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Geoffrey L Smith

Publications and source records attributed to Geoffrey L Smith.

53 records · Page 3Linked to original sources
PubMed2002

The vaccinia virus C12L protein inhibits mouse IL-18 and promotes virus virulence in the murine intranasal model.

A bioassay that measured the interleukin (IL)-12-induced production of interferon (IFN)-gamma from mouse splenocytes was used to identify a soluble factor in the supernatants of vaccinia virus (VV)-infected cells that inhibited the production of IFN-gamma. This soluble factor was expressed by 14 out of 16 VV strains including the Western Reserve (WR) strain, but strains Copenhagen and Tashkent and a mutant of strain WR called 6/2 lacked this activity. The gene encoding this activity was identified as C12L by transferring DNA present in VV WR but missing in VV WR 6/2 into VV Copenhagen and testing for expression of the soluble factor. The C12L protein shows amino acid similarity to IL-18 binding proteins that are encoded by poxviruses, mice and humans, and C12L protein produced from VV or baculovirus inhibited the biological activity of mouse IL-18 in vitro. Thus the inhibition of IL-12-induced IFN-gamma production was due to indirect effects of C12L on IL-18, illustrating the synergistic action of these pro-inflammatory cytokines. To study the role of the C12L protein in the virus life-cycle, we constructed a deletion mutant lacking the C12L gene and a revertant virus in which the gene was reinserted into the deletion mutant. In vitro the replication and plaque size of these viruses were indistinguishable. However, infection of BALB/c mice by the intranasal route showed that the deletion mutant was attenuated and induced lower weight loss and signs of illness compared to controls.

Administration, Intranasal↗
PubMed2002

The formation and function of extracellular enveloped vaccinia virus.

Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably, in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.

Animals↗
PubMed2002

Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface.

A vaccinia virus (VV) recombinant is described in which the outer envelope of extracellular enveloped virus (EEV), cell-associated enveloped virus (CEV) and intracellular enveloped virus (IEV) is labelled with the enhanced green fluorescent protein (EGFP) derived from Aequorea victoria. To construct this virus, EGFP was fused to the VV B5R protein from which the four short consensus repeats (SCRs) of the extracellular domain had been deleted. Cells infected with the recombinant virus expressed a B5R-EGFP fusion protein of 40 kDa that was present on IEV, CEV and EEV, but was absent from IMV. The recombinant virus produced 2- and 3-fold reduced levels of IMV and EEV, respectively. Analysis of infected cells by confocal microscopy showed that actin tail formation by the mutant virus was reduced by 86% compared to wild-type (WT). The virus formed a small plaque compared to WT, consistent with a role for actin tails in promoting cell-to-cell spread of virus. However, the enveloped virions were still transported to the cell surface, confirming that this process is independent of actin tail formation. Lastly, we compared the mutant virus with a recombinant VV in which the B5R SCR domains were deleted and show that, contrary to a previous report, the plaque size of the latter virus was reduced compared to WT. This observation reconciles an inconsistency in the field and confirms that viruses deficient in formation of actin tails form small plaques.

Actins↗
PubMed2002

The vaccinia virus soluble interferon-gamma receptor is a homodimer.

The vaccinia virus (VV) interferon (IFN)-gamma receptor (IFN-gammaR) is a 43 kDa soluble glycoprotein that is secreted from infected cells early during infection. Here we demonstrate that the IFN-gammaR from VV, cowpox virus and camelpox virus exists naturally as a homodimer, whereas the cellular IFN-gammaR dimerizes only upon binding the homodimeric IFN-gamma. The existence of the virus protein as a dimer in the absence of ligand may provide an advantage to the virus in efficient binding and inhibition of IFN-gamma in solution.

Animals↗
PubMed2002

The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox.

Camelpox virus (CMPV) and variola virus (VAR) are orthopoxviruses (OPVs) that share several biological features and cause high mortality and morbidity in their single host species. The sequence of a virulent CMPV strain was determined; it is 202182 bp long, with inverted terminal repeats (ITRs) of 6045 bp and has 206 predicted open reading frames (ORFs). As for other poxviruses, the genes are tightly packed with little non-coding sequence. Most genes within 25 kb of each terminus are transcribed outwards towards the terminus, whereas genes within the centre of the genome are transcribed from either DNA strand. The central region of the genome contains genes that are highly conserved in other OPVs and 87 of these are conserved in all sequenced chordopoxviruses. In contrast, genes towards either terminus are more variable and encode proteins involved in host range, virulence or immunomodulation. In some cases, these are broken versions of genes found in other OPVs. The relationship of CMPV to other OPVs was analysed by comparisons of DNA and predicted protein sequences, repeats within the ITRs and arrangement of ORFs within the terminal regions. Each comparison gave the same conclusion: CMPV is the closest known virus to variola virus, the cause of smallpox.

Animals↗
PubMed2002

The vaccinia virus B9R protein is a 6 kDa intracellular protein that is non-essential for virus replication and virulence.

Vaccinia virus (VV) strain Western Reserve gene B9R is shown to encode an intracellular 6 kDa protein that is expressed late during the infectious cycle. In vitro transcription and translation produced two polypeptides in the presence of microsomal membranes, but only the larger protein in the absence of membranes. The smaller protein sedimented with microsomes during centrifugation, suggesting it was inserted into the lipid membrane or into the microsomal lumen via the N-terminal hydrophobic signal sequence that was subsequently cleaved proteolytically. A VV mutant lacking B9R was constructed and found to replicate normally in cell culture and two in vivo models.

Base Sequence↗
PubMed2002

A study of the vaccinia virus interferon-gamma receptor and its contribution to virus virulence.

Vaccinia virus (VV) strain Western Reserve gene B8R encodes a 43 kDa glycoprotein that is secreted from infected cells early in infection as a homodimer. This protein has amino acid similarity with the extracellular domain of cellular IFN-gamma receptor (IFN-gammaR) and binds and inhibits IFN-gamma from a wide range of species. Here we demonstrate that the B8R protein also inhibits equine IFN-gamma. The 5' end of the B8R mRNA has been mapped by primer extension analysis and the contribution of IFN-gammaRs to VV virulence was studied by the construction of a deletion mutant lacking the B8R gene (vDeltaB8R) and a revertant virus (vB8R-R) in which the B8R gene was re-inserted into the deletion mutant. A recombinant virus that expressed a soluble form of the mouse IFN-gammaR was also constructed and studied. The virulence of these viruses was tested in rodent models of infection. In mice, the loss of the VV IFN-gammaR did not affect virulence compared with WT and revertant viruses, consistent with the low affinity of the VV IFN-gammaR for mouse IFN-gamma. However, expression of the mouse soluble IFN-gammaR increased virus virulence slightly. In rabbit skin, loss of the VV IFN-gammaR produced lesions with histological differences compared with WT and revertant viruses. Lastly, the affinity constants of the VV IFN-gammaR for human and mouse IFN-gamma were determined by surface plasmon resonance.

Amino Acid Sequence↗
PubMed2002

The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence.

The vaccinia virus (VV) N1L gene encodes a protein of 14 kDa that was identified previously in the concentrated supernatant of virus-infected cells. Here we show that the protein is present predominantly (>90%) within cells rather than in the culture supernatant and it exists as a non-glycosylated, non-covalent homodimer. The N1L protein present in the culture supernatant was uncleaved at the N terminus and was released from cells more slowly than the VV A41L gene product, a secreted glycoprotein that has a conventional signal peptide. Bioinformatic analyses predict that the N1L protein is largely alpha-helical and show that it is conserved in many VV strains, in other orthopoxviruses and in members of other chordopoxvirus genera. However, database searches found no non-poxvirus proteins with significant amino acid similarity to N1L. A deletion mutant lacking the N1L gene replicated normally in cell culture, but was attenuated in intranasal and intradermal murine models compared to wild-type and revertant controls. The conservation of the N1L protein and the attenuated phenotype of the deletion mutant indicate an important role in the virus life-cycle.

Animals↗
PubMed2002

Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes.

Previously, we developed a model for testing the virulence and immunogenicity of vaccinia virus (VV) mutants based on the intradermal injection of BALB/c mouse ear pinnae. The model is characterized by a local infection in the inoculated skin without signs of systemic illness, mimicking dermal vaccination with VV. Here a further characterization of this model is presented, including the responses of mice to infectious virus doses as low as 10 p.f.u., a quantification of the infiltrate at the site of infection and use of different virus and mouse strains. The model was then used to compare the pathogenesis of six mutants of VV strain Western Reserve (WR) lacking genes A36R, A40R, A44L, B12R, B13R or B15R with that of appropriate control viruses. All of these genes except B12R and B15R influence the outcome of dermal infection with WR and for A40R and B13R this is the first role that has been reported after infection of mammals. A comparison of new and published results from intradermal and intranasal models is presented, showing that out of 16 gene deletion or insertion mutants of VV, half have phenotypes distinct from controls in only one of these models. Thus, the intranasal and intradermal models are complementary tools for dissecting the genetic basis of VV virulence.

Age Factors↗
PubMed2002

The African swine fever virus protein j4R binds to the alpha chain of nascent polypeptide-associated complex.

The African swine fever virus (ASFV) j4R protein is expressed late during the virus replication cycle and is present in both the nucleus and the cytoplasm of infected cells. By using the yeast two-hybrid system, direct binding, and coprecipitation from cells, we showed that the j4R protein binds to the alpha chain of nascent polypeptide-associated complex (alpha NAC). Confocal microscopy indicated that a proportion of j4R and alpha NAC interact in areas close to the plasma membrane, as well as through the cytoplasm in cells. In vitro binding studies suggested that binding of j4R to alpha NAC did not interfere with the binding of alpha- and beta NAC subunits (the BTF3 transcription factor).

African Swine Fever Virus↗
PubMed2001

Differential processing and presentation of the H-2D(b)-restricted epitope from two different strains of influenza virus nucleoprotein.

The influenza virus strains A/NT/60/68 and A/PR/8/34 both have an immunodominant D(b)-restricted epitope in their nucleoprotein (NP) at amino acid residues 366-374, with two amino acid differences between the epitopes. Cross-reactive cytotoxic T lymphocytes (CTLs) were generated by priming mice with the influenza virus A/NT/60/68 NP and restimulating in vitro with influenza virus A/PR/8/34. CTLs that gave high levels of specific lysis recognized target cells infected with either strain of influenza virus with similar efficiency. Surprisingly, when target cells were infected with recombinant vaccinia viruses (VV) expressing the two different NPs, presentation of the D(b)-restricted epitope from the A/NT/60/68 NP was extremely poor, whereas presentation of the equivalent epitope from the A/PR/8/34 NP was as efficient as in influenza virus-infected cells. This difference was observed in spite of the fact that the two NP sequences show 94% identity at the amino acid sequence level. Experiments with additional cross-reactive CTL cell lines which recognized target cells less efficiently revealed a similar difference in presentation between the two NP epitopes in influenza virus-infected cells and showed a difference in the efficiency of presentation of the D(b)-restricted epitope from the two NP molecules independent of VV infection. The results show that two equivalent epitopes in highly similar proteins are processed with very different efficiency, even though they are both immunodominant epitopes. They also suggest that the previously described inhibition of antigen presentation by VV is a general, non-specific effect, which is more apparent for epitopes that are processed and presented less efficiently.

Animals↗
PubMed2001

A mutational analysis of the vaccinia virus B5R protein.

A mutational analysis of the vaccinia virus (VV) B5R protein is presented. This protein is related to the regulators of complement activation (RCA) superfamily, has four short consensus repeats (SCRs) that are typical of this superfamily and is present on extracellular enveloped virus (EEV) particles. Here we have constructed VV mutants in which the cytoplasmic tail (CT) of the B5R protein is progressively truncated, and domains of the B5R protein [the SCR (short consensus repeat) domains, the transmembrane anchor region or the CT] are substituted by corresponding domains from the VV haemagglutinin (HA), another EEV protein. Analysis of these mutant viruses showed that loss of the B5R CT did not affect the formation of intracellular enveloped virus (IEV), actin tails, EEV or virus plaque size. However, if the SCR domains of the B5R protein were replaced by the corresponding region of the HA, the virus plaque size was diminished, the formation of actin tails was decreased severely and the titre of infectious EEV released from cells was reduced approximately 25-fold compared to wild-type virus and 5-fold compared to a virus lacking the entire B5R gene. Thus the linkage of HA to the B5R transmembrane and CT is deleterious for the formation and release of EEV and for cell-to-cell virus spread. In contrast, deletion or substitution of the B5R CT did not affect virus replication, although the amount of cell surface B5R was reduced compared to control.

Actins↗
PubMed2001

Vaccinia virus semaphorin A39R is a 50-55 kDa secreted glycoprotein that affects the outcome of infection in a murine intradermal model.

Vaccinia virus (VV) protein A39R has amino acid similarity to the extracellular domain of a glycosylphosphatidylinositol-linked cell surface semaphorin (SEMA7A/CDw108) that has an immunological expression profile and binding properties, thereby implicating A39R as an immunomodulator. Previously, a closely related A39R protein expressed by ectromelia virus was shown to induce cytokine production and up-regulate ICAM-1 expression in mouse monocytes in vitro. In this study, we show that the A39R gene of VV strain Copenhagen (COP) encodes a 50-55 kDa secreted glycoprotein and is expressed late during infection. The A39R protein was secreted by eight of 15 strains of VV, but not by strain Western Reserve (WR). To analyse the VV A39R function, several recombinant viruses were made, including an A39R deletion mutant of VV COP and a WR mutant containing the A39R sequence from COP. Loss of the gene from COP did not affect virus growth in vitro, or VV virulence in a mouse intranasal model, and had only a slight effect on lesion size in an intradermal model. In contrast, expression of COP A39R by VV WR was associated with an increase in the severity and persistence of skin lesions after intradermal infection of mice. Finally, a histological examination of mouse skin infected with recombinant viruses suggested that A39R has direct or indirect pro-inflammatory properties.

Amino Acid Sequence↗
PubMed2001

The vaccinia virus A41L protein is a soluble 30 kDa glycoprotein that affects virus virulence.

Vaccinia virus (VV) gene A41L encodes an acidic protein with amino acid similarity to the 35 kDa protein of VV strain Lister, a soluble protein called vCKBP that binds CC chemokines, and to a protein from orf virus, called GIF, that binds GM-CSF and IL-2. However, despite the similarity, recombinant A41L protein was found not to bind these ligands or a variety of other chemoattractant molecules when tested using surface plasmon resonance. The A41L gene is expressed early and late during infection and encodes a 30 kDa protein that contains both N- and O-linked carbohydrate and is secreted from the infected cell. All 16 strains of VV and 2 strains of cowpox virus that were tested express the A41L protein, implying it has an important function for orthopoxviruses. Nonetheless, a VV strain Western Reserve deletion mutant lacking the A41L gene (vDeltaA41L) formed normal sized plaques and replicated to the same titre as wild-type and revertant viruses. The importance of the A41L protein in vivo was demonstrated in a mouse intradermal model in which infection with vDeltaA41L caused more severe lesions compared to wild-type and revertant viruses. Further examination in this model revealed that deletion of A41L enhanced clearance of infectious virus, suggesting that A41L expression reduces immunopathology. Consistent with this, histological examination of infected rabbit skin showed that the A41L protein could reduce the infiltration of inflammatory cells into the infected area. Together, these data suggest that the A41L protein constitutes a novel immunomodulatory protein.

Amino Acid Sequence↗
PubMed1999

A model for vaccinia virus pathogenesis and immunity based on intradermal injection of mouse ear pinnae.

Vaccinia virus (VV) proteins that interfere with the host response to infection are of interest because they provide insight into virus-host relationships and may affect the safety and immunogenicity of recombinant VV (rVV) vaccines. Such vaccines need assessment in animal models and with this aim a model of VV infection based on intradermal injection of BALB/c ear pinnae was developed and characterized. In this model, the outcome of infection is affected by the dose of virus inoculated but virus spread is minimal and the mice suffer no signs of systemic illness. Cellular and humoral immune responses to these infections were measured readily and were independent of virus dose over a 100-fold range. Thus the model seems suitable for the analysis of the safety and immunogenicity of VV mutants lacking specific immunomodulatory proteins or bearing foreign antigens.

Animals↗
PubMed1999

The vaccinia virus A4OR gene product is a nonstructural, type II membrane glycoprotein that is expressed at the cell surface.

Gene A40R from vaccinia virus (VV) strain Western Reserve has been characterized. The open reading frame (ORF) was predicted to encode a 159 amino acid, 18152 Da protein with amino acid similarity to C-type animal lectins and to the VV A34R protein, a component of extracellular enveloped virus (EEV). Northern blotting and S1 nuclease mapping showed that gene A40R is transcribed early during infection from a position 12 nucleotides upstream of the ORF, producing a transcript of approximately 600 nucleotides. Rabbit anti-sera were raised against bacterial fusion proteins containing parts of the A40R protein. These were used to identify an 18 kDa primary translation product and N- and O-glycosylated forms of 28, 35 and 38 kDa. The A40R proteins were detected early during infection, formed higher molecular mass complexes under non-reducing conditions and were present on the cell surface but absent from virions. The proteins partitioned with integral membrane proteins in Triton X-114. Canine pancreatic microsomal membranes protected in vitro-translated A40R from proteinase K digestion, suggesting the A40R protein has type II membrane topology. A mutant virus with the A40R gene disrupted after amino acid 50, so as to remove the entire lectin-like domain, and a revertant virus were constructed. Disruption of the A40R gene did not affect virus plaque size, in vitro growth rate and titre, EEV formation, or virus virulence in a murine intranasal model.

Administration, Intranasal↗