THE PRODUCT OF THE HERPES-SIMPLEX VIRUS TYPE-1 UL25 GENE IS REQUIRED FOR ENCAPSIDATION BUT NOT FOR CLEAVAGE OF REPLICATED VIRAL-DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino -acid open reading frame that codes for an essential protein, Previous studies have shown that the UL25 gene product is a virion component ( M.A. Ali et al., Virology 216:278-283, 1996) involved in virus penetra tion and capsid assembly (C. Addison et al., Virology 138:246-259, 198 4). In this study we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL 25 open reading frame and propagated on a complementing cell line. Alt hough the mutant was capable of synthesis of viral DNA, it did not for m plaques or produce infectious virus in noncomplementing cells. Antib odies specific for the UL25 protein were used to demonstrate that KUL2 5NS-infected Vero cells did not express the UL25 protein, Western immu noblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy in dicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of i nfected cells by sucrose gradient sedimentation analysis confirmed tha t the ratio of A to B capsids was elevated in KUL25NS-infected Vero ce lls. Following restriction enzyme digestion, specific terminal fragmen ts were observed in DNA isolated from KUL25NS-infected Vero cells, ind icating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electr ophoresis (PFGE), which showed the presence of genome-size viral DNA i n KUL25NS-infected Vero cells, DNase I treatment prior to PFGE demonst rated that monomeric HSV DNA was not packaged in the absence of the UL 25 protein, Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.