Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.

A herpes simplex virus type 2 (HSV-2) UL24 ␤-glucuronidase (UL24-␤gluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-␤gluc yielded cytopathic effect with syncytium formation. UL24-␤gluc replicated to wild-type viral titers in three different cell lines. UL24-␤gluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD 50 ) of the parental virus. Furthermore, few UL24-␤gluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-␤gluc at a dose equivalent to the LD 50 of parental virus (Ϸ5 ؋ 10 3 PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-␤gluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.

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