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.     

Comparison of upstream sequence requirements for positive and negative regulation of a herpes simplex virus immediate-early gene by three virus-encoded trans-acting factors.

Using a short-term cotransfection system with recombinant chloramphenicol acetyltransferase (CAT) target genes and intact genes for regulatory proteins, we previously demonstrated that expression from the promoter-regulatory region of the gene for the immediate-early 175,000-molecular-weight (IE175K) protein of herpes simplex virus type 1 was subject to trans-acting effects by three different virus-encoded components. In the present work we have attempted to delineate the upstream cis-acting requirements within the IE175K promoter-regulatory region for stimulation by the late structural protein Vmw65, stimulation by the IE110K protein, and repression by its own gene product, the IE175K protein. Our results augment previous reports of others by demonstrating that a construct containing only the single TAATGARAT consensus sequence, TAATGGAAT, between -115 and -106 was efficiently induced by Vmw65. Deletion to -108 effectively abolished the response to Vmw65. However, this latter construct remained responsive to IE110K stimulation and was induced as efficiently as the parental construct which contained sequences to -1900. Furthermore, not only basal levels of expression, but also Vmw65 activation of the parental construct and deletion mutants delta 380, delta 330, delta 300, and delta 160 and IE110K-activated expression of the delta 108 construct were all subject to dominant repression by the IE175K protein. Finally, we show that expression from each of the deletions was open to stimulation by linkage to the simian virus 40 enhancer region. Enhancer-stimulated expression from each construct, including the -108 deletion, was efficiently repressed by the IE175K protein. In contrast, expression from the simian virus 40 enhancer when linked to its own promoter was unaffected by IE175K. These results place sequence requirements for both IE110K stimulation and IE175K autoregulation within the minimal promoter region -108 to +30, separate from the major requirements for Vmw65 activation located further upstream.     

Recombinants between herpes simplex virus types 1 and 2: analyses of genome structures and expression of immediate early polypeptides.

Recombinants between temperature-sensitive mutants of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) were constructed. Using restriction endonucleases, we analyzed the genome composition of 17 intertypic recombinants and detected crossovers in every region of the genome. The virion DNA of one recombinant appeared to be largely "frozen" in two of the four possible genome arrangements of HSV. Knowledge of the genome structures of recombinants enabled us to physically map immediate early polypeptides. We present evidence that the immediate early polypeptide Vmw IE 110 of HSV-1 and its functionally equivalent polypeptide, Vmw IE 118, of HSV-2 may map in the repetitive sequences bounding the long unique region of HSV.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.     

Physical mapping of two herpes simplex virus type 1 host shutoff loci: rescue of each ts mutation occurs with two unique cloned regions of the viral genome.

Two complementing temperature-sensitive (ts) herpes simplex virus type 1 (HSV-1) mutants, PAA1rts1 and ts199, were defective in viral DNA synthesis and in the shutoff of cellular macromolecular synthesis at 39.5 degrees C, the nonpermissive temperature. PAA1sts1 and PAA1rts1+ recombinants and PAA1rts1+ revertants were used to examine the contributions of the PAA1r mutation and the ts1 mutation of PAA1rts1 in affecting the levels of viral and cellular DNA synthesized at 34 and 39.5 degrees C. The results of this study suggests an interaction between the viral DNA polymerase and the ts1+ gene product during HSV-1 DNA replication and possibly in the inhibition of host DNA synthesis by HSV-1. Physical mapping of the ts mutations present in ts199 and the PAA1sts1 recombinant ts1-8 were performed by intratypic marker rescue experiments. Surprisingly, both the ts1-8 and ts199 mutations were rescued by two cloned fragments: ts1-8 by BglII-K (map coordinates 0.095 to 0.163) and BglII-I (map coordinates 0.314 to 0.417), while ts199 was rescued by BglII-K and BglII-O (map coordinates 0.163 to 0.197). In more refined mapping experiments, the regions between coordinates 0.347 to 0.378 and 0.126 to 0.163 were able to rescue the ts1-8 mutation. Southern hybridization analysis confirmed that the fragments that rescued ts1-8 and those that rescued ts199 had homology, as predicted by the physical mapping results.     

Physical mapping of paar mutations of herpes simplex virus type 1 and type 2 by intertypic marker rescue.

Mutations (paar) in herpes simplex virus (HSV) which confer resistance to phosphonoacetic acid involve genes associated with virus-induced DNA polymerase activity. Two mutants of HSV (HSV-1 tsH and HSV-2 ts6) produce a thermolabile DNA polymerase activity. In this study, the ts lesions present in these mutants and those present in two independent phosphonoacetic acid-resistant mutants of HSV-1 and HSV-2 (paar-1 and paar-2) have been physically mapped by restriction endonuclease analysis of recombinants produced between HSV-1 and HSV-2 by intertypic marker rescue. All four mutations mapped within a 3.3-kilobase pair region around map unit 40. The accuracy of the method is reflected by the mapping results for tsH and paar-2, which were found to lie in the same 1.3-kilobase pair region. paar-1 was found to lie to the right of ts6. Virus-induced DNA polymerase is thought to have a molecular weight of 150,000, necessitating a gene with a coding capacity of 4.6 kilobase pairs. The four mutations mapped in this study all lie within a region smaller than this, but the results do not yet prove that all four lesions reside in this or any single gene.     

Molecular genetics of herpes simplex virus. VII. Characterization of a temperature-sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of gamma polypeptides.

We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 39 degrees C relative to that at 33.5 degrees C was 5 X 10(-6). The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that alpha and beta polypeptides were produced, whereas most gamma polypeptides were either reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43, and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 10(3) base pairs between 0.383 and 0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 molecular weight), which was previously identified as a beta polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells.     

Genetic and phenotypic analysis of herpes simplex virus type 1 mutants conditionally resistant to immune cytolysis

Nine temperature-sensitive (ts) mutants of herpes simplex virus type 1 selected for their inability to render cells susceptible to immune cytolysis after infection at the nonpermissive temperature have been characterized genetically and phenotypically. The mutations in four mutants were mapped physically by marker rescue and assigned to functional groups by complementation analysis. In an effort to determine the molecular basis for cytolysis resistance, cells infected with each of the nine mutants were monitored for the synthesis of viral glycoprotein in total cell extracts and for the presence of these glycoproteins in plasma membranes. The four mutants whose ts mutations were mapped were selected with polypeptide-specific antiserum to glycoproteins gA and gB; however, three of the four mutations mapped to DNA sequences outside the limits of the structural gene specifying these glycoproteins. Combined complementation and phenotypic analysis indicates that the fourth mutation also lies elsewhere. The ts mutations in five additional cytolysis-resistant mutants could not be rescued with single cloned DNA fragments representing the entire herpes simplex virus type 1 genome, suggesting that these mutants may possess multiple mutations. Complementation tests with the four mutants whose ts lesions had been mapped physically demonstrated that each represents a new viral gene. Examination of mutant-infected cells at the nonpermissive temperature for the presence of viral glycoproteins in total cell extracts and in membranes at the cell surface demonstrated that (i) none of the five major viral glycoproteins was detected in extracts of cells infected with one mutant, suggesting that this mutant is defective in a very early function; (ii) cells infected with six of the nine mutants exhibited greatly reduced levels of all the major viral glycoproteins at the infected cell surface, indicating that these mutants possess defects in the synthesis or processing of viral glycoproteins; and (iii) in cells infected with one mutant, all viral glycoproteins were precipitable at the surface of the infected cell, despite the resistance of these cells to cytolysis. This mutant is most likely mutated in a gene affecting a late stage in glycoprotein processing, leading to altered presentation of glycoproteins at the plasma membrane. The finding that the synthesis of both gB and gC was affected coordinately in cells infected with six of the nine mutants suggests that synthesis of these two glycoproteins, their transport to the cell surface, or their insertion into plasma membranes is coordinately regulated.