Biomedical subjects

Geoffrey L Smith

Publications and source records attributed to Geoffrey L Smith.

At least 37 records · Page 2Linked to original sources

PubMed2004

Yaba-like disease virus protein Y144R, a member of the complement control protein family, is present on enveloped virions that are associated with virus-induced actin tails.

Yaba-like disease virus (YLDV) is a yatapoxvirus, a group of slow-growing poxviruses from primates. Analysis of the growth cycle of YLDV in tissue culture showed that maximum virus titres were reached 3 days post-infection and at this time only 3.3 % of infectious progeny was extracellular. The intracellular and extracellular virions have different buoyant densities and are separable on CsCl density gradients. They are also distinguishable by electron microscopy with the extracellular virions having an additional lipid envelope. In YLDV-infected cells, thick actin bundles with virions at their tips were seen protruding from the cell surface, despite the fact that YLDV lacks a protein comparable to Vaccinia virus A36R, which is required for VV-induced actin tail formation. In addition to these observations, the YLDV gene Y144R was characterized. This gene is predicted to encode a transmembrane protein containing three short consensus repeat (SCR) motifs common to members of the complement control protein family. Antibody generated against recombinant Y144R recognized products of 36, 41 and 48-55 kDa in YLDV-infected cells and purified extracellular enveloped virus (EEV) but not intracellular mature virus (IMV). Y144R protein is a glycoprotein with type I membrane topology that is synthesized early and late during infection. By immunoblot, indirect immunofluorescence and immuno-cryoelectron microscopy the Y144R protein was detected on the intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and EEV. This represents the first study of a YLDV IEV, CEV and EEV protein at the molecular level.

Actins↗

PubMed2004

A new member of the interleukin 10-related cytokine family encoded by a poxvirus.

Poxviruses express numerous proteins involved in manipulating the host immune response. Analysis of the primary sequence and predicted structure of the 134R protein of Yaba-like disease virus (Y134R) indicated that it is similar to cellular proteins of the IL-10 family, specifically IL-19, IL-20 and IL-24. A flag-tagged Y134R was expressed from mammalian cells and identified as a secreted, monomeric glycoprotein that stimulated signal transduction from class II cytokine receptors IL-20Ralpha/IL-20Rbeta (IL-20R type1) and IL-22R/IL-20Rbeta (IL-20R type 2). Y134R induced phosphorylation of signal transducers and activators of transcription, their translocation to the nucleus and the induction of reporter gene expression. In contrast, Y134R was unable to induce similar responses from either the IL-22 or IFN-lambda (IL-28A, IL-28B, IL-29) class II cytokine receptors. To examine the role Y134R plays during a poxvirus infection, a vaccinia virus recombinant expressing Y134R was constructed and tested in a murine intranasal infection model. Compared with control viruses, the virus expressing Y134R had a reduced virulence, manifested by reduced weight loss, signs of illness and virus titres in infected organs. These results demonstrate that Y134R is a new viral member of the IL-10-related cytokine family and that its activity in vivo affects virus virulence.

Amino Acid Sequence↗

PubMed2004

Studying the binding and entry of the intracellular and extracellular enveloped forms of vaccinia virus.

This chapter describes the methods for the study of binding and entry of the two different forms of vaccinia virus (VV)-the intracellular mature virus (IMV) and extracellular enveloped virus (EEV)-using immunofluorescent staining and confocal microscopy. After binding to or penetration of the cells, IMV, EEV, and virus cores are distinguished by different antibodies. Bound virus or penetrated cores are visualized and recorded by confocal microscopy and can be accurately counted. Although specific antibodies to IMV, EEV, and virus cores are required, this method is highly quantitative and also allows the recognition of virus aggregates, which would not be possible using other techniques, such as flow cytometry and radiolabeling of virus particles. Furthermore, this method bypasses the need for EEV purification that may damage the EEV membrane and release an IMV particle.

Animals↗

PubMed2003

Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans.

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.

Animals↗

PubMed2003

Steroid hormone synthesis by vaccinia virus suppresses the inflammatory response to infection.

The 3beta-hydroxysteroid dehydrogenase (3beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Vaccinia virus (VV) also synthesizes steroid hormones with a 3beta-HSD enzyme (v3beta-HSD) encoded by gene A44L. Here we examined the effects of v3beta-HSD in VV disease using wild-type (vA44L), deletion (vDeltaA44L), and revertant (vA44L-rev) viruses in a murine intranasal model. Loss of A44L was associated with an attenuated phenotype. Early (days 1-3) after infection with vDeltaA44L or control viruses the only difference observed between groups was the reduced corticosterone level in lungs and plasma of vDeltaA44L-infected animals. Other parameters examined (body weight, signs of illness, temperature, virus titres, the pulmonary inflammatory infiltrate, and interferon [IFN]-gamma levels) were indistinguishable between groups. Subsequently, vDeltaA44L-infected animals had reduced weight loss and signs of illness, and displayed a vigorous pulmonary inflammatory response. This was characterized by rapid recruitment of CD4+ and CD8+ lymphocytes, enhanced IFN-gamma production and augmented cytotoxic T lymphocyte activity. These data suggest that steroid production by v3beta-HSD contributes to virus virulence by inhibiting an effective inflammatory response to infection.

3-Hydroxysteroid Dehydrogenases↗

PubMed2003

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense.

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Adaptor Proteins, Signal Transducing↗

PubMed2003

A soluble chemokine-binding protein from vaccinia virus reduces virus virulence and the inflammatory response to infection.

Many poxviruses express a secreted protein that binds CC chemokines with high affinity and has been called viral CC chemokine inhibitor (vCCI). This protein is unrelated to any known cellular protein, yet can compete with host cellular CC chemokine receptors to modulate host inflammatory and immune responses. Although several strains of vaccinia virus (VV) express a vCCI, the best characterized VV strains Western Reserve and Copenhagen do not. In this study, we have expressed the vCCI from VV strain Lister in a recombinant Western Reserve virus (v Delta B8R-35K) and characterized its binding properties in vitro and its effect on virulence in vivo relative to wild-type virus (v Delta B8R) or a revertant virus (v Delta B8R-R) where Lister 35-kDa had been removed. Cells infected with v Delta B8R-35K secreted a 35-kDa protein that bound the CC chemokine macrophage-inflammatory protein 1 alpha. Expression of vCCI attenuated the virus in a murine intranasal model, characterized by reduced mortality and weight loss, decreased virus replication and spread, and a reduced recruitment of inflammatory cells into the lungs of VV-infected mice. The CC chemokines macrophage-inflammatory protein 1 alpha, eotaxin, and macrophage chemotactic protein 1 were detected in bronchoalveolar lavage fluids from v Delta B8R-infected mice; however, bronchoalveolar lavage fluids from v Delta B8R-35K-infected mice had lower levels of chemokines and a reduced chemotactic activity for murine leukocytes in vitro. These observations suggest that vCCI plays an important role in regulating leukocyte trafficking to the lungs during VV infection by binding to CC chemokines and blocking their chemotactic activities.

Animals↗

PubMed2003

Vaccinia virus cores are transported on microtubules.

Infection with Vaccinia virus (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK(2) cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop-start manner with an average speed of 51.8 (+/-3.9) microm min(-1), consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.

Cell Line↗

PubMed2003

A kinetic analysis of immune mediators in the lungs of mice infected with vaccinia virus and comparison with intradermal infection.

The early inflammatory response to a virus may be critical in restricting infection and in shaping the subsequent adaptive immune response. In this study we have examined the early inflammatory response of mice following infection with vaccinia virus (VV) strain Western Reserve (WR). Respiratory challenge of BALB/c mice with VV led to early virus replication in the lung and upper respiratory tract followed by dissemination of virus to other visceral organs and to the brain. The number of inflammatory cells, largely macrophages and T lymphocytes, recovered from bronchoalveolar lavage (BAL) fluid increased markedly during infection and coincided with the expression of CC chemokine ligands (CCL) 3, 2 and 11 and CXC chemokine ligands (CXCL) 1 and 2/3 in BAL. The peak of the inflammatory response occurred around day 10 and declined thereafter. The antiviral cytokines IFN-gamma and TNF-alpha, and the reactive nitrogen intermediate nitric oxide (NO), were also detected in BAL from VV-infected mice. A markedly different inflammatory response was observed after intradermal inoculation of WR into the ear pinnae of mice. Intradermal challenge was followed by highly localized virus replication and by a cellular influx, consisting largely of neutrophils and T lymphocytes, into the dermal compartment of the infected ear. Together these findings highlight differences in the pathogenesis and in the cellular inflammatory response to WR following intranasal and intradermal inoculation of mice.

Administration, Intranasal↗

PubMed2003

The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model.

Chordate poxviruses encode several uncharacterized POZ-kelch proteins and three of these are present in Vaccinia virus (VV) strain Western Reserve. VV gene C2L is predicted to encode a protein of 512 amino acid residues with a POZ/BTB domain in the N-terminal region and three kelch motifs in the C-terminal half of the protein. We have identified the C2L gene product as an intracellular protein of 56 kDa and constructed and characterized a VV mutant lacking the C2L gene (v Delta C2L). Compared to wild-type and revertant viruses, v Delta C2L had unaltered growth in vitro, but had a different plaque morphology due to an altered cytopathic effect (CPE) of infected cells. Deleting C2L had no effect on VV-induced formation of actin tails or enhanced cell motility, but affected the development of VV-induced cellular projections and the Ca(2+)-independent cell/extracellular matrix adhesion late during infection. In an intranasal mouse model, C2L did not contribute to virus virulence. However, in an intradermal mouse model, infection with v Delta C2L resulted in larger lesions and more cell infiltration into the infected ears during recovery from infection. Thus, in this model, C2L protein inhibits inflammation and reduces immunopathology. In summary, we found that C2L is not required for virus replication in vitro but contributes to aspects of VV-induced CPE and reduces immunopathology in vivo.

Animals↗

PubMed2003

Yaba-like disease virus protein 7L is a cell-surface receptor for chemokine CCL1.

Yaba-like disease virus (YLDV) genes 7L and 145R are located on opposite ends of the genome and are predicted to encode 7-transmembrane proteins (7-TM) that share 53 and 44 % amino acid identity, respectively, to human CC chemokine receptor 8 (hCCR8). In this report, we demonstrate that early after infection with YLDV, cells acquire the ability to bind human CCL1. By expression of genes 7L and 145R in vaccinia virus, we demonstrated that each protein is glycosylated and is exposed on the cell surface with the N terminus outside the cell. Protein 7L, but not 145R, is able to bind hCCL1 (K(d)=0.6+/-0.13 nM) and couple to heterotrimeric G-proteins and to activate the extracellular signal-regulated kinases (ERK1/2). 7L binds several chemokines including the viral chemokines vMIPI and vMIPII and hCCL7/MCP3. This binding seems species-specific as 7L does not bind the murine orthologues of CCL1 and CCL7 in the assays used. This represents the first example of a poxviral 7-TM chemokine receptor that has functional interactions with a human chemokine.

Amino Acid Sequence↗

PubMed2003

Vaccinia virus interleukin-18-binding protein promotes virulence by reducing gamma interferon production and natural killer and T-cell activity.

Interleukin-18 (IL-18) is a proinflammatory cytokine that promotes natural killer (NK) and T-cell activation. Several poxviruses, including vaccinia virus (VV), encode a soluble IL-18-binding protein (IL-18bp). The role of the VV IL-18bp (gene C12L) in vivo was studied with wild-type (vC12L), deletion mutant (vDeltaC12L), and revertant (vC12L-rev) viruses in a murine intranasal model of infection. The data show that vDeltaC12L was markedly attenuated, characterized by a mild weight loss and reduced virus titers in lungs, brain, and spleen. Three days after infection, NK cytotoxic activity was augmented in the lung, spleen, and mediastinal lymph nodes (MLNs) of vDeltaC12L-infected mice compared to controls. Seven days after infection, vDeltaC12L-infected mice displayed heightened VV-specific cytotoxic T-lymphocyte (CTL) responses in the lungs, spleen, and MLNs. Gamma interferon (IFN-gamma) levels were also dramatically elevated in lavage fluids and cells from lungs of mice infected with vDeltaC12L. Finally, we demonstrate that IL-18 is produced in vitro and in vivo after VV infection. Taken together, these data demonstrate a role for the vIL-18bp in counteracting IL-18 in both the innate and the specific immune response to VV infection and indicate that the ability of IL-18 to promote vigorous T-cell responses (cytotoxic activity and IFN-gamma production) is a critical factor in the accelerated clearance of the vDeltaC12L mutant.

Animals↗

PubMed2003

Vaccinia virus motility.

Vaccinia virus (VV), the virus smallpox vaccine, replicates in the cytoplasm of infected cells. The intracellular movement of this large virus would be inefficient without specific transport mechanisms; therefore, VV uses microtubules for movement during both entry and egress. In addition, the dissemination of virus from infected cells to adjacent cells is promoted by the polymerization of actin beneath cell surface virions to drive virus particles away from the cell. Last, the roles of different VV particles in virus movement within and between hosts are discussed.

Biological Transport↗

PubMed2002

Vaccinia virus CrmE encodes a soluble and cell surface tumor necrosis factor receptor that contributes to virus virulence.

Poxviruses encode soluble cytokine receptors to interfere with host immune functions. Cells infected with vaccinia virus (VV) strains USSR, Lister, and Evans express soluble and cell surface tumor necrosis factor receptors (vTNFRs). We have characterized vTNFR activity in VV USSR and identified an open reading frame that encodes both soluble and cell surface activity, hereafter referred to as VV cytokine response modifier E (VV CrmE). Expression and characterization from recombinant VV and baculovirus showed VV CrmE to be an 18-kDa protein that bound human, mouse, and rat TNF-alpha, but not human LT alpha. VV CrmE inhibited the cytotoxic and apoptotic activities of human, but not mouse or rat, TNF in vitro. Nonetheless, in a murine intranasal model, USSR recombinants lacking CrmE were attenuated, demonstrating a role in vivo. Furthermore, expression of VV or cowpox virus vTNFRs from VV strain WR (which itself does not express a vTNFR) was shown to enhance virulence in the murine model.

Animals↗

PubMed2002

Smallpox: anything to declare?

Smallpox was eradicated in 1977, but it remains a concern owing to the potential use of the causative agent variola virus in bioterrorism. This article provides an overview of the World Health Organization's spectacular success in achieving the eradication of smallpox. It discusses how variola virus could potentially re-emerge and how prepared we are to counter such a re-emergence. Finally, the potential threat from other orthopoxviruses that exist naturally or that have been genetically engineered is considered. In the words of Rep. Christopher Shay, 'Better to be scared by the improbable possibility than to be unprepared for the catastrophic reality'.

Bioterrorism↗

PubMed2002

The vaccinia virus F12L protein is associated with intracellular enveloped virus particles and is required for their egress to the cell surface.

The vaccinia virus (VV) F12L gene encodes a 65 kDa protein that is expressed late during infection and is important for plaque formation, EEV production and virulence. Here we have used a recombinant virus (vF12LHA) in which the F12L protein is tagged at the C terminus with an epitope recognized by a monoclonal antibody to determine the location of F12L in infected cells and whether it associates with virions. Using confocal and electron microscopy we show that the F12L protein is located on intracellular enveloped virus (IEV) particles, but is absent from immature virions (IV), intracellular mature virus (IMV) and cell-associated enveloped virus (CEV). In addition, F12L shows co-localization with endosomal compartments and microtubules. F12L did not co-localize with virions attached to actin tails, providing further evidence that actin tails are associated with CEV but not IEV particles. In vDeltaF12L-infected cells, virus morphogenesis was arrested after the formation of IEV particles, so that the movement of these virions to the cell surface was inhibited and CEV particles were not found. Previously, virus mutants lacking IEV- or EEV-specific proteins were either unable to make IEV particles (vDeltaF13L and vDeltaB5R), or were unable to form actin tails after formation of CEV particles (vDeltaA36R, vDeltaA33R, vDeltaA34R). The F12L deletion mutant therefore defines a new stage in the morphogenic pathway and the F12L protein is implicated as necessary for microtubule-mediated egress of IEV particles to the cell surface.

Animals↗

PubMed2002

Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread.

The roles of vaccinia virus (VV) intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV) and their associated proteins in virus spread were investigated. The plaques made by VV mutants lacking individual IEV- or EEV-specific proteins (vDeltaA33R, vDeltaA34R, vDeltaA36R, vDeltaA56R, vDeltaB5R, vDeltaF12L and vDeltaF13L) were compared in the presence of IMV- or EEV-neutralizing antibodies (Ab). Data presented show that for long-range spread, the comet-shaped plaques of VV were caused by the unidirectional spread of EEV probably by convection currents, and for cell-to-cell spread, VV uses a combination of Ab-resistant and Ab-sensitive pathways. Actin tails play a major role in the Ab-resistant pathway, but mutants such as vDeltaA34R and vDeltaA36R that do not make actin tails still spread from cell to cell in the presence of Ab. Most strikingly, the Ab-resistant pathway was abolished when the A33R gene was deleted. This effect was not due to alterations in the efficiency of neutralization of EEV made by this mutant, nor due to a deficiency in IMV wrapping to form IEV, which was indispensable for EEV formation by vDeltaA33R and vDeltaA34R. We suggest a role for A33R in promoting Ab-resistant cell-to-cell spread of virus. The roles of the different virus forms in the VV life-cycle are discussed.

Animals↗

PubMed2002

An investigation of incorporation of cellular antigens into vaccinia virus particles.

Vaccinia virus (VV) infection produces several types of virus particle called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). Some cellular antigens are associated with EEV and these vary with the cell type used to grow the virus. To investigate if specific cell antigens are associated with VV particles, and to address the origin of membranes used to envelope IMV and IEV/CEV/EEV, we have studied whether cell antigens and foreign antigens expressed by recombinant VVs are incorporated into VV particles. Membrane proteins that are incorporated into the endoplasmic reticulum (ER), intermediate compartment (IC), cis/medial-Golgi, trans-Golgi network (TGN) or plasma membrane were not detected in purified IMV particles. In contrast, proteins present in the TGN or membrane compartments further downstream in the exocytic pathway co-purify with EEV particles when analysed by immunoblotting. Immunoelectron microscopy found only low levels of these proteins in IEV, CEV/EEV. The incorporation of foreign antigens into VV particles was not affected by loss of individual IEV or EEV-specific proteins or by redirection of B5R to the ER. These data suggest that (i) host cell antigens are excluded from the lipid envelope surrounding the IMV particle and (ii) membranes of the ER, IC and cis/medial-Golgi are not used to wrap IMV particles to form IEV. Lastly, the VV haemagglutinin was absent from one-third of IEV and CEV/EEV particles, whereas other EEV antigens were present in all these virions.

Animals↗