Biomedical subjects

Bernard Moss

Publications and source records attributed to Bernard Moss.

At least 37 records · Page 2Linked to original sources

PubMed2005

Poxvirus multiprotein entry-fusion complex.

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.

Cell Line↗

PubMed2005

The enhanced tumor selectivity of an oncolytic vaccinia lacking the host range and antiapoptosis genes SPI-1 and SPI-2.

The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.

Animals↗

PubMed2005

Physical and immunological characterization of a recombinant secreted form of the membrane protein encoded by the vaccinia virus L1R gene.

We reported that immunization with recombinant proteins derived from vaccinia virus (VV) particles could provide protection against infection. Here we describe the physical and antigenic properties of the L1R membrane protein. The recombinant protein (L1R(185t)) was secreted as a monomer and correct folding was suggested by the presence of three intramolecular disulfide bonds and binding to conformation-specific monoclonal antibodies (MAbs). Furthermore, anti-L1R(185t) rabbit antisera exhibited potent virus-neutralizing activity against the IMV form of VV. We raised six MAbs against L1R(185t). Three recognized linear epitopes (residues 118--128) and neutralized IMV infectivity. These MAbs blocked binding of each other to L1R(185t) but failed to block binding of two previously described neutralizing anti-L1R MAbs, 7D11 and 2D5. The latter two antibodies blocked each other in binding L1R(185t). Thus, two antigenic sites on L1R overlap functional domains and based on recent structural studies these are found in accessible regions of the IMV L1R protein.

Amino Acid Sequence↗

PubMed2005

Comparative immunogenicity in rhesus monkeys of multi-protein HIV-1 (CRF02_AG) DNA/MVA vaccines expressing mature and immature VLPs.

We developed an AIDS vaccine for Western and West-Central Africa founded on HIV-1 subtype CRF02_AG. Rhesus macaques were primed with Gag-Pol-Env-expressing plasmid DNA and boosted with a recombinant modified vaccinia virus Ankara (rMVA), expressing matched proteins. Two DNA vaccine constructs (IC1-90 and IC48) that differed by point mutations in gag and pol were compared. IC1-90 produces primarily immature (core comprises unprocessed Pr55Gag) HIV-like particles (VLPs) and IC48 produces mature VLP with processed Pr55Gag, immature VLP, and intracellular protein aggregates. Both vaccines raised significant cellular responses for Gag, Pol, and Env. Approximate twofold higher ELISPOT responses to Gag and Env epitopes were observed for IC48 animals than for IC1-90 animals at the peak post-MVA effector (P = 0.028) and late memory (P = 0.051) phases, respectively. Greater breadth for IC48-primed animals was observed than for IC1-90-primed animals at peak response (P = 0.03). Our results indicated that the vaccines elicited high frequency T cell responses and primed anti-Env antibody. They also suggest that expression of different forms of VLP has a significant effect on elicited cellular and humoral immunity.

AIDS Vaccines↗

PubMed2005

External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice.

During morphogenesis, poxviruses undergo a remarkable transition from spherical immature forms to brick-shaped infectious particles lacking helical or icosahedral symmetry. In this study, we show that the transitory honeycomb lattice coating the lipoprotein membrane of immature vaccinia virus particles is formed from trimers of a 62-kD protein encoded by the viral D13L gene. Deep-etch electron microscopy demonstrated that anti-D13 antibodies bound to the external protein coat and that lattice fragments were in affinity-purified D13 preparations. Soluble D13 appeared mostly trimeric by gel electrophoresis and ultracentrifugation, which is consistent with structural requirements for a honeycomb. In the presence or absence of other virion proteins, a mutated D13 with one amino acid substitution formed stacks of membrane-unassociated flat sheets that closely resembled the curved honeycombs of immature virions except for the absence of pentagonal facets. A homologous domain that is present in D13 and capsid proteins of certain other lipid-containing viruses support the idea that the developmental stages of poxviruses reflect their evolution from an icosahedral ancestor.

Capsid Proteins↗

PubMed2005

Neutralizing antibody and protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein.

A secreted, glycosylated polypeptide containing amino acids 14 to 762 of the SARS coronavirus (SARS-CoV) spike protein and a polyhistidine tag was expressed in recombinant baculovirus-infected insect cells. Mice received the affinity-purified protein with either a saponin (QS21) or a Ribi (MPL + TDM) adjuvant subcutaneously and were challenged intranasally with SARS-CoV. Both regimens induced binding and neutralizing antibodies and protection against SARS-CoV intranasal infection. However, the best results were obtained with QS21 and protein, which provided the highest antibody as well as complete protection of the upper and lower respiratory tract.

Animals↗

PubMed2005

Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors.

BACKGROUND: Replication of the vaccinia virus genome occurs in cytoplasmic factory areas and is dependent on the virus-encoded DNA polymerase and at least four additional viral proteins. DNA synthesis appears to start near the ends of the genome, but specific origin sequences have not been defined. Surprisingly, transfected circular DNA lacking specific viral sequences is also replicated in poxvirus-infected cells. Origin-independent plasmid replication depends on the viral DNA polymerase, but neither the number of additional viral proteins nor the site of replication has been determined. RESULTS: Using a novel real-time polymerase chain reaction assay, we detected a >400-fold increase in newly replicated plasmid in cells infected with vaccinia virus. Studies with conditional lethal mutants of vaccinia virus indicated that each of the five proteins known to be required for viral genome replication was also required for plasmid replication. The intracellular site of replication was determined using a plasmid containing 256 repeats of the Escherichia coli lac operator and staining with an E. coli lac repressor-maltose binding fusion protein followed by an antibody to the maltose binding protein. The lac operator plasmid was localized in cytoplasmic viral factories delineated by DNA staining and binding of antibody to the viral uracil DNA glycosylase, an essential replication protein. In addition, replication of the lac operator plasmid was visualized continuously in living cells infected with a recombinant vaccinia virus that expresses the lac repressor fused to enhanced green fluorescent protein. Discrete cytoplasmic fluorescence was detected in cytoplasmic juxtanuclear sites at 6 h after infection and the area and intensity of fluorescence increased over the next several hours. CONCLUSION: Replication of a circular plasmid lacking specific poxvirus DNA sequences mimics viral genome replication by occurring in cytoplasmic viral factories and requiring all five known viral replication proteins. Therefore, small plasmids may be used as surrogates for the large poxvirus genome to study trans-acting factors and mechanism of viral DNA replication.

Cell Line↗

PubMed2005

The 1.51-Angstrom structure of the poxvirus L1 protein, a target of potent neutralizing antibodies.

Although eradicated from nature more than two decades ago, the threat of smallpox has reemerged because of concerns over its use as a biological weapon. We present the structure of the poxvirus L1 protein, a molecule that is conserved throughout the poxvirus family and is nearly identical in vaccinia virus and in variola virus, which causes smallpox. L1 is a myristoylated envelope protein that is a potent target for neutralizing antibodies and an important component of current experimental vaccines. The L1 structure reveals a hydrophobic cavity located adjacent to its N terminus. The cavity would be capable of shielding the myristate moiety, which is essential for virion assembly. The structure of L1 is a step in the elucidation of molecular mechanisms common to all poxviruses that may stimulate the design of safer vaccines and new antipoxvirus drugs.

Amino Acid Sequence↗

PubMed2005

Simian immunodeficiency virus (SIV) envelope quasispecies transmission and evolution in infant rhesus macaques after oral challenge with uncloned SIVmac251: increased diversity is associated with neutralizing antibodies and improved survival in previously immunized animals.

BACKGROUND: Oral infection of infant macaques with simian immunodeficiency virus (SIV) is a useful animal model to test interventions to reduce postnatal HIV transmission via breast-feeding. We previously demonstrated that immunization of infant rhesus macaques with either modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol and Env, or live-attenuated SIVmac1A11 resulted in lower viremia and longer survival compared to unimmunized controls after oral challenge with virulent SIVmac251 (Van Rompay et al., J. Virology 77:179-190, 2003). Here we evaluate the impact of these vaccines on oral transmission and evolution of SIV envelope variants. RESULTS: Limiting dilution analysis of SIV RNA followed by heteroduplex mobility assays of the V1-V2 envelope (env) region revealed two major env variants in the uncloned SIVmac251 inoculum. Plasma sampled from all infants 1 week after challenge contained heterogeneous SIV env populations including one or both of the most common env variants in the virus inoculum; no consistent differences in patterns of env variants were found between vaccinated and unvaccinated infants. However, SIV env variant populations diverged in most vaccinated monkeys 3 to 5 months after challenge, in association with the development of neutralizing antibodies. CONCLUSIONS: These patterns of viral envelope diversity, immune responses and disease course in SIV-infected infant macaques are similar to observations in HIV-infected children, and underscore the relevance of this pediatric animal model. The results also support the concept that neonatal immunization with HIV vaccines might modulate disease progression in infants infected with HIV by breast-feeding.

Animals↗

PubMed2005

Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV.

An infant macaque model was developed to test pediatric vaccine candidates aimed at reducing HIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral inoculations with virulent SIVmac251 at 4 weeks of age, significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure; unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV or MVA-SIV. When infected, ALVAC-SIV- and MVA-SIV-vaccinated animals also had reduced viremia compared with unimmunized animals. The results of these investigations suggest that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection through breastfeeding.

Administration, Oral↗

PubMed2005

Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda-based recombination.

The large capacity of vaccinia virus (VAC) for added DNA, cytoplasmic expression and broad host range make it a popular choice for gene delivery, despite the burdensome need for multiple plaque purifications to isolate recombinants. Here we describe how a bacterial artificial chromosome (BAC) containing the entire VAC genome can be engineered in Escherichia coli by homologous recombination using bacteriophage lambda-encoded enzymes. The engineered VAC genomes can then be used to produce clonally pure recombinant viruses in mammalian cells without the need for plaque purification.

Bacteriophage lambda↗

PubMed2005

Studies in macaques on cross-clade T cell responses elicited by a DNA/MVA AIDS vaccine, better conservation of CD8 than CD4 T cell responses.

One of the unknowns faced by an HIV/AIDS vaccine is the ability of a single clade vaccine to protect against the multiple genetic subtypes and recombinant forms of HIV-1 present in the current pandemic. Here, we use a macaque model to investigate the ability of our clade B vaccine that consists of DNA priming and modified vaccinia Ankara (MVA) virus boosting to elicit T cell responses that recognize an A/G recombinant of HIV-1. To test for cross-reactive T cells, intracellular cytokine staining was conducted using five pools of Gag and six pools of Env peptides representing B or A/G sequences. Studies using the peptide pools revealed essentially complete conservation of the CD8 response but only approximately 50% conservation of the CD4 response. Thus, the ability of an HIV vaccine for one clade to protect against other clades may be more limited by the ability to provide CD4 T cell help than the ability to elicit CD8 effector functions.

AIDS Vaccines↗

PubMed2005

Epitope-mapping studies define two major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein B5R.

Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.

Antibodies, Monoclonal↗

PubMed2005

Vaccinia virus nonstructural protein encoded by the A11R gene is required for formation of the virion membrane.

The vaccinia virus A11R gene has orthologs in all known poxvirus genomes, and the A11 protein has been previously reported to interact with the putative DNA packaging protein A32 in a yeast two-hybrid screen. Using antisera raised against A11 peptides, we show that the A11 protein was (i) expressed at late times with an apparent mass of 40 kDa, (ii) not incorporated into virus particles, (iii) phosphorylated independently of the viral F10 kinase, (iv) coimmunoprecipitated with A32, and (v) localized to the viral factory. To determine the role of the A11 protein and test whether it is indeed involved in DNA packaging, we constructed a recombinant vaccinia virus with an inducible A11R gene. This recombinant was dependent on inducer for single-cycle growth and plaque formation. In the absence of inducer, viral late proteins were produced at normal levels, but proteolytic processing and other posttranslational modifications of some proteins were inhibited, suggesting a block in virus particle assembly. Consistent with this observation, electron microscopy of cells infected in the absence of inducer showed virus factories with abnormal electron-dense viroplasms and intermediate density regions associated with membranes and containing the D13 protein. However, no viral membrane crescents, immature virions, or mature virions were produced. The requirement for nonvirion protein A11 in order to make normal viral membranes was an unexpected and exciting finding, since neither the origin of these membranes nor their mechanism of formation in the cytoplasm of infected cells is understood.

Animals↗

PubMed2005

Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly.

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.

Amino Acid Sequence↗

PubMed2005

Vaccinia virus A21 virion membrane protein is required for cell entry and fusion.

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-beta-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.

Amino Acid Sequence↗

PubMed2005

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.

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.

Amino Acid Sequence↗

PubMed2005

Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge.

Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections.

Animals↗