Genetic and cell biological characterization of the vaccinia virus A30 and G7 phosphoproteins

The vaccinia virus proteins A30 and G7 are known to play essential roles in early morphogenesis, acting prior to the formation of immature virions. Their repression or inactivation results in the accumulation of large virosomes, detached membrane crescents, and empty immature virions. We have undertaken further study of these proteins to place them within the context of the F10 kinase, the A14 membrane protein, and the H5 phosphoprotein, which have been the focus of previous studies within our laboratory. Here we confirm that both A30 and G7 undergo F10 kinase-dependent phosphorylation in vivo and recapitulate that modification of A30 in vitro. Although the detached crescents observed upon loss of A30 or G7 echo those seen upon repression of A14, no interaction between A30/G7 and A14 could be detected. We did, however, determine that the A30 and G7 proteins are unstable during nonpermissive tsH5 infections, suggesting that the loss of A30/G7 is the underlying cause for the formation of lacy or curdled virosomes. We also determined that the temperature-sensitive phenotype of the Cts11 virus is due to mutations in two codons of the G7L gene. Phenotypic analysis of nonpermissive Cts11 infections indicated that these amino acid substitutions compromise G7 function without impairing the stability of either G7 or A30. Utilizing Cts11 in conjunction with a rifampin release assay, we determined that G7 acts at multiple stages of virion morphogenesis that can be distinguished both by ultrastructural analysis and by monitoring the phosphorylation status of several viral proteins that undergo F10-mediated phosphorylation.     

Vaccinia virus A6L encodes a virion core protein required for formation of mature virion

Vaccinia virus A6L is a previously uncharacterized gene that is conserved in all sequenced vertebrate poxviruses. Here, we constructed a recombinant vaccinia virus encoding A6 with an epitope tag and showed that A6 was expressed in infected cells after viral DNA replication and packaged in the core of the mature virion. Furthermore, we showed that A6 was essential for vaccinia virus replication by performing clustered charge-to-alanine mutagenesis on A6, which resulted in two vaccinia virus mutants (vA6L-mut1 and vA6L-mut2) that displayed a temperature-sensitive phenotype. At 31 degrees C, both mutants replicated efficiently; however, at 40 degrees C, vA6L-mut1 grew to a low titer, while vA6L-mut2 failed to replicate. The A6 protein expressed by vA6L-mut2 exhibited temperature-dependent instability. At the nonpermissive temperature, vA6L-mut2 was normal at viral gene expression and viral factory formation, but it was defective for proteolytic processing of the precursors of several major virion proteins, a defect that is characteristic of a block in virion morphogenesis. Electron microscopy further showed that the morphogenesis of vA6L-mut2 was arrested before the formation of immature virion with nucleoid and mature virion. Taken together, our data show that A6 is a virion core protein that plays an essential role in virion morphogenesis.     

Temperature-sensitive mutants with lesions in the vaccinia virus F10 kinase undergo arrest at the earliest stage of virion morphogenesis.

Vaccinia virus encodes two protein kinases; the B1 kinase is expressed early and appears to play a role during DNA replication, whereas the F10 kinase is expressed late and is encapsidated in virions. Here we report that the F10 kinase gene is the locus affected in a complementation group of temperature-sensitive mutants composed of ts15, ts28, ts54, and ts61. Although these mutants have a biochemically normal phenotype at the nonpermissive temperature, directing the full program of viral gene expression, they fail to form mature virions. Electron microscopic analysis indicates that morphogenesis undergoes arrest at a very early stage, prior to the formation of membrane crescents or immature virions. An essential role for the F10 protein kinase in orchestrating the onset of virion assembly is implied.     

Vaccinia virus morphogenesis: a13 phosphoprotein is required for assembly of mature virions

The 70-amino-acid A13L protein is a component of the vaccinia virus membrane. We demonstrate here that the protein is expressed at late times of infection, undergoes phosphorylation at a serine residue(s), and becomes encapsidated in a monomeric form. Phosphorylation is dependent on Ser40, which lies within the proline-rich motif SPPP. Because phosphorylation of the A13 protein is only minimally affected by disruption of the viral F10 kinase or H1 phosphatase, a cellular kinase is likely to be involved. We generated an inducible recombinant in which A13 protein expression is dependent upon the inclusion of tetracycline in the culture medium. Repression of the A13L protein spares the biochemical progression of the viral life cycle but arrests virion morphogenesis. Virion assembly progresses through the formation of immature virions (IVs); however, these virions do not acquire nucleoids, and DNA crystalloids accumulate in the cytoplasm. Further development into intracellular mature virions is blocked, causing a 1,000-fold decrease in the infectious virus yield relative to that obtained in the presence of the inducer. We also determined that the temperature-sensitive phenotype of the viral mutant Cts40 is due to a nucleotide transition within the A13L gene that causes a Thr(48)-->Ile substitution. This substitution disrupts the function of the A13 protein but does not cause thermolability of the protein; at the nonpermissive temperature, virion morphogenesis arrests at the stage of IV formation. The A13L protein, therefore, is part of a newly recognized group of membrane proteins that are dispensable for the early biogenesis of the virion membrane but are essential for virion maturation.     

Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions

Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigmaNS and core protein mu2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigmaNS and mu2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigmaNS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigmaNS-mutant virus tsE320, sigmaNS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigmaNS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigmaNS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigmaNS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.     

Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus.

The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32Pi into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.     

The phenotype in vitro and in infected cells of herpes simplex virus 1 alpha trans-inducing factor (VP16) carrying temperature-sensitive mutations introduced by substitution of cysteines.

alpha trans-inducing factor (alpha TIF, VP16, Vmw65) is an essential structural protein of herpes simplex virus, being required for virion assembly. The protein also forms complexes with host proteins and a response element and transactivates the alpha genes which carry this element. The protein contains an acidic carboxyl terminus required for transactivation and a much larger amino-terminal domain required for promoter recognition. We report the first set of temperature-sensitive (ts) mutations deliberately introduced into the protein by substitution of the cysteine codons with those specifying glycine at positions 78, 102, and 176, either singly or in combinations. We report the following results. (i) All mutated proteins synthesized in vitro formed complexes with the DNA response element at room temperature. However, the mutant with the triple substitution and two mutants with substitutions in two of the three cysteines exhibited a ts phenotype at 33 and 37 degrees C, and one exhibited a ts phenotype only at 37 degrees C. (ii) Replacement of wild-type alpha TIF with genes carrying substitutions in any two cysteines conferred a ts phenotype for replication at 39.5 degrees C. Shift-down experiments indicated that the 10(4)- to 10(5)-fold reduction in virus yield at the nonpermissive temperature was due to the disfunction of alpha TIF late in infection, presumably in virion maturation. (iii) The alpha TIF expressed in cells infected with mutant viruses exhibited the same ts phenotype in protein-DNA complex formation as those expressed in vitro from mutated plasmids. Although the virus carrying the alpha TIF substitutions at Cys-102 and Cys-176 failed to induce a reporter gene linked to the alpha 4 promoter at 39.5 degrees C, it replicated as well as the parent virus in cells maintained for the first 10 h of infection at 39.5 degrees C. We conclude the following. (i) Formation of DNA-protein complexes containing alpha TIF is a poor prognosticator of alpha TIF function. (ii) The data presented here and in the literature strongly support the hypothesis that the secondary structure of the alpha TIF is very sensitive to deletions or insertions which probably affect the interaction of alpha TIF with both viral proteins in the virion and cellular proteins during infection. As a consequence, deletion-insertion mutagenesis may not shed useful information on the role of transactivating function of alpha TIF in infection. (iii) Since cysteines may play a role in stabilizing the secondary structure of proteins, substitutions of cysteines may be a powerful technique for site-specific construction of ts mutants in essential viral proteins.     

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).     

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.     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.     

Genetic analysis of the vaccinia virus I6 telomere-binding protein uncovers a key role in genome encapsidation

The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle.     

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.     

Molecular genetics of herpes simplex virus. VIII. further characterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of the viral reproductive cycle.

Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39 degrees C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39 degrees C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis.     

The vaccinia virus I1 protein is essential for the assembly of mature virions.

The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis.