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Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.

The RNA species synthesized in vitro by a transcribing nucleoprotein (TNP) complex of vesicular stomatitis virus (VSV) were translated with high efficiency in a fractionated cell-free system derived from reticulocytes. The use of TNP complexes isolated from VSV Indiana, VSV New Jersey, and Chandipura viruses showed that in each case the predominant polypeptides synthesized had electrophoretic mobilities identical to their virion N, NS, and M polypeptides in proportions reflecting those found in infected cells rather than purified virions. A minor polypeptide corresponding to unglycosylated polypeptide G was also observed, but the in vitro synthesis of polypeptide L was not detected. The addition of RNase inhibitor to transcription mixtures markedly increased the rate of RNA synthesis. Furthermore, the messenger activity of the RNA was significantly enhanced. The inclusion of S-adenosyl L-methionine during transcription substantially increased the messenger activity of the product RNA, suggesting a requirement for methylation. Fractionation by oligodeoxythymidylic acid-cellulose chromatography revealed that the RNA required a polyadnylic acid tract for messenger activity.

mRNA's homologous to the herpes simplex virus type 1 DNA restriction endonuclease fragment BamHI p, which contains the thymidine kinase gene, have been identified and mapped by hybrid-arrested translation and mRNA selection. Such mRNA's, when translated in vitro, directed the synthesis of polypeptides of apparent molecular weights 43,000 (VI43) and 39,000 (VI39). mRNA for enzymatically active thymidine kinase was enriched by more than 20-fold after selection. Mapping was carried out with restriction endonuclease fragments of BamHI p, and locations of the 5' and 3' termini of VI43 mRNA were deduced. Analysis of nucleotide sequences around the 5' terminus revealed several consensus sequences commonly found at the start of eucaryotic mRNA's and which are presumably involved in initiation of transcription by RNA polymerase II. Translation of mRNA's for VI43, VI39, and the thymidine kinase enzyme was arrested only by a 1,170-base-pair region of BamHI p. Since this region is insufficient for adjacent genes, coding sequences for VI43 and VI39 must overlap; the possible relationship of these two polypeptides is discussed. A virus-induced product equivalent to VI39 was detected in infected cells.

The BglII N fragment of herpes simplex virus type 2 (HSV-2) DNA (approximately 0.58 to 0.63 map unit) was examined for encoded products. Using plasmid pGZ59, which consists of BglII-N cloned in pAT153, in conjunction with hybrid arrested translation, mRNA selection, and in vitro protein synthesis, we found that the major translated product of this region has an approximate molecular weight of 37,800. By further mapping, coding sequences for this polypeptide were located within the region of BglII-N representing approximately 0.58 to 0.61 genome map unit. To demonstrate immunological specificity, we used staphylococcal A protein immunoprecipitation with rabbit anti-HSV-1 or HSV-2 sera and antigens from HSV-1 or HSV-2 total mRNA translated in vitro and BglII-N-selected mRNA. The results show that the 37,800-dalton polypeptide has HSV-2 immunological specificity, as it is precipitated with anti-HSV-2 sera but not with anti-HSV-1 or control sera.

Previous studies (R. J. Watson and J. B. Clements, Virology 91:364--379, 1978; C. M. Preston, J. Virol. 29:275--284, 1979) have shown that the herpes simplex virus type 1 (HSV-1) mutant tsK has a temperature-sensitive lesion in an immediate early polypeptide whose function is to induce synthesis of new viral transcripts, including mRNA, for pyrimidine deoxyribonucleoside kinase. The studies presented here examine the properties of immediate early polypeptides in wild-type HSV-1 and tsK-infected cells at 31 and 38.5 degrees C. The overall pattern of immediate early protein synthesis was similar in wild-type HSV-1- and tsK-infected cells when radiolabeled with [35S]methionine or 14C-amino acid mixture. Further investigation, however, revealed two aberrant properties of the polypeptide Vmw 175 in tsK-infected cells at 38.5 degrees C. Upon cell fractionation, large amounts of this polypeptide were recovered in the cytoplasmic fraction, in contrast to tsK-infected cells at 31 degrees C or wild-type HSV-1-infected cells at either temperature. Furthermore, at 38.5 degrees C tsK-induced Vmw 175 was not processed normally to forms of lower electrophoretic mobility. Both of these defects were reversible upon downshift of tsK-infected cells, even in the absence of further protein synthesis, but were not observed in cells infected with a revertant of tsK. Coinfection of tsK-infected cells with wild-type HSV-1 did not alleviate these lesions, suggesting that they resulted from an abnormal Vmw 175 polypeptide rather than from a defective processing enzyme. Temperature upshift of tsK-infected cells caused reversion of Vmw 175 to the mutant form. The progression to synthesis of late polypeptides was also arrested; therefore, a functional lesion was also reversible upon temperature changes between 31 and 38.5 degrees C during the early stages of infection. The identification of a polypeptide with abnormal properties in tsK-infected cells and the demonstration that these properties, and the functional lesion, are reversible may provide an important system for investigation of HSV-1 transcriptional control.

Polyadenylated immediate-early transcripts of herpes simplex virus type 1, made in BHK cells infected and maintained in the presence of cycloheximide, have been separated on denaturing agarose gels containing methyl mercuric hydroxide. Three virus-specific mRNA bands of estimated sizes 4.7, 3.0, and 2.0 kilobases (kb) were detected, and these mRNA's were mapped on the virus genome and also used to direct protein synthesis in vitro. The 4.7- and 3.0-kb mRNA's hybridized predominantly to certain DNA fragments which are located in the short and long repetitive regions of the genome, respectively, whereas the 2.0-kb mRNA's mapped to three discrete regions of the virus DNA. In vitro translation of these separated mRNA size classes indicated that the 3.0-kb mRNA specified the synthesis of virus polypeptide Vmw 110, whereas the 2.0-kb mRNA's specified Vmw 68, 63, and 12. The synthesis of small amounts of Vmw 175 was specified by the 4.7-kb mRNA. In contrast with the mRNA's which specify these other immediate-early polypeptides, that specifying Vmw 12 is much larger than required for its coding sequences.