Uninfected cell polymerase efficiently transcribes early but not late herpes simplex virus type 1 mRNA.

Abstract

The sequences ofthe 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 ofthe DNA contains recognizable T-A-T-A and C-A-T boxes upstream from their respective 5 The question ofwhat factors control the temporal regulation of DNA virus gene expression is an active field ofresearch. The development of faithful uninfected cell transcription systems (3), as well as other techniques, has led to the following observations concerning this regulation in the smaller DNA viruses. In the papovaviruses (3, 4), the uninfected cell polymerase system favors the recognition of promoters for early genes. However, this preference is not absolute; at relatively high template DNA concentrations, the late promoters are efficiently recognized. In adenovirus (5), the situation is somewhat different in that the major late promoter appears to be readily recognized during the early phases of gene expression. Here, the appearance of abundant late mRNA involves changes in processing patterns for the late mRNA precursors.We have recently reported the detailed characterization of a number of early and late HSV-1 mRNAs (6-8). Interestingly, although HSV-1 mRNAs share general properties of cellular mRNA, splicing of HSV-1 mRNAs is rare. To date, only two well-characterized viral mRNAs, one a (9) and one y (8), have recognizable splices in them. Such a low incidence of splicing suggests that posttranscriptional processing of intervening sequences does not play a major role in the biogenesis of many HSV-1 mRNAs.In this study, we have examined the nucleotide sequences of the DNAs encoding the 5' ends of two well-characterized major unspliced HSV-1 mRNAs (6, 7). The DNA sequences upstream of these mRNAs share features with the HSV-1 thymidine kinase (tk) gene (10, 11) and with many other eukaryotic mRNAs (12, 13). The promoter for the 5.2-kilobase (kb) early mRNA was readily recognizable by the "Manley" HeLa cell polymerase system (3), while that for the 6-kb late mRNA was not. Preliminary data indicate that this difference between early and late HSV-1 mRNA recognition is general.MATERIALS AND METHODS Cells and Virus. Monolayer cultures of HeLa cells were grown at 370C in Eagle's minimal essential medium/10% calf serum without antibiotics. Plaque-purified virus of the KOS strain of HSV-1 was used for all infections.Enzymes. All enzymes were obtained from Bethesda Research Laboratories (Rockville, MD) and digestion was carried out in buffers recommended by that supplier.Isolation, Labeling, and Size Fractionation ofPolyribosomal RNA. Viral RNA synthesized in the absence of HSV-1 DNA synthesis (early RNA) was prepared as described (6). The drugs were a gift of C. Shipman of the University of Michigan.Polyribosomes were isolated from the cytoplasm of HSV-1-infected cells by the magnesium precipitation method of Palmiter (14) as described (6-8). Details of the isolation of poly(A)...

Transactivation of a late herpes simplex virus promoter.

“…We assume that this also is the case with the unmodified VP5 promoter. This conclusion is in agreement with the data of Frink et al (12), who demonstrated that the unmodified VP5 promoter is not recognized by an in vitro transcription system prepared from uninfected HeLa cells. It is not obvious what features account for the inactivity of the VP5 promoter in uninfected cells.…”

“…This current classification may be somewhat oversimplified, because some late genes (e.g., that for VP5) are detectably expressed before replication (12), whereas others are not (18). In the former case, (the so-called 3--y class) it seems possible that the observed pattern of transcription can be accounted for by two distinct processes: first, the 13-y promoters are activated by ICP4 with delayed-early kinetics and second, template amplification, the result of DNA replication, boosts the level of expression further.…”

“…Recently, Frink et al (12) have reported that the promoter for the gene encoding the major capsid protein, VP5 (a delayed-early/late or 1-ry gene) was not recognized by an in vitro transcription system prepared from uninfected cells. This result was of interest to us as it was in marked contrast to the results obtained by Read and Summers (36) with the delayed-early TK promoter and the promoter of another late gene, both of which are recognized in vitro by extracts from uninfected cells.…”

“…Frink et al (12) have shown that the unspliced 6.0-kilobase mRNA encoding VP5 is transcribed from right to left on the prototype arrangement of HSV-1 DNA and that the 5' end of the transcript is located close to the BamHI cleavage site separating fragments F' and B' (Fig. 1 VOL.…”

DNA Sequence of the Major Capsid Protein Gene of Herpes Simplex Virus Type 1

“…Alternatively, the transcript specifying 24K may have been present in low abundance, and therefore not detected by Costa et al (1981). Earlier versions of the DNA sequence encoding the carboxy terminus of 24K and the non-coding region at the 5' end of the 149K gene in HSV-1 strain KOS (Frink et aL, 1981 ;Costa et aL, 1984) contain several errors which were corrected without comment by Costa et al, (1985a). The remaining differences between the HSV-1 strain 17 and strain KOS sequences are located between the 24K and 149K coding regions, and thus may represent genuine sequence differences between the two strains.…”