The Equine Herpesvirus 1 U_S2 Homolog Encodes a Nonessential Membrane-Associated Virion Component

Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 U_S2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 U_S2 protein specifically detected a protein with an M_r of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-M_r U_s2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr U_s2 polypeptide. Irrespective of its size, the U_S2 protein was incorporated into virions. The EHV-1 U_S2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 U_S2 protein or to a truncated U_S2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 U_S2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the U_S2 protein in the viral envelope and plasma membrane of infected cells, a U_S2-negative RacL11 mutant (L11DU_S2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a U_S2-repaired virus. After infection of BALB/c mice with L11DU_S2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 U_S2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.