recombinant MVA as a RSV vaccine.

Animals↗

PubMed1999

Vaccinia virus serpin-1 deletion mutant exhibits a host range defect characterized by low levels of intermediate and late mRNAs.

Orthopoxviruses encode three serpin homologs-SPI-1, SPI-2 and SPI-3-of which SPI-2 has been well characterized as an inhibitor of ICE-like proteases. A rabbitpox virus SPI-1 deletion mutant exhibited a host range restriction in human lung A549 and pig kidney 15 cell lines that was attributed to apoptosis. Here we report that replication of a vaccinia virus SPI-1 deletion mutant (DeltaSPI-1) was restricted in primary human keratinocytes as well as A549 cells. Although chromatin condensation was detected in some A549 cells, other morphological or biochemical signs of apoptosis including DNA fragmentation, cleavage of poly(ADP-ribose)polymerase or nuclear mitotic apparatus protein, or caspase 3 activation were not found. Moreover, DeltaSPI-1 protected A549 cells from apoptosis induced by tumor necrosis factor, whereas the corresponding DeltaSPI-2 mutant did not. Further studies indicated undiminished amounts of vaccinia virus early mRNA and replicated DNA in the absence of the SPI-1 product. However, there were reduced amounts of viral intermediate and late mRNAs, viral late proteins, cleaved core proteins, and virus particles. These data suggested that apoptosis is not the determining factor in the host range restriction of DeltaSPI-1 and that the SPI-1 gene product is needed to allow efficient expression of intermediate and late genes in A549 cells.

Apoptosis↗

PubMed1999

IL-18 binding and inhibition of interferon gamma induction by human poxvirus-encoded proteins.

Molluscum contagiosum virus (MCV) is a common, human poxvirus that causes small papular skin lesions that persist for long periods without signs of inflammation. Previous studies revealed that MCV encodes a family of proteins with homology to mammalian IL-18 binding proteins. IL-18 is a proinflammatory cytokine that induces synthesis of interferon gamma, activates NK cells, and is required for a T-lymphocyte helper type 1 response. We expressed and purified the proteins encoded by the MC53L and MC54L genes of MCV, as well as their human and murine homologs. All four recombinant proteins were able to bind with high affinity to human and murine IL-18 molecules and inhibited IL-18 mediated interferon gamma production in a dose-dependent manner. The pirating of IL-18 binding proteins by poxviruses and their use as decoy receptors is consistent with the critical role of IL-18 in defense against virus infections and provides a mechanism for evasion of the immune system by MCV.

Animals↗

PubMed1999

Membrane topology of the vaccinia virus A17L envelope protein.

The formation of a lipoprotein membrane within specialized areas of the cytoplasm is the first visible step in poxvirus morphogenesis. The A17L viral protein, an essential nonglycosylated membrane component, was predicted to have four centrally located alpha-helical membrane-spanning domains. The gene was expressed as a 23-kDa protein in a cell-free transcription/translation system containing canine pancreatic microsomes. The N- and C-terminal ends of the membrane-associated protein were susceptible to proteinase digestion, whereas the central region was resistant, consistent with a model in which the first and fourth hydrophobic domains are membrane spanning. This topology was supported by the sizes of the major proteinase-resistant membrane-associated products of genes containing one or more deleted hydrophobic domains and by evidence that the C-terminus was intraluminal and glycosylated on deletion of the second, third, and fourth domains, the third and fourth domains, or just the fourth domain. Moreover, glycosylation also occurred when an N-glycosylation site was introduced into the second hydrophobic domain of the full-length A17L protein. The data indicated a predominant topology in which the N- and C-termini are cytoplasmic, the first and fourth hydrophobic domains span the microsomal membrane, and the second and third hydrophobic domains are intraluminal. This arrangement has important implications for interactions of the A17L protein with other membrane components.

Amino Acid Sequence↗

PubMed1999

Histidine codons appended to the gene encoding the RPO22 subunit of vaccinia virus RNA polymerase facilitate the isolation and purification of functional enzyme and associated proteins from virus-infected cells.

Vaccinia virus encodes a eukaryotic-like RNA polymerase composed of two large and six small subunit protein species. A replication-competent virus with 10 histidine codons added to the single endogenous J4R open reading frame was constructed. The altered migration of the 22-kDa subunit of RNA polymerase on SDS-polyacrylamide gel electrophoresis confirmed that J4R encoded the RPO22 subunit and that the mutant virus was genetically stable. The histidine-tagged RNA polymerase bound quantitatively to metal-affinity resins and was eluted in an active form upon addition of imidazole. Glycerol gradient sedimentation of the eluted fraction indicated that most of the RPO22 in infected cells is associated with RNA polymerase. Using stringent washing conditions, metal-affinity chromatography resulted in a several hundred-fold increase in RNA-polymerase-specific activity, and substantially pure enzyme was obtained with an additional conventional chromatography step. When mild conditions were used for washing the metal-affinity resin, the vaccinia virus-encoded capping enzyme, early transcription factor, and nucleoside triphosphate phosphohydrolase I specifically co-eluted with the tagged RNA polymerase, consistent with their physical association. The ability to selectively bind RNA polymerase to an affinity column provided a simple and rapid method of concentrating and purifying active enzyme and protein complexes.

Animals↗

PubMed1999

Identification of human and mouse homologs of the MC51L-53L-54L family of secreted glycoproteins encoded by the Molluscum contagiosum poxvirus.

Molluscum contagiosum virus (MCV) is a human poxvirus that produces small, benign skin tumors primarily in young children and encodes proteins that modulate the host immune response. The MC51L, MC53L, and MC54L open reading frames of MCV have significant amino acid sequence similarities and related proteins are encoded by other poxviruses. These three MCV genes were individually expressed in mammalian cells as glycosylated and secreted proteins. A database search detected partial sequences of homologous human and mouse cDNAs; determination of the complete sequences confirmed the homology and indicated potential signal peptides and N-glycosylation sites as well as a pattern of cysteines that are conserved in the viral proteins. The human gene, which was mapped to chromosome 11q13, was highly expressed in spleen and lymph nodes, suggesting an immune modulatory role. The latter finding is consistent with a recent report (Novick et al., 1999, Immunity 10, 127-136) that the human protein binds IL-18, suggesting an anti-inflammatory role for the poxvirus homologs.

Amino Acid Sequence↗

PubMed1999

Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity.

Overcoming preexisting immunity to vaccinia virus in the adult population is a key requirement for development of otherwise potent recombinant vaccinia vaccines. Based on our observation that s.c. immunization with vaccinia induces cellular and antibody immunity to vaccinia only in systemic lymphoid tissue and not in mucosal sites, we hypothesized that the mucosal immune system remains naive to vaccinia and therefore amenable to immunization with recombinant vaccinia vectors despite earlier vaccinia exposure. We show that mucosal immunization of vaccinia-immune BALB/c mice with recombinant vaccinia expressing HIV gp160 induced specific serum antibody and strong HIV-specific cytotoxic T lymphocyte responses. These responses occurred not only in mucosal but also in systemic lymphoid tissue, whereas systemic immunization was ineffective under these circumstances. In this context, intrarectal immunization was more effective than intranasal immunization. Boosting with a second dose of recombinant vaccinia was also more effective via the mucosal route. The systemic HIV-specific cytotoxic T lymphocyte response was enhanced by coadministration of IL-12 at the mucosal site. These results also demonstrate the independent compartmentalization of the mucosal versus systemic immune systems and the asymmetric trafficking of lymphocytes between them. This approach to circumvent previous vaccinia immunity may be useful for induction of protective immunity against infectious diseases and cancer in the sizable populations with preexisting immunity to vaccinia from smallpox vaccination.

AIDS Vaccines↗

PubMed1999

Identification of a transcription factor, encoded by two vaccinia virus early genes, that regulates the intermediate stage of viral gene expression.

Vaccinia virus early, intermediate, and late stage genes are sequentially transcribed by the viral RNA polymerase within the cytoplasm of infected cells. We found that the 34- and 45-kDa polypeptides encoded by vaccinia virus ORFs A8R and A23R, respectively, were necessary to reconstitute transcription of a template with an intermediate stage promoter. Coexpression of the A8R and A23R genes in Escherichia coli was required for in vitro activity. In addition, the two polypeptides copurified, indicating their association as protein subunits of a vaccinia virus intermediate transcription factor. This factor, which we named VITF-3, complemented three viral proteins-namely, the RNA polymerase, capping enzyme, and a 30-kDa protein called VITF-1 that is also a subunit of the RNA polymerase-and an unidentified cell factor called VITF-2. Expression of the A8R and A23R genes occurred between 1 and 5 h after vaccinia virus infection and was not prevented by an inhibitor of DNA replication, consistent with a role for VITF-3 in specifically regulating intermediate transcription in vivo. The vaccinia virus A8R and A23R genes are highly conserved among vertebrate poxviruses, but no other viral or cellular homologs were identified.

Gene Expression Regulation, Viral↗