Temperature-sensitive Mutants Of Vaccinia Virus Research Articles

Vaccinia Virus Phospholipase Protein F13 Promotes Rapid Entry of Extracellular Virions into Cells.

The vaccinia virus protein F13, encoded by the F13L gene, is conserved across the subfamily Chordopoxvirinae and is critical among orthopoxviruses to produce the wrapped form of virus that is required for cell-to-cell spread. F13 is the major envelope protein on the membrane of extracellular forms of virus; however, it is not known if F13 is required in steps postwrapping. In this report, we utilize two temperature-sensitive vaccinia virus mutants from the Condit collection of temperature-sensitive viruses whose small plaque phenotypes have been mapped to the F13L gene. Despite the drastic reduction in plaque size, the temperature-sensitive viruses were found to produce levels of extracellular virions similar to those of the parental strain, Western Reserve (WR), at the permissive and nonpermissive temperatures, suggesting that they are not defective in extracellular virion formation. Analyses of extracellular virions produced by one temperature-sensitive mutant found that those produced at the nonpermissive temperature had undetectable levels of F13 and bound cells with efficiency similar to that of WR but displayed delayed cell entry kinetics. Additionally, low-pH treatment of cells bound by extracellular virions produced at the nonpermissive temperature by the temperature-sensitive reporter virus was unable to overcome a block in infection by bafilomycin A1, suggesting that these virions display increased resistance to dissolution of the extracellular virion envelope. Taken together, our results suggest that F13 plays a role both in the formation of extracellular virions and in the promotion of their rapid entry into cells by enhancing the sensitivity of the membrane to acid-induced dissolution.IMPORTANCE Vaccinia virus (VACV) is an orthopoxvirus and produces two infectious forms, mature virions (MV) and extracellular virions (EV). EV are derived from MV and contain an additional membrane that must first be removed prior to cell entry. F13 is critical for the formation of EV, but a postenvelopment role has not been described. Here, two temperature-sensitive VACV mutants whose deficiencies were previously mapped to the F13L locus are characterized. Both viruses produced EV at the nonpermissive temperature at levels similar to those of a virus that has F13L, yet they had a small plaque phenotype and rate of spread similar to that of an F13L deletion virus. F13 was undetectable on the EV membrane at the nonpermissive temperature, and these EV exhibited delayed cell entry kinetics compared to EV containing F13. This study is the first to conclusively demonstrate a novel role for F13 in cell entry of the EV form of the virus.

Marker rescue mapping of the combined Condit/Dales collection of temperature-sensitive vaccinia virus mutants

Complementation analysis of the combined Condit/Dales collection of vaccinia virus temperature-sensitive mutants has been reported (Lackner, C.A., D'Costa, S.M., Buck, C., Condit, R.C., 2003. Complementation analysis of the Dales collection of vaccinia virus temperature-sensitive mutants. Virology 305, 240–259), however not all complementation groups have previously been assigned to single genes on the viral genome. We have used marker rescue to map at least one representative of each complementation group to a unique viral gene. The final combined collection contains 124 temperature-sensitive mutants affecting 38 viral genes, plus five double mutants.

The vaccinia virus E8R gene product is required for formation of transcriptionally active virions

Two vaccinia virus temperature-sensitive mutants were mapped to the E8R gene and subjected to phenotypic characterization. Dts23 contains a missense mutation in the coding region of E8R (L81F), and in Cts19 the initiating methionine codon is changed from ATG to ATA (M1I). The two ts mutants display normal patterns of gene expression and DNA replication during infection. The E8 protein is synthesized exclusively late during infection and packaged into virion cores Western blot analysis revealed that E8 synthesis is reduced in Dts23 infected cells at permissive (31 °C) and non-permissive temperature (39.7 °C) and absent in Cts19 infection under both conditions. Dts23 virions produced at 39.7 °C were indistinguishable in appearance from wt virions. Cts19 fails to produce identifiable viral structures when incubated at 39.7 °C. Purified Dts23 virions produced at 39.7 °C contain reduced amounts of E8 and have a high particle to infectivity ratio; purified Cts19 virions grown at 31 °C also show reduced infectivity and do not contain detectable E8. Dts23 grown at 39.7 °C could enter cells but failed to synthesize early mRNA or produce CPE. Soluble extracts from mutant virions were active in a promoter dependent in vitro transcription assay, however intact mutant cores were defective in transcription. We suggest that E8 plays a subtle role in virion core structure that impacts directly or indirectly on core transcription.

Vaccinia virus temperature-sensitive mutants in the A28 gene produce non-infectious virions that bind to cells but are defective in entry

The vaccinia virus temperature-sensitive mutations Cts6 and Cts9 were mapped by marker rescue and DNA sequencing to the A28 gene. Cts6 and Cts9 contain an identical 2-bp deletion truncating the A28 protein and removing the fourth conserved cysteine near the C-terminus. Cts9 mutant virions produced at 40 °C were non-infectious and unable to cause cytopathic effect. However, the mutant A28 protein localized to purified mature virions (MV) at 31 °C and 40 °C. MV of Cts9 produced at 40 °C bound to cells but did not enter cells. Low pH treatment of Cts9-infected cells at 18 h p.i. failed to produce fusion from within at 40 °C, but gave fusion at 31 °C. Adsorption of Cts9 mutant virions to cells followed by low pH treatment showed a defect in fusion from without. The Cts9 phenotype suggests that the A28 protein is involved in both virus entry and cell–cell fusion, and supports the linkage between the two processes.

Temperature-sensitive mutants in the vaccinia virus 4b virion structural protein assemble malformed, transcriptionally inactive intracellular mature virions

Two noncomplementing vaccinia virus temperature-sensitive mutants, Cts8 and Cts26, were mapped to the A3L gene, which encodes the major virion structural protein, 4b. The two ts mutants display normal patterns of gene expression, DNA replication, telomere resolution, and protein processing during infection. Morphogenesis during mutant infections is normal through formation of immature virions with nucleoids (IVN) but appears to be defective in the transition from IVN to intracellular mature virus (IMV). In mutant infections, aberrant particles that have the appearance of malformed IMV accumulate. The mutant particles are wrapped in Golgi-derived membranes and exported from cells. Purified mutant particles are indistinguishable from wt particles in protein and DNA composition; however, they are defective in a permeabilized-virion-directed transcription reaction despite containing significant (Cts8) or even normal (Cts26) levels of specific transcription enzymes. These results indicate that the 4b protein is required for proper metamorphosis of IMV from IVN and that proper organization of the IMV structure is required to produce a transcriptionally active virion particle.

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.

Analysis of a Temperature-Sensitive Vaccinia Virus Mutant in the Viral mRNA Capping Enzyme Isolated by Clustered Charge-to-Alanine Mutagenesis and Transient Dominant Selection

We have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994)Proc. Natl. Acad. Sci. USA91, 4554–4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus capping enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis.In vitroassays of various capping enzyme activities revealed that in permeabilized virions, enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the capping enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis.

Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.

Mutations in ORF D13L and Other Genetic Loci Alter the Rifampicin Phenotype of Vaccinia Virus

Phenotypic analysis of vaccinia virus temperature-sensitive mutants identified several virus isolates that were either resistant or hypersensitive to rifampicin. Temperature-sensitive mutants 1085, 7743, 1085R, and 7743R were found to be rifampicin resistant (Rif R). Each virus contained at least one mutation in ORF D13L which resulted in a change of amino acid sequence. Analysis of each point mutation by marker rescue demonstrated that mutations gly to asp at residue 80, pro to phe at 458, lys to asn at 484, and ile to val at 485 conferred the resistance phenotype, while the mutation changing ser to leu at 176 did not affect the rifampicin phenotype. Temperature-sensitive mutants C6, C17, and C43 were found to be rifampicin hypersensitive. Only C43, however, was found to contain a lesion in ORF D13L. This mutation, ser to asn at 169 was confirmed by marker rescue to confer the hypersensitive phenotype. The results presented here along with those from previous reports show that known mutations conferring rifampicin resistance cluster at the C or N-terminus of D 13L while a mutation mapping at residue 169 confers rifampicin hypersensitivity. Further, the finding that the hypersensitive mutants C6 and C17 contain a wild-type D 13L ORF suggests a possible role of other vital functions in the interaction of rifampicin with vaccinia virus.

Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

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°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° and 40°C. With the DNA synthesis-defective vector virus, foreign gene expression controlled by the p11 DNA synthesis-dependent late promoter was inhibited at 40°C, but could be turned on by shift to 33°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.

Structure and expression of the vaccinia virus gene which prevents virus-induced breakdown of RNA

Three noncomplementing vaccinia virus temperature-sensitive mutants, ts4, ts22, and ts23, exhibit an abortive late phenotype characterized by the simultaneous cessation of protein synthesis, the breakdown of rRNA and viral mRNA, and an increase in intracellular concentrations of 2'-5'-linked oligoadenylates late during infection at the nonpermissive temperature (R.F. Pacha and R.C. Condit, J. Virol. 56:395-403, 1985; R.J. Cohrs, R.C. Condit, R.F. Pacha, C.L. Thompson, and O.K. Sharma, J. Virol. 63:948-951, 1989). We have identified the virus gene affected by the abortive late mutants, determined its DNA sequence, and analyzed its transcription. The gene resides in the HindIII A DNA fragment, it has a predicted coding capacity of 57 kilodaltons, and it is transcribed both early and late during infection. The early transcript of the abortive late gene is unusual; it contains a 426-nucleotide 5' untranslated region, and it must be synthesized by transcription through an early transcription termination signal which is located in the middle of the gene in a hairpin loop structure. DNA sequence and transcription analysis of two flanking genes is also presented.

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the is mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these is mutants is dependent upon the cell type.

Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication

Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection.

Identification of temperature-sensitive mutants of vaccinia virus that are defective in conversion of concatemeric replicative intermediates to the mature linear DNA genome.

Pulsed-field gel electrophoresis was used to screen temperature-sensitive mutants of vaccinia virus for the ability to convert replicated viral DNA into mature linear 185-kilobase hairpin-terminated genomes. Of 30 mutually noncomplementing mutants tested, 5 displayed a temperature-sensitive defect in the resolution of the telomere fusion configuration within concatemeric replicative intermediates, resulting in a failure to convert such intermediates to the linear monomeric genome. Adjacent genomic units in the concatemeric arrays generated in these mutants were arranged in both tandem and inverted orientations. The observation that four of the five mutants had a severe general defect in the synthesis of the late class of viral proteins suggests that at least one late protein is directly required to resolve the telomere fusion intermediate to hairpin termini. The identification of such telomere resolution proteins should be facilitated by genetic and molecular characterization of resolution-defective mutants, such as C63, in which late protein synthesis is not severely affected.

Intramolecular homologous recombination in cells infected with temperature-sensitive mutants of vaccinia virus

I have used a plasmid containing two copies of the Saccharmyces cerevisiae his3 gene to study intramolecular homologous recombination in vaccina virus-infected cells. Recombination of the plasmid was monitored by restriction enzyme digestion and Southern blot hybridization in cells infected with representatives from each of 32 complementation groups of temperature-sensitive mutants ts42 and ts17 did not replicate nor detectably recombine the input plasmid. All except one of the mutants that synthesized normal amounts of viral DNA and protein replicated and recombined the plasmid in a manner indistinguishable from wild-type virus. The remaining mutant, ts13, only poorly replicated and recombined the input plasmid. Thus, the processes of replication and recombination could not be separated by using this battery of mutants. Viral mutants defective in late protein synthesis were unable to resolve the vaccinia virus concatemer junction in plasmids but carried out intramolecular homologous recombination with plasmids as efficiently as did wild-type virus at the conditionally lethal temperature. This result distinguishes homologous recombination, which requires early gene products, from resolution of concatemer junctions, which requires additional late gene products.

Modulation of ppp(A2'p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus.

Activation of the ppp(A2'p)nA (2-5A)-dependent RNase was investigated during the abortive infection of BSC40 cells by a temperature-sensitive mutant of vaccinia virus, ts22. At the nonpermissive temperature, ts22 has an abortive late phenotype. At the onset of late-viral-gene expression, viral mRNA is degraded and rRNA is cleaved into discrete fragments in the absence of prior interferon treatment (R. F. Pacha and R. C. Condit, J. Virol. 56:395-403, 1985). Concomitant with rRNA cleavage, an increase in 2-5A occurred late during infection. Discrete 18S- and 28S-rRNA degradation products from BSC40 cells infected with ts22 at the nonpermissive temperature comigrated in denaturing agarose gels with rRNA cleaved fragments produced by the activation of 2-5A-dependent RNase in uninfected cells transfected with exogenous 2-5A. An increase in 2-5A levels and a similar discrete and characteristic degradation of rRNA were observed in BSC40 cells infected with wild-type vaccinia virus in the presence of isatin-beta-thiosemicarbazone. The results show that the ts22 lesion and the action of isatin-beta-thiosemicarbazone may affect the same pathway, leading to the activation of latent 2-5A-dependent RNase and resulting in indiscriminate RNA degradation and inhibition of viral replication.

Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase.

The phenotypic defects of three temperature-sensitive (ts) mutants of vaccinia virus, the ts mutations of which were mapped to the gene for one of the high-molecular-weight subunits of the virion-associated DNA-dependent RNA polymerase, were characterized. Because the virion RNA polymerase is required for the initiation of the viral replication cycle, it has been predicted that this type of mutant is defective in viral DNA replication and the synthesis of early viral proteins at the nonpermissive temperature. However, all three mutants synthesized both DNA and early proteins, and two of the three synthesized late proteins as well. RNA synthesis in vitro by permeabilized mutant virions was not more ts than that by the wild type. Furthermore, only one of three RNA polymerase activities that was partially purified from virions assembled at the permissive temperature displayed altered biochemical properties in vitro that could be correlated with its ts mutation: the ts13 activity had reduced specific activity, increased temperature sensitivity, and increased thermolability under a variety of preincubation conditions. Although the partially purified polymerase activity of a second mutant, ts72, was also more thermolabile than the wild-type activity, the thermolability was shown to be the result of a second mutation within the RNA polymerase gene. These results suggest that the defects in these mutants affect the assembly of newly synthesized polymerase subunits into active enzyme or the incorporation of RNA polymerase into maturing virions; once synthesized at the permissive temperature, the mutant polymerases are able to function in the initiation of subsequent rounds of infection at the nonpermissive temperature.

Marker rescue mapping of vaccinia virus temperature-sensitive mutants using overlapping cosmid clones representing the entire virus genome

A set of 11 overlapping cosmid clones of wild type (wt) vaccinia virus DNA was constructed. The clones together span almost the entire vaccinia virus genome. The clones were used to map temperature-sensitive ( ts) mutants of vaccinia virus by marker rescue. Map positions were obtained for mutants representing 29 of 32 complementation groups tested.

Fine structure marker rescue of temperature-sensitive mutations of vaccinia virus within a central conserved region of the genome.

Fine structure marker rescue involving the use of subfragments of vaccinia virus HindIII DNA fragments L, J, and H has been used to map the mutations in eight temperature-sensitive mutants of vaccinia virus representing four complementation groups. Comparison of their map locations with the positions of the open reading frames and RNA transcripts that have been mapped within this region has allowed the identification of one or two polypeptides as the temperature-sensitive gene product of each mutant.