The N-terminal glycine of EHV-1 UL11 is essential for the localization of UL11 and EHV-1 replication in cultured cells.

Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid (aa) protein encoded by ORF51. UL11 is modified by acylation including myristoylation and palmitoylation. Myristoylation of EHV-1 UL11 is assumed to occur on the N-terminal glycine, while palmitoylation is assumed to occur on the seventh and ninth cysteines. ORF51, which encodes the first 24 aa, overlaps ORF50 encoding UL12. We previously demonstrated that UL11 was essential for EHV-1 replication in cultured cells and that UL11 was localized at the Golgi apparatus where herpesviruses obtain their final envelope. It is unclear whether the acylation is related to the localization of EHV-1 UL11 and viral replication. In this study, we investigated the role of UL11 acylation in the intracellular localization and viral growth and replication of EHV-1. We constructed seven UL11 acylation mutant plasmids and seven UL11 acylation mutant BAC DNAs; then, we analysed the localizations of the mutant UL11s and attempted virus rescue. We found that both the N-terminal glycine and the seventh or ninth cysteine, especially N-terminal glycine, were involved in the localization of UL11 and viral replication. Taken together, these results suggest that EHV-1 viral growth requires that UL11 is modified by myristoylation of an N-terminal glycine and by palmitoylation of at least one of the cysteines, and that UL11 is localized at the Golgi apparatus. This study shows that a single amino acid in EHV-1 can determine the fate of viral replication.

Prevention of fatal equine herpesvirus type 1 encephalitis in mice by immunization with a limited-replication cycle virus.

Equine herpesvirus type 1 (EHV-1) is a devastating pathogen of horses, their natural hosts, and causes fatal encephalitis in non-natural hosts. We previously demonstrated that acylation of the tegument protein UL11 is required for viral replication in cultured cells. We created a mutant virus (EHV-1 UL12 trunc UL11 G2AC7AC9A), in which glycyl and cysteinyl residues at positions 2, 7 and 9 of UL11 that are normally acylated were replaced with alanyl residues. This virus, designated the 2/7/9 mutant, has a limited-replication cycle (LRC), in which replication stops after just a few cycles. Here, we tested whether the 2/7/9 mutant could be used as a vaccine against fatal encephalitis in a mouse model. A virulence test showed that the 2/7/9 mutant was not pathogenic in mice and elicited an antibody response. We also attempted to use the 2/7/9 mutant to immunize mice against a zebra-borne EHV-1, 94-137. Two trials were conducted, each with five immunized mice, five non-immunized and five control mice. In both trials, clinical signs and fatalities were much lower in the immunized mice than in the non-immunized mice. In addition, none of the mice in either trial developed neutralizing antibodies, indicating that the immunity induced by the 2/7/9 mutant was not due to neutralizing activity. The results indicate that the 2/7/9 LRC mutant has promise as a vaccine against EHV-1 infection non-natural hosts.