within Its Second Exon a Novel Open Reading Frame That Is Translated in Frame with the UL15 Gene Product

The UL15 gene of herpes simplex virus type 1 is composed of two exons. A mutation previously shown to preclude viral DNA cleavage and packaging at the nonpermissive temperature was identified as a change from a highly conserved serine to proline at codon 653. Separate viral mutants that contained stop codons inserted into exon I of UL15 (designated S648) or an insertion of the Escherichia coli lacZ gene into a truncated UL15 exon II [designated HSV-1(DUL15ExII)] were constructed. Recombinant viruses derived from S648 and HSV-1(DUL15ExII) and containing restored UL15 genes were constructed and designated S648R and HSV-1(DUL15ExIIR), respectively. Unlike HSV-1(DUL15ExIIR) and S648R, the viruses containing mutant UL15 genes failed to cleave and package viral DNA when propagated on noncomplementing cells. As revealed by electron microscopy, large numbers of enveloped capsids lacking viral DNA accumulated within the cytoplasm of cells infected with either S648 or HSV-1(DUL15ExII) but not in cells infected with HSV-1(DUL15ExIIR) or S648R. Thus, one function of the UL15 gene is to effectively prevent immature particles lacking DNA from exiting the nucleus by envelopment at the inner lamella of the nuclear membrane. Cells infected with HSV-1(DUL15ExII) did not express the 75,000- or 35,000-apparent-Mr proteins previously shown to be prod-ucts of the UL15 open reading frame, whereas the 35,000-apparent-Mr protein was readily detectable in cells infected with S648. We conclude that at least the 75,000-Mr protein is required for viral DNA cleavage an