The herpes simplex virus UL33 gene product is required for the assembly of full capsids

Phenotypic analysis of the herpes simplex virus type 1 temperature-sensitive DNA-positive mutant, ts1233, revealed that the mutant had a structural defect at the nonpermissive temperature (NPT). Cells infected with ts1233 at the NPT contained large numbers of intermediate capsids, lacking dense cores but possessing some internal structure. No full capsids or enveloped virus particles were detected. In contrast to the defect in another packaging-deficient mutant ts1201, the block in the formation of dense-cored, DNA-containing capsids in tsl233-infected cells at the NPT could not be reversed by transferring the cells to the permissive temperature in the presence of a protein synthesis inhibitor. Furthermore, the capsids produced by ts1233 at the NPT had more compact internal structures than those of the gene UL26 mutant ts1201. Southern blot analysis of viral DNA in ts1233-infected cells confirmed that the mutant DNA was not encapsidated at the NPT and showed that the unpackaged DNA was not cleaved into genome-length molecules. The ts1233 mutation was mapped by marker rescue to the vicinity of genes UL32 and UL33. Sequence analysis of the DNA in this region from the mutant and two independently isolated revertants for growth revealed that ts1233 had a single base-pair change at the amino-terminal end of UL33, resulting in the substitution of an isoleucine with an asparagine. The nucleotide sequence of the revertants in this part of the genome was identical to that of wild-type virus

Processing of the herpes simplex virus assembly protein ICP35 near its carboxy terminal end requires the product of the whole of the UL26 reading frame

The herpes simplex virus (HSV) type 1 assembly protein ICP35 consists of a family of polypeptides, ranging in molecular weight from about 45,000–39,000. The lower molecular weight forms of ICP35 are derived from the higher molecular weight species by slow post-translational modification. The reading frame of gene UL26 and the region within this gene which exhibited homology to the cytomegalovirus assembly protein, the analogous protein to ICP35, were expressed separately under immediate-early (IE) gene regulation in a HSV vector containing a temperature-sensitive mutation in the major transcriptional regulator Vmwt 75. Monoclonal antibody specific for ICP35 immunoprecipitated several polypeptides with molecular weights around 75,000 from extracts of cells infected with a recombinant expressing the IE gene UL26 at the nonpermissive temperature (NPT). These results suggested that the UL26 gene specified a protein distinct from ICP35 but which had some antigenic sites in common with ICP35. In extracts of cells infected at the NPT with a recombinant expressing only the carboxy terminal half of UL26 coding sequences, the monoclonal antibody immunoprecipitated large amounts of the high molecular weight forms of ICP35. The lower molecular weight processed forms of ICP35, however, were not detectable. When cells were coinfected with both recombinants ICP35 was processed to its lower molecular weight forms. This processing step, which occurred near the carboxy terminus of ICP35, was not dependent on capsid formation. The work, together with previous information on the processing of the CMV assembly protein, suggests that UL26 product may be a protease