Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA

Abstract

Previous reports from this laboratory (Honess and Roizman, 1974) have operationally defined alpha polypeptides as the viral proteins that are synthesized first in HEp-2 cells treated with cycloheximide from the time of infection with herpes simplex virus type 1 until the withdrawal of the drug 12 to 15 h after infection. It has also been shown that the viral RNA (designated alpha RNA) that accumulates in the cytoplasm during cycloheximide treatment and on polyribosomes immediately upon withdrawal of the drug is homologous to 10 to 12% of viral DNA, whereas the viral RNA accumulating in the cytoplasm of untreated cells at 8 to 14 h after infection is homologous to 43% of viral DNA (Kozak and Roizman, 1974). In the present study, alpha RNA and cytoplasmic RNA extracted from untreated cells 8 h after infection were each hybridized in liquid to in vitro labeled restriction endonuclease fragments generated by cleavage of herpes simplex virus type 1 DNA with Hsu I, with Bgl II, and with both enzymes simultaneously. The data show that only a subset of the fragments hybridized to alpha RNA, and these are scattered within both the L and S components of the DNA. There are at least five noncontiguous regions in the DNA homologous to alpha RNA; two of these are located partially within the reiterated sequences in the S component. All fragments tested hybridized more extensively with 8-h cytoplasmic RNA than with alpha RNA. Four adjacent fragments, corresponding to 30% of the DNA and mapping within the L component, hybridized exclusively with the cytoplasmic RNA extracted from cells 8 h after infection.

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

“…A 10-pl sample (-104 U) of S1 nuclease was added, and digestion was carried out for 30 min at 450C. At this time, 20 ug of tRNA was added as carrier to each sample, and the material was ethanol precipitated.…”

Separation and characterization of herpes simplex virus type 1 immediate-early mRNA's

“…The locations on the genome of transcripts which accumulate in cells infected with herpes simplex virus type 1 (HSV-1) at various times postinfection, in the presence or absence of DNA or protein synthesis inhibitors, have been analyzed by hybridization to the fragments of HSV-1 DNA generated by digestion with a number of restriction endonucleases (3,10). These, and previous studies (6,22), have indicated that transcription of the HSV-1 genome is subject to temporal regulation.…”

“…Inhibition of virus protein synthesis with cycloheximide was found to result in accumulation of RNA sequences which hybridized to certain DNA fragments only (3,10), and these restricted RNA sequences, transcribed by a preexisting aamanitin-sensitive host RNA polymerase (4), have been defined as immediate-early (IE) RNA. IE RNAs accumulate also in cells infected at the nonpermissive temperature with certain HSV-1 temperature-sensitive (ts) mutants, and are the only virus transcripts detectable in ts Kinfected cells (24).…”

Genome Variation and Evolution Among Herpes Viruses*

“…In addition, studies on the functional arrangements of the HSV, CMV, and PRV genomes have shown that the genes expressed immediately after infection map within and nearby the reiterated sequences Weststrate, personal communication) .79 **. 23 Although the functional arrangement of the EBV genome is not known, the genes expressed in the restringently infected cells and presumed to be early functions also map at or near the internal, tandemly reiterated sequences.27 Subject to error due to variation in genome size, the circular representations therefore are aligned with respect to the long stretch of quasi unique sequences, high G+C regions, and location of early functions.…”