Vaccinia Virus Mutant Research Articles

Construction of a novel set of transfer vectors to study vaccinia virus replication and foreign gene expression

Vaccinia virus (VV) is a useful expression vector for many laboratory applications. To date, approximately 60% (∼120) of the VV genes remain uncharacterized. The thought of smallpox being used as a biological weapon has gained attention. In light of this, it is imperative that we continue to study the basic replication of VV, a poxvirus that is closely related to smallpox. A set of plasmid vectors were constructed to generate gene deletions (pZIPPY-NEO/GUS) in VV or for foreign gene expression (pBR-EXPRESS). The vectors contain the Escherichia coli neomycin resistance ( neo) and β-glucuronidase ( gusA) genes as selectable markers to facilitate isolation of recombinant viruses. These are the first transfer vectors to use a neo/gusA selection system. We used these vectors to successfully generate a recombinant D9R deletion mutant of VV and to express E. coli lacZ gene. Results indicate that both vectors are highly suited for their designed purpose.

The role of the cytoskeleton in the life cycle of viruses and intracellular bacteria: tracks, motors, and polymerization machines.

Recent advances in microbiology implicate the cytoskeleton in the life cycle of some pathogens, such as intracellular bacteria, Rickettsia and viruses. The cellular cytoskeleton provides the basis for intracellular movements such as those that transport the pathogen to and from the cell surface to the nuclear region, or those that produce cortical protrusions that project the pathogen outwards from the cell surface towards an adjacent cell. Transport in both directions within the neuron is required for pathogens such as the herpesviruses to travel to and from the nucleus and perinuclear region where replication takes place. This trafficking is likely to depend on cellular motors moving on a combination of microtubule and actin filament tracks. Recently, Bearer et al. reconstituted retrograde transport of herpes simplex virus (HSV) in the giant axon of the squid. These studies identified the tegument proteins as the viral proteins most likely to recruit retrograde motors for the transport of HSV to the neuronal nucleus. Similar microtubule-based intracellular movements are part of the biological behavior of vaccinia, a poxvirus, and of adenovirus. Pathogen-induced surface projections and motility within the cortical cytoplasm also play a role in the life cycle of intracellular pathogens. Such motility is driven by pathogen-mediated actin polymerization. Virulence depends on this actin-based motility, since virulence is reduced in Listeria ActA mutants that lack the ability to recruit Arp2/3 and polymerize actin and in vaccinia virus mutants that cannot stimulate actin polymerization. Inhibition of intracellular movements provides a potential strategy to limit pathogenicity. The host cell motors and tracks, as well as the pathogen factors that interact with them, are potential targets for novel antimicrobial therapy.

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.

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.

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.

A Mitogenic Signal Triggered at an Early Stage of Vaccinia Virus Infection: IMPLICATION OF MEK/ERK AND PROTEIN KINASE A IN VIRUS MULTIPLICATION

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.

Vaccinia virus utilizes microtubules for movement to the cell surface

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 μm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.

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.

Role of vaccinia virus A20R protein in DNA replication: construction and characterization of temperature-sensitive mutants.

Previous analyses of randomly generated, temperature-sensitive vaccinia virus mutants led to the mapping of DNA synthesis negative complementation groups to the B1R, D4R, D5R, and E9L genes. Evidence from the yeast two-hybrid system that the D4R and D5R proteins can interact with the A20R protein suggested that A20R was also involved in DNA replication. We found that the A20R gene was transcribed early after infection, consistent with such a role. To investigate the function of the A20R protein, targeted mutations were made by substituting alanines for charged amino acids occurring in 11 different clusters. Four mutants were not isolated, suggesting that they were lethal, two mutants exhibited no temperature sensitivity, two mutants exhibited partial temperature sensitivity, and two mutants formed no plaques or infectious virus at 39 degrees C. The two mutants with stringent phenotypes were further characterized. Temperature shift-up experiments indicated that the crucial period was between 6 and 12 h after infection, making it unlikely that the defect was in virus entry, early gene expression, or a late stage of virus assembly. Similar patterns of metabolically labeled viral early proteins were detected at permissive and nonpermissive temperatures, but one mutant showed an absence of late proteins under the latter conditions. Moreover, no viral DNA synthesis was detected when cells were infected with either stringent mutant at 39 degrees C. The previous yeast two-hybrid analysis together with the present characterization of A20R temperature-sensitive mutants suggested that the A20R, D4R, and D5R proteins are components of a multiprotein DNA replication complex.

Antibody Neutralization of the Extracellular Enveloped Form of Vaccinia Virus

The extracellular enveloped form (EEV) of vaccinia virus (VV) is important for the long-range dissemination of the virus inside the host. Early work suggested that both IMV and EEV infectivity could be inhibited by antibodies, although two other studies reported that EEV was resistant to neutralization. Here, we readdressed this question, using four VV-immune antisera and their purified IgG, and showed that EEV infectivity can be inhibited by antibody produced from a live infection in plaque-reduction assays, although EEV is more resistant to neutralization by convalescent antibodies than is IMV. In parallel, indirect immunofluorescent staining and confocal microscopy showed that antibody aggregated EEV and prevented it from binding to cells. Using the IgG and Fab fragments prepared from this antiserum, we tested whether EEV made by VV mutants lacking genes A33R, A34R, A36R, A56R, B5R, F12L, or F13L can be inhibited in plaque-reduction assays. Although vΔB5R was slightly more resistant than other mutants, none of these mutants escaped neutralization completely, suggesting that multiple virus proteins are involved in the inhibition. Using an antibody specific to B5R protein and B5R mutants with consecutive short consensus repeat (SCR) domains deleted, the neutralization epitopes on B5R were mapped to within the SCR domain 1.

Establishment of a rescue system for canine distemper virus.

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.

Induction of necrotic-like cell death by tumor necrosis factor alpha and caspase inhibitors: novel mechanism for killing virus-infected cells.

Induction of apoptotic cell death generally requires the participation of cysteine proteases belonging to the caspase family. However, and similar to most cell types, mouse fibroblasts are normally resistant to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Surprisingly, TNF-alpha treatment of vaccinia virus-infected mouse fibroblasts resulted in necrotic-like cell death, which was significantly reduced in cells infected with a vaccinia virus mutant lacking the caspase inhibitor B13R. Furthermore, TNF-alpha also induced necrotic-like cell death of fibroblasts in the presence of peptidyl caspase inhibitors. In both cases, necrosis was accompanied by generation of superoxide species. Caspase inhibitors also sensitized fibroblasts to killing by double-stranded RNA and gamma interferon. In all cases, cell death was efficiently blocked by antioxidants or mitochondrial respiratory chain inhibitors. These results define a new mitochondrion-dependent mechanism which may be important in the killing of cells infected with viruses encoding caspase inhibitors.

The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles.

The vaccinia virus (VV) A27L gene encodes a 14 kDa protein that is required for the formation of intracellular enveloped virus (IEV) and, consequently, normal sized plaques. Data presented here show that A27L plays an additional role in VV assembly. When cells were infected with the VV WR32-7/Ind 14K, under conditions that repress A27L expression, transport of intracellular mature virus (IMV) from virus factories was inhibited and some IMV was found in aberrant association with virus crescents. In contrast, other VV mutants (vDeltaB5R and vDeltaF13L) that are defective in IEV formation produce IMV particles that are transported out of virus factories. This indicated a specific role for A27L in IMV transport. Induction of A27L expression at 10 h post-infection promoted the dispersal of clustered IMV particles, but only when microtubules were intact. Formation of IEV particles was also impaired when cells were infected with WR32-7/14K, a VV strain expressing a mutated form of the A27L protein; however, this mutation did not inhibit intracellular transport of IMV particles. Collectively, these data define two novel aspects of VV morphogenesis. Firstly, A27L is required for both IMV transport and the process of envelopment that leads to IEV formation. Secondly, movement of IMV particles between the virus factory and the site of IEV formation is microtubule-dependent.

The Effects of Targeting the Vaccinia Virus B5R Protein to the Endoplasmic Reticulum on Virus Morphogenesis and Dissemination

The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K2X2) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K2X4) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vΔB5R. Similar levels of B5R protein were made by each virus, but the B5R-K2X2 protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K2X4 protein colocalised with 1,4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K2X2 was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K2X2 were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K2X2 EEV.

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.

Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy.

Tumor-directed gene therapy faces many obstacles. Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations for a successful therapeutic outcome. A thymidine kinase-deleted mutant vaccinia virus has been shown in marker studies to replicate selectively in tumor tissue in animal models. Purine nucleoside phosphorylase (PNP), from E. coli, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) to the toxic purine 6-methylpurine. In this study, we investigated the cytotoxic properties of PNP, expressed by an optimized synthetic early/late promoter in a vaccinia virus (vMPPNP). In vitro cytotoxicity of psoralen-inactivated vMPPNP (1 microg of psoralen, 4 min of LWUV [365 nm]) at the maximum tolerated dose (MTD) of 6-MPDR (80 microM) reduced cell viability by day 3 to 1.7%. At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 microM) caused reduction of cell viability to 19.8% within 4 days. Furthermore, there was complete abrogation of viral replication after intracellular conversion of prodrug into the active toxin. The potency of such a system was similar among all histologies tested. Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system. When virus was delivered intraperitoneally into athymic mice with hepatic metastases, followed by administration of prodrug, there was a significant prolongation of survival and a 30% cure rate. In summary, owing to its tumor-targeting capabilities, high transduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy.

Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein.

The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus.

Poxviral/Retroviral Chimeric Vectors Allow Cytoplasmic Production of Transducing Defective Retroviral Particles

Defective vaccinia viruses were constructed that express functional Moloney murine leukemia virus-based vector genomes, giving rise to substantial titers of transduction-competent retrovirus particles after infection of a retroviral packaging cell line. For this purpose, the proviral retrovirus genome, engineered into the vaccinia virus mutant, was subjected to several modifications, including the replacement of retroviral promoter sequences by vaccinia virus sequences and the precise fusion of the transcription stop signal downstream of and the removal of such signals within the transcription unit, allowing cytoplasmic transcription of distinct full-length retroviral transcripts. Vaccinia-mediated expression of retroviral vector particles could be observed as early as 3 h postinfection and resulted in stable transduction of NIH/3T3 target cells at higher titers than the control performed by conventional plasmid transfections. Thus at least part of the vaccinia life cycle and retroviral assembly can occur concomitantly. Due to the favorable properties of vaccinia vectors, including high coding capacity, stability, and wide host range, defective vaccinia viral/retroviral chimeric vectors are promising tools for gene therapy applications.

Functional Analysis of Vaccinia Virus B5R Protein: Role of the Cytoplasmic Tail

Vaccinia extracellular enveloped virus (EEV) is important for cell-to-cell and long-range virus spread bothin vitroandin vivo. Six genes have been identified that encode protein constituents of the EEV outer membrane, and some of these proteins are critical for EEV formation. The B5R gene encodes an EEV-specific type I membrane protein, and deletion of this gene markedly decreases EEV formation and results in a small plaque phenotype. Data suggest that the transmembrane domain, cytoplasmic tail, or both contain the EEV localization signals that are required for targeting of the B5R protein to EEV and for EEV formation. Here, we report the construction of mutant vaccinia viruses in which the wild-type B5R gene was replaced with a mutated one that encodes a protein with the putative cytoplasmic tail deleted. The mutated protein showed normal intracellular distribution and was properly incorporated into EEV. Vaccinia viruses expressing the B5R protein lacking the cytoplasmic tail formed plaques that were similar in type and size to those formed by wild-type viruses and produced equivalent amounts of infectious EEV. These results indicate that the B5R cytoplasmic tail is not necessary for EEV formation and points to the transmembrane domain as the major determinant for targeting the B5R protein to the outer membrane of EEV and for supporting EEV formation.

Analysis of a vaccinia virus mutant expressing a nonpalmitylated form of p37, a mediator of virion envelopment.

Vaccinia virus encodes a 37-kDa palmitylated protein (p37) that is required for envelopment, translocation, and cell-to-cell spread of virions. We have analyzed the biological significance of the palmitate modification by constructing a recombinant vaccinia virus that expresses a nonpalmitylated p37 and comparing its biological activity to that of the wild-type virus. The mutant virus is inefficient at cell-to-cell spread and does not produce or release enveloped virions, although it produces normal amounts of nonenveloped virions. Furthermore, the mutant virus is not able to nucleate actin to propel itself through and out of the cell, a function requiring the indirect participation of p37. The deficiency in protein function appears to result from a lack of appropriate targeting to the membranes of the trans-Golgi network (TGN) which leaves p37 soluble in the cytoplasm. We conclude that the palmitate moiety is necessary for targeting or anchoring p37 to the TGN membrane, where, along with other vaccinia virus-encoded proteins, p37 is involved in the complex process of virion envelopment and release.