High-resolution characterization of herpes simplex virus type 1 transcripts encoding alkaline exonuclease and a 50,000-dalton protein tentatively identified as a capsid protein

Abstract

Four partially overlapping mRNAs (1.9, 2.3, 3.9, and 4.5 kilobases [kb]) were located between 0.16 and 0.19 map units on the herpes simplex virus type 1 genome. Their direction of transcription was found to be from right to left. The 2.3-kb mRNA was found to be early (1), whereas the others were late (P3y). Partial sequence analysis of the DNA encoding these genes indicated that the promoter for the 2.3-kb mRNA shares structural features with other early (1) promoters. In vitro translation of hybrid-selected mRNA indicated that among the proteins these mRNAs encode are an 82,000-dalton (d) polypeptide reactive with a monoclonal antibody against herpes simplex virus type 2 alkaline exonuclease and a 50,000-d polypeptide weakly reactive with a polyclonal antibody made against the capsid protein VP19C. Further experiments suggested that the 2.3-kb mRNA encodes the 82,000-d polypeptide, whereas one (or both) of the larger mRNAs encodes the 50,000-d protein. A novel finding was that the 1.9-kb mRNA appears to share part of the translational reading frame for alkaline exonuclease, but any polypeptide it encodes does not react with the monoclonal antibody to this enzyme.

Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates

“…At later time points (13,24, and 48 h postinfection), the 186and 152-kb bands are very prominent in well DNA from KOSand AN-1-2a-infected cells (Fig. 4, lanes 5, 7, 9, 11, 13, and 15), Samples for PFGE were prepared by suspending infected cells in a block containing melted low-melting-point agarose as described previously (51).…”

“…HSV alkaline nuclease (UL12) is a relatively abundant 85-kDa phosphoprotein in infected cells (2,3). UL12 is encoded by a 2.3-kb member of a family of unspliced 3Ј coterminal mRNAs (13,15). We have recently demonstrated that, as originally proposed by Costa et al (13), another member of this family of mRNAs (1.9 kb) encodes an N-terminally truncated protein (UL12.5) which shares its carboxy terminus with the alkaline nuclease protein (38).…”

“…UL12 is encoded by a 2.3-kb member of a family of unspliced 3Ј coterminal mRNAs (13,15). We have recently demonstrated that, as originally proposed by Costa et al (13), another member of this family of mRNAs (1.9 kb) encodes an N-terminally truncated protein (UL12.5) which shares its carboxy terminus with the alkaline nuclease protein (38). A viral frameshift mutant (AN-F1) that was constructed is predicted to abolish the gene product of the 2.3-kb mRNA (full-length alkaline nuclease) but leave intact the UL12.5 product of the 1.9-kb mRNA.…”

The product of the UL12.5 gene of herpes simplex virus type 1 is a capsid-associated nuclease

“…These include enzymes that are involved in general nucleotide metabolism, enzymes that direct the synthesis of viral DNA, and enzymes that promote the maturation of newly replicated DNA and its packaging into capsids. One viral protein which appears to play a critical role in the last two processes is a deoxyribonuclease encoded by the UL12 gene (4,7,13). The UL12 gene product is commonly referred to as alkaline nuclease (AN) since it requires an alkaline environment for optimal in vitro activity (1,8,18).…”

“…Previous transcript-mapping studies identified five 3Ј-coterminal mRNAs which localized to the region of the HSV-1 genome containing the UL12 open reading frame (4,7). AN was shown to be encoded by the 2.3-kb member of this family, whereas the 1.9-kb transcript in this group was predicted to encode an amino-terminally truncated form of AN.…”