K P Anderson

Viral DNA synthesis is required for the efficient expression of specific herpes simplex virus type 1 mRNA species

Inhibition of HSV-1 DNA synthesis with either ~abinosyladenine plus the adenosine deaminase inhibitor pentostatin, or with arabinosylthymine, showed a viral mRNA population identical to that seen prior to viral DNA replication (early) by the criteria of quantitative hybridization, specific mRNA species identifiable by Southern blot hybridization of size~aetionated RNA, and migration of polypeptides resolved by in vitro translation of purified viral mRNA. The amount of viral mRNA associated with infected cell polyribosomes was determined by quantitative DNA excess solution hybridization. At 2 hr postinfection (p.i.) (before viral DNA synthesis) and in drug-treated cells at 6 hr p.i., the majority of the polyadenylated RNA was cell specific with some virus-specific RNA detectable. In contrast, at 6 hr p.i., in the absence of drugs (during m~imum viral DNA synthesis), nearly all the polyadenylated polyribosomal RNA was viral. Blot hybridization of size-fractionated viral RNA confirmed several specific differences between the viral mRNA species occurring before and after HSV-1 DNA synthesis, which have been reported previously from this laboratory. These differences also were reflected in the in &ro translation products encoded by the viral mRNAs. The mRNA species and the eneoded polypeptides that were present in the absence of viral DNA synthesis are a subset of those viral mRNA species and polypeptides expressed in the presence of viral DNA synthesis. The viral mRNA species fall into several groups based on their relative abundance at various times of infection. These data suggest that, in the normal virus infection cycle, the onset of viral DNA synthesis is necessary for normal expression of later viral genes.

Isolation and characterization of HSV-1 mRNA

We have reviewed our methods for the isolation, mapping, and characterization of HSV-1 mRNA abundant at the three stages of infection. The complexity of viral mRNA increases with each stage of infection. By use of HSV-1 DNA restriction fragments bound to cellulose, we have been able to examine in some detail the time of appearance and properties of viral mRNA homologous to specific regions of the genome.