Expression and Characterization of Equine Herpesvirus 1 (EHV-1) ORF19 Product, the Virion-Associated Host Shutoff (VHS) Homolog

Abstract

The ability of herpes simplex virus types 1 and 2 (HSV-1 and -2) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of one or more viral proteins. These viral regulatory proteins are components of infecting virions and influence gene expression during the early stage of viral infection. One of the most obvious effects is rapid suppression of host protein synthesis, which has been termed virion host shutoff (vhs). It has been demonstrated that virion host shutoff is due to disruption of host polyribosomes and accelerated turnover of cellular mRNAs. In general, HSV-1 strains inhibit host protein synthesis somewhat more slowly than HSV-2 strains. This inhibition has been demonstrated using multiplicities of infection (m.o.i.) as low as 4 plaque forming units (pfu) and is apparent in infected cultures within 3 hr postinfection. The phenomenon of HSV-1 virion-associated host shutoff has been studied extensively and shown to be mediated by a tegument protein encoded by the UL41 gene. This process occurs in the absence of de novo viral gene expression. Several HSV-1 vhs mutant viruses have been selected and characterized which are defective in both protein synthesis and cellular mRNA degradation, indicating that the vhs protein is responsible for host protein synthesis shutoff. HSV-1 UL41 homologs have been identified in the genomes of several other alphaherpesviruses, such as pseudorabies virus (PRV), varicella zoster virus (VZV), and equine herpesvirus type 1 (EHV-1). However, little is known about the expression of these vhs homolog genes or the functions of their putative products. To begin investigating EHV-1 vhs activity, initial studies were performed to compare the effect of EHV-1 and HSV-1 infection on cellular protein synthesis and mRNA degradation in four actinomycin-D treated cell types, including equine dermis (ED) cells, rabbit kidney (RK) cells, baby hamster kidney (BHK) cells, and primary hamster embryo (HE) cells. Interestingly, neither virion-associated suppression of cellular protein synthesis nor degradation of mRNA during EHV-1 infection at 10 or 50 pfu/cell was observed, whereas comparable HSV-1 infections resulted in dose-dependent inhibition of host protein synthesis and increased degradation of cellular mRNA. These data demonstrated that EHV-1 infection does not result in virion-associated host shutoff activity comparable to that of HSV-1. There are several possible explanations for the observed differences in host protein synthesis and cellular mRNA stabilities between EHV-1 and HSV-1 infections: (1) EHV-1 ORF19, the HSV-1 UL41 homolog, is not expressed in EHV-1 infection, therefore no ORF19 protein is available for packaging into virions; (2) ORF19 is expressed as an active vhs protein, but that protein is not efficiently packaged into EHV-1 virions; (3) the ORF19 protein is expressed and packaged similarly to HSV-1 vhs, but it is structurally divergent from HSV-1 vhs such that it lacks strong vhs activity; (4) the ORF19 protein is present in EHV-1 viral particles and has intrinsic vhs activity, but that activity is abrogated during viral infection. The purpose of this study was to analyze EHV-1 ORF19 with respect to: (1) expression of ORF19 and its temporal regulation during lytic viral infection; (2) detection of ORF19 protein expression during EHV-1 infection and localization of ORF19 protein in viral particles; (3) determination of the intrinsic vhs activity of ORF19 protein compared to that of HSV-1 vhs; (4) structural, functional, and mutational comparisons of EHV-1 ORF19 protein and HSV-1 vhs; and (5) examination of potential physical association of ORF19 protein with another viral tegument protein, EHV-1 a-TIF, the agene transinducing factor, and of potential regulation of ORF19's vhs activity by a-TIF via this interaction. The results of these analyses will aid in determining which, if any, of these four postulated explanations appears most valid, and provide the first extensive characterization of vhs expression and function in an alphaherpesvirus other than herpes simplex virus.