Expression and function of the equine herpesvirus 1 virion-associated host shutoff homolog

Abstract

The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virionassociated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-2143Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 1923Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).

Peptide transport activity of the transporter associated with antigen processing (TAP) is inhibited by an early protein of equine herpesvirus-1

“…It could be argued that the inhibition of TAP activity observed in EHV-1-infected cells might be due to the inhibition of synthesis of TAP proteins as a result of virus-encoded virion host shut-off activity. However, this scenario is highly unlikely because EHV-1, in contrast to BHV-1 and HSV, does not exhibit early shut-off of protein synthesis (Feng et al, 1996).…”

Mutational analysis of the virion host shutoff gene (UL41) of herpes simplex virus (HSV): characterization of HSV type 1 (HSV-1)/HSV-2 chimeras

“…One possible explanation is that the homologous sequences in UL41 and the cellular proteins are motifs common to a number of DNases and RNases and that the domain responsible for the specificity of UL41 for RNA lies elsewhere. One candidate for such a domain is a region of UL41 centered on amino acid 180, which is similar to the RNA recognition or RNP motif found in yeast and human poly(A) binding proteins (7,37). Although there are no biochemical data indicating that the Vhs protein binds RNA, it is tempting to speculate that this motif is important for recognition of the target mRNA.…”

“…vhs (UL41) homologs have been identified in a number of alphaherpesviruses, including HSV types 1 and 2 (HSV-1 and -2) (6,8,9,11,25), varicella-zoster virus (5), pseudorabies virus (2), bovine herpesvirus, gallid herpesvirus, and equine herpesvirus 1 (7,40). Of these, the most closely related, and also the most extensively studied, are the vhs genes of HSV-1 and HSV-2.…”

Molecular characterization of duck enteritis virus UL41 protein

“…The UL41 gene is highly conserved in alphaherpesviruses [ 7 , 8 ], and UL41-encoded proteins have been described in other Alphaherpesvirinae subfamily members, including bovine herpesvirus 1 (BHV-1) [ 9 , 10 ], monkey B virus (macacine herpesvirus 1; BV) [ 11 ], equine herpesvirus-1 (EHV-1) [ 12 ], pseudorabies virus (PRV) [ 7 ], and varicella-zoster virus (VZV) [ 13 , 14 ]. The HSV-1 virion host shutoff (VHS) protein, a late (γ2) tegument protein, is encoded by the UL41 gene and is packaged into the virion; essential for viral infection [ 15 – 19 ], VHS is released into the host cell cytoplasm, selectively degrading cellular mRNAs via its RNase activity and contributing to the shutoff of host cell protein synthesis early in infection [ 20 , 21 ].…”