A temperature-sensitive mutation in a herpes simplex virus type 1 gene required for viral DNA synthesis maps to coordinates 0.609 through 0.614 in UL

ts701 is a temperature-sensitive mutant of herpes simplex virus type 1 strain KOS induced by hydroxylamine mutagenesis (C.T. Chu, D. S. Parris, R. A. F. Dixon, F. E. Farber, and P. A. Schaffer, Virology 98:168-181, 1979). In the present study, the mutation rendering ts701 temperature sensitive was mapped to coordinates 0.609 through 0.614 in the UL region of the genome. At the nonpermissive temperature, ts701 (i) failed to induce the synthesis of viral DNA, (ii) exhibited a dramatically reduced ability to drive replication of a plasmid containing the herpes simplex virus origin of viral DNA synthesis, oriS, (iii) generated no viral polypeptides of the late (gamma 2) kinetic class, and (iv) produced virions with electron-translucent cores. Northern (RNA) blot hybridization demonstrated that two mRNAs--one of the beta kinetic class and one of the gamma kinetic class--hybridized to a 1.3-kilobase viral DNA fragment that rescued the mutation in ts701. Based on the phenotype and mapping of ts701, it is likely that its mutation lies in the gene specifying the 65,000-Mr DNA-binding protein (65KDBP) recently described by Marsden et al. (H.S. Marsden, M.E.M. Campbell, L. Haarr, M. C. Frame, D. S. Parris, M. Murphy, R. G. Hope, M. T. Muller, and C. M. Preston, J. Virol. 61:2428-2437, 1987).     

Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly.

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).     

Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2.

BHK cells infected with the temperature-sensitive mutant ts13 of herpes simplex virus type 2 at a nonpermissive temperature lack the alkaline nuclease activity, which is induced by the mutant at a permissive temperature and by wild-type virus at either temperature. For ts13, enzyme activity could be induced by a temperature shift to permissive conditions, but not in the presence of cycloheximide. After a shift from permissive to nonpermissive conditions in the presence of cycloheximide, the activity was stable in wild-type, but not in mutant-infected, cells. After extensive purification, the wild-type nuclease was fourfold more heat stable in the presence of substrate than was the mutant enzyme. Mixtures of both purified enzymes showed the predicted intermediate stabilities. The results strongly suggest that the enzyme is virus coded and that the mutant possesses a lesion in the structural gene of the enzyme.     

Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus.

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848, 1992). By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Alb1, maps to the N gene. Sequencing of the Alb1 N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Alb1 mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.     

Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability.

The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosylase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia virus ts4149-infected cells in order to assess the recombination events that allowed virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.     

Phenotypic revertants of temperature-sensitive M protein mutants of vesicular stomatitis virus: sequence analysis and functional characterization.

Twenty-five spontaneous temperature-stable revertants of four different temperature-sensitive (ts) M protein mutants (complementation group III: tsG31, tsG33, tsO23, and tsO89) were sequenced and tested for their ability to inhibit vesicular stomatitis virus RNA polymerase activity in vitro. Consensus sequences of the coding region of each M protein gene were determined, using total viral RNA as template. Fifteen different sequences were found among the 25 revertants; 14 differed from their ts parent by a single amino acid (one nucleotide), and 1 differed by two amino acids (two nucleotides). Amino acids were altered in various positions between residues 64 and 215, representing over 60% of the polypeptide chain. Resequencing of the Glasgow and Orsay wild types and the four ts mutants confirmed previously published differences (Y. Gopalakrishana and J. Lenard, J. Virol., 56:655-659, 1985), and one or two additional differences were found in each. The relative charges of the revertant M proteins, as determined by nonequilibrium pH gradient electrophoresis, were consistent with the deduced sequences in every case. The ability of each revertant M protein to inhibit the RNA polymerase activity of nucleocapsids prepared from its parent ts mutant was also tested. Only 13 of the 25 revertants had M protein with high (wild type-like) polymerase-inhibiting activity, while 5 had low (ts-like) activity, and 7 had intermediate activity, demonstrating that this property is not an essential concomitant of the temperature-stable phenotype. It is concluded that the high reversion frequency observed for these mutants arises from a very high incidence of pseudoreversion, i.e., many different molecular changes can repair the ts phenotype.     

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.     

A temperature-sensitive lesion in the small subunit of the vaccinia virus-encoded mRNA capping enzyme causes a defect in viral telomere resolution.

Using pulsed-field gel electrophoresis, we demonstrated that the temperature-sensitive (ts) conditional lethal mutant ts9383 is, at the nonpermissive temperature, defective in the resolution of concatemeric replicative intermediate DNA to linear 185-kb monomeric DNA genomes. The resolution defect was shown to be the result of a partial failure of the mutant virus to convert the replicated form of the viral telomere to hairpin termini. In contrast to other mutants of this phenotype, pulse-labeling of viral proteins at various times postinfection revealed no obvious difference in the quantity or temporal appearance of members of the late class of polypeptides. Using the marker rescue technique, we localized the ts lesion in ts9383 to an approximately 1-kb region within the HindIII D fragment. Both the ts phenotype and the resolution defect were shown to be caused by a single-base C----T point mutation resulting in the conversion of the amino acid proline to serine in codon 23 of open reading frame D12. This gene encodes a 33-kDa polypeptide which is known to be the small subunit of the virus-encoded mRNA capping enzyme (E. G. Niles, G. J. Lee-Chen, S. Shuman, B. Moss, and S. S. Broyles, Virology 172:513-522, 1989). The data are consistent with a role for this capping enzyme subunit during poxviral telomere resolution.     

Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins.

Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein. We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T. Y. Shih, M. O. Weeks, H. A. Young, and E. M. Scolnick, J. Virol. 31:546-556, 1979). The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C. The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21. A second difference from the published sequence for wt v-Ki-ras (N. Tsuchida, T. Ryder, and E. Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found. However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37. Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed.     

Alterations in Substrate Specificity and Physicochemical Properties of Deoxythymidine Kinase of a Drug-Resistant Herpes Simplex Virus Type 1 Mutant

The deoxythymidine kinase (dTK) activity of a 5-methoxymethyldeoxyuridine-resistant mutant (MMdU(r)-20) of herpes simplex virus type 1 was compared with that of the parental wild-type (WT) virus. The dTK activity induced by the mutant was consistently less than that induced by the WT virus, was inhibited by antibody specific for herpes simplex virus dTK, and was more thermostable than the WT dTK. Further, it was inhibited to a lesser degree than the WT dTK by the nucleoside analogs MMdU and arabinosylthymine (araT), which suggests that one of the effects of the mutation was a selective alteration in substrate recognition by the dTK. The loss of ability to inhibit the mutant dTK by E-(2)-5-bromovinyldeoxyuridine was not as great as that seen with araT and MMdU. This agrees well with our previous observation that the MMdU(r)-20 mutant of herpes simplex virus is only partially resistant to this analog, as compared with araT and MMdU (V. Veerisetty and G. A. Gentry, Virology 114:576-579, 1981). [2-(14)C]araT was used to explore further the resistance to araT. Extracts of cells infected with the mutant, although producing a small amount of [(14)C]araTMP, were unable to produce [(14)C]araTTP, in contrast to extracts of cells infected with the WT virus. Both extracts, however, produced [(14)C]dTTP from [(14)C]deoxyribosylthymine. Finally, the ability of the extracts to phosphorylate [(14)C]dTMP was examined. It was found that this activity was greatly reduced relative to dTK activity in the case of the mutant. These findings suggest that a mutation in the dTK polypeptide has affected recognition not only of nucleoside substrates but of the nucleotide substrate dTMP as well, which agrees with the suggestion of Chen et al. that both activities are located on the same polypeptide (M. S. Chen and W. H. Prusoff, J. Biol. Chem. 253:1325-1327, 1978; M. S. Chen, J. Walker, and W. H. Prusoff, J. Biol. Chem. 254:10747-10753, 1979; M. S. Chen, W. P. Summers, J. Walker, W. C. Summers, and W. H. Prusoff, J. Virol. 30:942-945, 1979).     

Uukuniemi virus glycoproteins accumulate in and cause morphological changes of the Golgi complex in the absence of virus maturation.

We have studied the transport of the Uukuniemi virus membrane glycoproteins in baby hamster kidney and chick embryo cells by using a temperature-sensitive mutant (ts12). Uukuniemi virus assembles in the Golgi complex, where both glycoproteins G1 and G2 and nucleocapsid protein N accumulate (E. Kuismanen, B. B?ng, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984). At the restrictive temperature (39 degrees C), the glycoproteins of ts12 were transported to the Golgi complex as in wild-type, virus-infected cells, whereas the nucleocapsid protein failed to accumulate there. Pulse-chase labeling followed by immunoprecipitation and treatment with endo-beta-N-acetylglucosaminidase H showed that G1 synthesized at 39 degrees C in ts12-infected cells had an altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a lack of terminal glycosylation. The typical Uukuniemi virus-induced vacuolization and expansion of the Golgi complex could be seen also in ts12-infected cells at 39 degrees C, although no virus particles were formed. This suggests that the morphological changes were induced by the Uukuniemi virus glycoproteins. In wild-type virus- or ts12-infected cells, G1 and G2 could not be chased out from the Golgi complex even after 6 h of treatment with cycloheximide. The glycoproteins were thus retained in the Golgi even under conditions when no virus maturation took place and when nucleocapsids did not accumulate in the Golgi region. Accordingly, the glycoproteins of Uukuniemi virus were found to have properties resembling those of Golgi-specific proteins. This virus model system may be useful in studying the synthesis and transport of membrane proteins that are transported to and retained in the Golgi.     

Novel approach to the development of effective H5N1 influenza A virus vaccines: use of M2 cytoplasmic tail mutants

Outbreaks of highly pathogenic H5N1 influenza viruses in avian species began in Asia and have since spread to other continents. Concern regarding the pandemic potential of these viruses in humans is clearly warranted, and there is an urgent need to develop effective vaccines against them. Previously, we and others demonstrated that deletions of the M2 cytoplasmic tail caused a growth defect in A/WSN/33 (H1N1) influenza A virus in vitro (K. Iwatsuki-Horimoto, T. Horimoto, T. Noda, M. Kiso, J. Maeda, S. Watanabe, Y. Muramoto, K. Fujii, and Y. Kawaoka, J. Virol. 80:5233-5240, 2006; M. F. McCown and A. Pekosz, J. Virol. 79:3595-3605, 2005; M. F. McCown and A. Pekosz, J. Virol. 80:8178-8189, 2006). We therefore tested the feasibility of using M2 tail mutants as live attenuated vaccines against H5N1 virus. First we generated a series of highly pathogenic H5N1 (A/Vietnam/1203/04 [VN1203]) M2 cytoplasmic tail deletion mutants and examined their growth properties in vitro and in vivo. We found that one mutant, which contains an 11-amino-acid deletion from the C terminus (M2del11 virus), grew as well as the wild-type virus but replicated in mice less efficiently. We then generated a recombinant VN1203M2del11 virus whose hemagglutinin (HA) gene was modified by replacing sequences at the cleavage site with those of an avirulent type of HA (M2del11-HAavir virus). This M2del11-HAavir virus protected mice against challenge with lethal doses of homologous (VN1203; clade 1) and antigenically distinct heterologous (A/Indonesia/7/2005; clade 2) H5N1 viruses. Our results suggest that M2 cytoplasmic tail mutants have potential as live attenuated vaccines against H5N1 influenza viruses.     

Restricted replication of mouse hepatitis virus A59 in primary mouse brain astrocytes correlates with reduced pathogenicity.

Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342-infected cells was similar. Electron microscopy showed in wild-type virus-infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild-type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild-type virus.     

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.