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A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.

The requirements for expression of genes under the control of early (alkaline exonuclease) and late (VP5) herpes simplex virus type 1 (HSV-1) gene promoters were examined in a transient expression assay, using the bacterial chloramphenicol acetyltransferase gene as an expression marker. Both promoters were induced, resulting in the production of high levels of the enzyme upon low-multiplicity infection by HSV-1. S1 nuclease analysis of hybrids between RNA isolated from infected cells containing HSV-1 promoter constructs and marker gene DNA demonstrated normal transcriptional initiation of the marker gene directed by the viral promoters. Viral DNA sequences no more than 125 bases 5' of the putative transcriptional cap site were sufficient for maximum activity of the late promoter. In contrast to expression controlled by the early gene, the late promoter was not active at a measurable level in uninfected cells until DNA sequences between 75 and 125 bases 5' of the transcriptional cap site were deleted. Cotransfection of cells with the expression marker controlled by HSV promoters and a cosmid containing HSV alpha (immediate-early) genes indicated that full expression of both early and late promoters requires the same virus-induced host cell modifications. Inhibition of viral DNA synthesis results in an increased rate of transient expression of marker genes under control of either early or late promoters in contrast to the situation in normal virus infection. These data provide evidence that the normal course of expression of late HSV genes involves negative modulation of potentially active promoters in the infected cell.

The basis for the inability of the macroplaque (MP) strain of herpes simplex virus type 1 to express mature glycoprotein C (gC) was examined. RNA transfer (Northern) blot analysis with hybridization probes from the region of the herpes simplex virus type 1 DNA known to encode the gC gene indicated that gC mRNA was produced in MP-infected HeLa cells at levels relative to other mRNAs comparable with that seen in KOS-infected cells. Comparative nucleotide sequence analysis of the gC gene from the MP and KOS strains, coupled with the results of recently reported marker rescue experiments, indicates that the inability of MP to produce gC is due to a frameshift mutation in the gC-coding sequence. Because two different (out-of-phase) open reading frames overlap the gC-coding sequence in the region of the mutation, MP mRNA can encode two gC-related polypeptides. Two polypeptides of the predicted size and precipitable by anti-gC antibodies were produced by in vitro translation of MP mRNA. These polypeptides have not been detected in extracts from infected cells with the same antibodies. Comparative nucleotide sequence analyses led to several corrections in the published sequence for the gC gene and the 17,800-molecular-weight polypeptide gene just to the right in KOS DNA. These relatively minor effects on the predicted amino code sequence of gC are tabulated.

The two partially colinear 6-kilobase (kb) and 1.5-kb mRNAs mapping between 0.23 and 0.27 map units on the herpes simplex virus type 1 genome were precisely located. The 5' end of the 6-kb mRNA was located 28 bases downstream of the sequence ATATATT and was 10 bases to the left of the BamHI site at 0.268. This position is ca. 90 bases to the left of our earlier reported sequence (R. J. Frink, K. G. Draper, and E. K. Wagner, Proc. Natl. Acad. Sci. U.S.A. 78:6139-6143, 1981). We used a polyclonal antibody made against purified herpes simplex virus type 1 VP5 to demonstrate that the 155,000-dalton translation product of the 6-kb mRNA is this capsid protein. The antibody did not react with the 35,000-dalton translation product of the 1.5-kb mRNA. We also confirmed our identification of VP5 as the translation product of the 6-kb mRNA by comparison of tryptic peptides of the in vitro-translated protein and authentic VP5.

Four partially overlapping mRNAs (1.9, 2.3, 3.9, and 4.5 kilobases [kb]) were located between 0.16 and 0.19 map units on the herpes simplex virus type 1 genome. Their direction of transcription was found to be from right to left. The 2.3-kb mRNA was found to be early (beta), whereas the others were late (beta gamma). Partial sequence analysis of the DNA encoding these genes indicated that the promoter for the 2.3-kb mRNA shares structural features with other early (beta) promoters. In vitro translation of hybrid-selected mRNA indicated that among the proteins these mRNAs encode are an 82,000-dalton (d) polypeptide reactive with a monoclonal antibody against herpes simplex virus type 2 alkaline exonuclease and a 50,000-d polypeptide weakly reactive with a polyclonal antibody made against the capsid protein VP19C. Further experiments suggested that the 2.3-kb mRNA encodes the 82,000-d polypeptide, whereas one (or both) of the larger mRNAs encodes the 50,000-d protein. A novel finding was that the 1.9-kb mRNA appears to share part of the translational reading frame for alkaline exonuclease, but any polypeptide it encodes does not react with the monoclonal antibody to this enzyme.

We described the detailed characterization and high-resolution mapping of nine herpes simplex virus type 1 mRNAs encoded in EcoRI fragment I. Four of these mRNAs are partially colinear and encode the same sized polypeptide in vitro. Nucleotide sequence analysis of the DNA around the 5' ends of these mRNAs suggested that the larger may encode a small (ca. 100-dalton) polypeptide not resolvable by in vitro translation.

A detailed sequence analysis of the herpes simplex virus type 1 (HSV-1) and HSV-2 DNA encoding the alkaline exonuclease mRNA clusters has been completed. Three partially colinear mRNAs (2.3, 1.9, and 0.9 kilobases) are completely encoded within the DNA sequence presented. The putative promoter regions of the transcripts were inserted upstream of a plasmid-borne chloramphenicol acetyl transferase (CAT) gene and assayed for their ability to induce transcription of the CAT gene upon low multiplicity of infection with HSV in transient expression assays. We conclude that the expression of all three transcripts appear to be controlled by individual promoters. The 2.3-kilobase mRNA contains an open translational reading frame sufficient to encode 626 amino acids for the HSV-1 alkaline exonuclease enzyme; this value is 620 amino acids for HSV-2. A comparison of the predicted amino acid sequences of the HSV-1 and HSV-2 alkaline exonuclease enzymes revealed significant amino acid differences in the N-terminal portions of the two proteins; however, computer analyses suggest that the three-dimensional structures of the HSV-1 and HSV-2 nuclease enzymes are very similar. The 0.9-kilobase mRNA contains an open reading frame which shares a small amount of out-of-phase overlap with the C-terminal portion of the alkaline nuclease open reading frame. This open reading frame has the capacity to encode a 96-amino-acid polypeptide (10,500 daltons).

High-resolution transcription mapping localized a spliced 2.7-kilobase herpes simplex virus type 1 mRNA. The 4-kilobase intron of this transcript encodes a nested set of transcripts on the opposite DNA strand. The nucleotide sequence of the DNA encoding the left-hand and right-hand exons of the spliced transcript was determined, and the salient features are presented here. Of major interest is that both exons contained regions within several hundred bases of the splice donor and acceptor sites which showed homology to two regions of the Epstein-Barr virus genome, which are themselves 3 kilobases apart. The spliced herpes simplex virus transcript encoded a translational reading frame which could encode a protein with an approximate size of 75,000 daltons. This value is in agreement with in vitro translation data. The predicted amino acid sequence of the herpes simplex virus protein had significant homology with putative amino acid sequences encoded by the homologous Epstein-Barr virus DNA sequences.