Search

We have used DNA bound to cellulose to isolate and translate in vitro herpes simplex virus type 1 (HSV-1) mRNA's encoded by HindIII fragment L (mapping between 0.592 and 0.647), and 8.450-base-pair (8.45-kb) portion of the long unique region of the viral genome. Readily detectable, late mRNA's 2.7 and 1.9 kb in size encoding 69,000- and 58,000-dalton polypeptides, respectively, were isolated. A very minor late mRNA family composed of two colinear forms, one 2.6 kb and one 2.8 kb, was isolated and found to encode only an 85,000-dalton polypeptide. A major early mRNA, 1.8 kb in size encoding a 64,000-dalton polypeptide, was also isolated. High-resolution mapping of these mRNA's by using S1 nuclease and exonuclease VII digestion of hybrids between them and 5' and 3' end-labeled DNA fragments from the region indicated that the major early mRNA contained no detectable splices, and about half of its 3' end was complementary to the 3' region of the very minor 2.6- to 2.8-kb mRNA's encoded on the opposite strand. These mRNA's also contained no detectable splices. The major late 2.7-kb mRNA was found to be a family made up of members with no detectable splices and members with variable-length (100 to 300 bases) segments spliced out very near (ca. 50 to 100 bases) the 5' end.

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 sequences of the DNAs encoding the 5' ends of one early and one late herpes simplex virus type 1 mRNA were analyzed, and the 5' ends of these mRNA species were precisely located. Neither mRNA species is spliced and the noncoding strand of the DNA contains recognizable T-A-T-A and C-A-T boxes upstream from their respective 5' ends. The early mRNA was efficiently transcribed by a commercially available uninfected cell lysate system, but the late mRNA was not. This difference between early and late mRNAs appears to be general in this virus.

We precisely localized the coding region and determined the nucleotide sequence of a 1.2-kilobase beta herpes simplex virus type 1 mRNA which underlies the 3' region of the 5.2-kilobase beta mRNA mapping in HindIII fragment K. This mRNA, which lacks readily detectable splices, has its own promoter by the criteria of identification of putative herpes simplex virus type 1 control sequences and in vitro transcription by a Manley polymerase system.

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.

The homology between herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, respectively) DNA between 0.58 and 0.674 map units was compared by Southern and dot blot analysis with DNA of one type of virus as a hybridization probe against the other type. Regions of high homology were interspersed with regions of detectably lower homology. However, only one region (between 0.647 and 0.653 map units) contained few or no homologous sequences. In situ RNA blot hybridization demonstrated that the mRNA species transcribed in the right-hand portion of the region are homologous between HSV-1 and HSV-2, as was previously found for the left-hand portion. A 2.7-kilobase HSV-2 transcript in the right-hand portion of the studied region was clearly that encoding HSV-2 glycoprotein C. Comparative nucleotide sequence analysis of specific regions demonstrated that homologous translational reading frames could be identified in the virus types. This analysis also demonstrated that homology could be abruptly lost outside such reading frames. Comparison of regions of homology with published HSV-1 transcription maps suggests that there can also be large divergence within translational reading frames. Some, but not complete, sequence homology was seen in the putative promoter sequence for the 730-base HSV-1 mRNA mapping to the right of glycoprotein C and the corresponding HSV-2 DNA. This suggests that the rather strict conservation of promoter sequences between homologous HSV-1 and HSV-2 transcripts seen in other regions of the genome may not be a necessary feature between these virus types.

We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this communication, we determined the nucleotide sequence of the DNA encoding this mRNA family and precisely located the mRNAs associated with this DNA sequence. The major mRNA species is unspliced and encoded by a 2.520-nucleotide region. Just upstream of the 5' end are TATA and CAT box sequences characteristic of HSV-1 promoters. The 3' end maps near a region containing a nominal polyadenylation signal. Three minor species (2,400, 2,200, and 1,900 bases, respectively) appear to share a very short leader sequence with the 5' end of the major mRNA and are then encoded by uninterrupted DNA sequences beginning about 100, 400, and 625 bases downstream of the 5' end of the major unspliced mRNA. These positions map at or very near positions which agree reasonably well with consensus splice acceptor sequences. The fourth mRNA is encoded by a contiguous 730-nucleotide sequence at the 3' end of the major unspliced mRNA and has its 5' end just downstream of recognizable TATA and CAT box sequences. We suggest that this mRNA is controlled by its own promoter. The nucleotide sequence data, in combination with the mRNA localization, demonstrate four potential polypeptides encoded by the region. The largest is 1,569 bases long and defines a 523-amino acid protein with sequence features characteristic of a glycoprotein. This was confirmed to be HSV-1 glycoprotein C by immune precipitation of the in vitro translation product of the major unspliced mRNA, performed with a polyspecific antibody to HSV-1 envelope glycoproteins (anti-env-1 serum), and by comparison of tryptic peptides of this translation product with those of authentic HSV-1 glycoprotein C. Polypeptides encoded by some of the minor species also were tentatively identified.