Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR.

One hundred temperature-sensitive mutants of vaccinia virus WR were isolated from virus that had been mutagenized with 5-bromodeoxyuridine or N-methyl-N'-nitro-N-nitrosoguanidine. A rapid screening procedure based on the ability of vaccinia virus to form plaques under liquid overlay medium was used to identify potential mutants among randomly picked plaque isolates or plaques preselected for their small size after temperature shift-up. The preselection technique resulted in a sixfold increase in the number of successful mutant isolations relative to the number of plaques picked. All of the mutants had efficiencies of plating at 39.5 degrees C relative to that at 33 degrees C of 10(-4) or less, and 33 of 40 produced 10% or less of the amount of virus at the nonpermissive temperature (39.5 degrees C) relative to that at the permissive temperature (33 degrees C). Experiments with the fluorescent DNA binding dye Hoechst 33258 demonstrated that 6 of the 100 mutants failed to form characteristic cytoplasmic DNA factories at 39.5 degrees C. To facilitate the functional grouping of such a large number of mutants, a rapid infectious center assay was developed. Thirty of the mutants were assigned to 16 or 17 complementation-recombination groups by using this assay. Recombination experiments have allowed the construction of a genetic map representing 22 mutants in 12 of these groups.     

How vaccinia virus has evolved to subvert the host immune response

Viruses are obligate intracellular parasites and are some of the most rapidly evolving and diverse pathogens encountered by the host immune system. Large complicated viruses, such as poxviruses, have evolved a plethora of proteins to disrupt host immune signalling in their battle against immune surveillance. Recent X-ray crystallographic analysis of these viral immunomodulators has helped form an emerging picture of the molecular details of virus-host interactions. In this review we consider some of these immune evasion strategies as they apply to poxviruses, from a structural perspective, with specific examples from the European SPINE2-Complexes initiative. Structures of poxvirus immunomodulators reveal the capacity of viruses to mimic and compete against the host immune system, using a diverse range of structural folds that are unique or acquired from their hosts with both enhanced and unexpectedly divergent functions.     

Conditional expression of foreign genes by temperature-sensitive mutants of vaccinia virus

To assess the utility of two temperature-sensitive (ts) mutant vaccinia viruses as vectors for the conditional in vitro expression of recombinant foreign genes, we have studied the kinetics of expression of foreign genes incorporated into these viruses. At nonpermissive temperature, 40 degrees C, these viruses were defective either in DNA synthesis or in virus assembly. Foreign gene expression was affected by the nature of the ts lesion and by the nature of the vaccinia promoter positioned upstream from the foreign gene. With both vector viruses, a foreign gene controlled by the p7.5 early-late promoter was expressed at both 33 degrees and 40 degrees C. With the DNA synthesis-defective vector virus, foreign gene expression controlled by the p11 DNA synthesis-dependent late promoter was inhibited at 40 degrees C, but could be turned on by shift to 33 degrees C. This ts expression system provides an alternative to use of drugs that inhibit DNA synthesis as a means for experimental manipulation of gene expression. Both vector viruses can be used with existing vaccinia virus expression technology.     

Isolation and characteristics of vaccinia virus proteins associated with cell membranes of infected cells]

The proteins of vaccinia virus associated with plasma membrane infected cells BHK-21, p60, p45, p42, p40,p35,p34,p28,p23 were isolated from plasma membranes using affinity chromatography, gel-electrophoresis and passive elution. An immunochemical characterization was carried out using specific antiserum to these proteins. Investigation of temporal regulation of proteins synthesis in infected cells showed that proteins p60, p45, p42, p40, p28 were late, and p35, p34, p23--early-late proteins. Immunochemical analysis of vaccinia virus mRNA cell-free translational products was carried out using specific antiserum. The polypeptide-precursors of viral proteins were identified.     

Differences and similarities in viral life cycle progression and host cell physiology after infection of human dendritic cells with modified vaccinia virus Ankara and vaccinia virus

Modified vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus (VV) that has attracted significant attention as a candidate viral vector vaccine for immunization against infectious diseases and treatment of malignancies. Although MVA is unable to replicate in most nonavian cells, vaccination with MVA elicits immune responses that approximate those seen after the administration of replication-competent strains of VV. However, the mechanisms by which these viruses elicit immune responses and the determinants of their relative immunogenicity are incompletely understood. Studying the interactions of VV and MVA with cells of the human immune system may elucidate these mechanisms, as well as provide a rational basis for the further enhancement of the immunogenicity of recombinant MVA vectors. Toward this end, we investigated the consequences of MVA or VV infection of human dendritic cells (DCs), key professional antigen-presenting cells essential for the generation of immune responses. We determined that a block to the formation of intracellular viral replication centers results in abortive infection of DCs with both VV and MVA. MVA inhibited cellular protein synthesis more rapidly than VV and displayed a distinct pattern of viral protein expression in infected DCs. MVA also induced apoptosis in DCs more rapidly than VV, and DC apoptosis after MVA infection was associated with an accelerated decline in the levels of intracellular Bcl-2 and Bcl-X(L). These findings suggest that antigen presentation pathways may contribute differentially to the immunogenicity of VV and MVA and that targeted modifications of virus-induced DC apoptosis may further increase the immunogenicity of MVA-vectored vaccines.     

Expression of the E3L gene of vaccinia virus in transgenic mice decreases host resistance to vaccinia virus and Leishmania major infections

The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2′-5′-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.     

Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis.

Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.     

Transient host range selection for genetic engineering of modified vaccinia virus Ankara

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei.

The replication of vaccinia virus is thought to take place exclusively in the cytoplasm of host cells. However, using DNA-DNA hybridization techniques, it can be shown that a significant fraction of the synthesis of vaccinia DNA takes place in the nucleus as well as the cytoplasm. The (3H) thymiding pulse-labeled vaccinia DNA synthesized in the nucleus reaches a maximum at about 3 h after infection, corresponding to the time of maximal DNA synthesis in infected cells. At this time host DNA synthesis drops to about 25% of the rate of the uninfected cells. Even with short labeling times (2 min) the nucleus is found to contain 60% of the incorporated (3H)thymidine, much of which is in vaccinia DNA. Prior inhibition of host nuclear DNA synthesis with mitomycin C, followed by removal of the antibiotic causes a subsequent inhibition of vaccinia DNA synthesis and complete suppression of mature virus. Purified nuclei, isolated from vaccinia-infected cells, also synthesize vaccinia DNA in vitro. Over 90% of the DNA synthesized in vitro by isolated nuclei contain vaccinia-specific sequences.     

Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus.

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11). By using ascites, one of the antibodies, mAbC3, neutralized (50%) virus infectivity with a titer of about 10(-4), whereas the others exhibited lower neutralization titers of 10(-2) to 10(-3). The binding of the mAbs to vaccinia virus did not alter virus attachment to cells. However, virus uncoating was extensively blocked by mAbC3, whereas mAbB11 and mAbF11 had little or no effect. The three mAbs recognized a similar 14K protein in cowpox, rabbitpox, and vaccinia Elstree strains, indicating a high degree of protein conservation among orthopoxviruses. Based on the binding of mAbs to V-8 protease cleavage products of the 14K protein, the extent of protein recognition for other poxviruses, and differences in the degree of virus neutralization and of virus uncoating into cells, we suggest that the three mAbs recognize different domains of vaccinia 14K viral envelope protein. Furthermore, our findings indicate that the 14K protein may play a role in virus penetration.     

The host phosphoinositide 5-phosphatase SHIP2 regulates dissemination of vaccinia virus

After fusing with the plasma membrane, enveloped poxvirus virions form actin-filled membranous protrusions, called tails, beneath themselves and move toward adjacent uninfected cells. While much is known about the host and viral proteins that mediate formation of actin tails, much less is known about the factors controlling release. We found that the phosphoinositide 5-phosphatase SHIP2 localizes to actin tails. Localization requires phosphotyrosine, Abl and Src family tyrosine kinases, and neural Wiskott-Aldrich syndrome protein (N-WASP) but not the Arp2/Arp3 complex or actin. Cells lacking SHIP2 have normal actin tails but release more virus. Moreover, cells infected with viral strains with mutations in the release inhibitor A34 release more virus but recruit less SHIP2 to tails. Thus, the inhibitory effects of A34 on virus release are mediated by SHIP2. Together, these data suggest that SHIP2 and A34 may act as gatekeepers to regulate dissemination of poxviruses when environmental conditions are conducive.     

Mapping the genomic location of the gene encoding alpha-amanitin resistance in vaccinia virus mutants.

To facilitate the determination of the genomic location of the vaccinia virus gene(s) encoding alpha-amanitin resistance (alpha r) (Villarreal et al., J. Virol. 51:359-366, 1984), a collection of alpha r, temperature-sensitive (ts) mutants were isolated. The premise of these experiments was that mutants might be found whose dual phenotypes were the result of a single or two closely linked mutations. Genetic analyses of the alpha rts mutant library revealed two mutants, alpha rts7 and alpha rts12, that apparently fit this criterion; in alpha rts7 the two lesions were indistinguishable, whereas in alpha rts12 the two mutations were closely linked but separable. Cloned vaccinia virus HindIII DNA fragments were used to marker rescue the temperature-sensitive phenotype of these two dual mutants. The temperature-sensitive lesion of alpha rts7 was rescued by the HindIII N fragment (1.5 kilobases), whereas alpha rts12 was rescued by the neighboring HindIII M fragment (2.0 kilobases). The progeny virions of the alpha rts7 HindIII-N rescue reverted to an alpha-amanitin-sensitive phenotype, whereas the alpha rts12 HindIII-M progeny were still resistant to the drug. Taken together, these data indicate that the gene encoding alpha-amanitin resistance maps to the HindIII N fragment and provides evidence for the existence of essential vaccinia virus genes in a region of the genome previously believed to be nonessential for replication in tissue culture. Biochemical analyses revealed that both mutants were capable of synthesizing DNA as well as early and late viral proteins at the permissive and nonpermissive temperatures. At the nonpermissive temperature alpha rts12 and alpha rts7 were unable to process the major core precursors P94 and P65 into VP62 and VP60.