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Previous results indicated that the herpes simplex virus 1 (HSV-1) UL31 gene is necessary and sufficient for localization of the UL34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon UL31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored UL31 gene. The replication of the UL31 deletion virus was restored on UL31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of UL34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas UL34 protein localized at the nuclear membrane in rabbit skin cells, and UL31 complementing CV1 cells infected with the UL31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of UL34 protein to the nuclear membrane. We speculate that this function partially complements that of UL31 and may explain why UL31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells. [Copyright &y& Elsevier]