Characterization of a major late herpes simplex virus type 1 mRNA.

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.

Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome.

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.

Isolation and translation of mRNA encoded by a specific region of the herpes simplex virus type 1 genome.

We have examined in detail the major mRNA species encoded by the region of the herpes simplex virus type 1 genome encoded by HindIII fragment K (0.53-0.59 from the left end of the prototype arrangement of the genome) by using this restriction fragment bound to cellulose as a reagent for isolation of this mRNA. Before viral DNA replication in infected cells (early), a major species of viral mRNA 5.2 kilobases (kb) in length is abundant. After the onset of viral DNA replication (late), four mRNA species are abundant: 7, 5.2, 3.8, and 1.8 kb in size. We have used reverse transcriptase from avian myeloblastosis virus to make DNA complementary to these RNA species and their 3' ends. We have shown by hybridization of this complementary DNA to Southern blots of herpes simplex virus type 1 DNA that the 7-, 5.2-, and 1.8-kb mRNA species have their 3' ends to the right of 0.59 and are at least partially colinear. The 3.8-kb mRNA has a 3' end mapping to the left of the 3' ends of these other species. In vitro translation of HindIII fragment K-specific mRNA in a reticulocyte lysate system yielded three major polypeptide products: 140,000, 122,000, and 54,000 daltons (d). Less prominent species of 86,000 and 65,000 d also were produced. Translation of size-fractionated HindIII fragment K-specific mRNA showed that the 7-, 5.2-, and 3.8-kb mRNA's encoded the 54,000-, 140,000-, and 122,000-d polypeptides, respectively. The 140,000-d polypeptide was the major polypeptide translated using early HindIII fragment K-specific mRNA as a template. The 3.8-kb mRNA also encoded the 86,000-d polypeptide, whereas the 1.8-kb mRNA encoded a polypeptide that was indistinguishable from the 54,000-d polypeptide encoded by the 7-kb mRNA, in addition to the 65,000-d polypeptide. The implications of the data are discussed.