The Herpes Simplex Virus Type 1 vhs-UL41 Gene Secures Viral Replication by Temporarily Evading Apoptotic Cellular Response to Infection: Vhs-UL41 Activity Might Require Interactions with Elements of Cellular mRNA Degradation Machinery

Abstract

HSV-1 vhs activity.The infection of cells with herpes simplex virus type 1 (HSV-1) is associated with a global shutoff of host protein synthesis early postinfection (p.i.) (15,25,26,38,46,48). Analyses of the infected cells have revealed the destruction of polyribosomes and the degradation of infected cell mRNAs following infection with the wild type (wt) virus (9,11,25,26,46,48,50). We have previously derived viral mutants which did not shut off host protein synthesis and degrade the mRNAs (38). Employing marker rescue analyses with fragments from the entire genome, we have mapped the virion host shutoff ("vhs") phenotype within a 265-bp segment of the UL41 gene (26). The mRNA degradation function was also found to map in this region. Sequencing analyses of the corresponding 265-bp segments within the wt and the vhs-1 mutant virus revealed a threonine-to-isoleucine mutation in amino acid 214 of the vhs protein (A. D. Kwong and N. Frenkel, unpublished results). All in all, the UL41 gene product corresponds to a 489-amino-acid phosphoprotein localized in the virion tegument, from where it is released upon viral entry into the cell (25,(38)(39)(40)(41). It is expressed as a late (␥1) gene product which is incorporated into the structural particles of progeny virus. We have shown that in the presence of actinomycin D the UL41 protein was involved in the degradation of mRNAs transcribed prior to and during infection, including the housekeeping genes ␤-actin and ␣-tubulin, which were persistently present in the cells, and the heat shock protein 70 (HSP70), induced p.i. (46). These mRNAs were stable in cells infected with the vhs-1 mutant virus.Several studies examined whether the vhs protein had an intrinsic nucleolytic activity. Read and coworkers (36) have shown that the transfection of cells with a plasmid containing the UL41 gene inhibited the expression of a cotransfecting chloramphenicol acetyltransferase reporter gene. This activity was absent in cells transfected with mutant plasmids (22,36). Furthermore, Everly and Read (12) provided genetic and biochemical evidence that the vhs protein has an mRNase activity. Smiley and coworkers (7,8) documented that the vhs protein produced in vitro employing the rabbit reticulocyte (RRL) translation system possessed an endonucleolytic activity on added exogenous RNA substrates. Furthermore, when vhs was expressed in the budding yeast Saccharomyces cerevisiae there was inhibition of colony formation, while vhs mutants tested in the system had no activity. Cell extracts of yeast expressing vhs displayed an endoribonuclease activity if the extracts were

Selective Ablation of Virion Host Shutoff Protein RNase Activity Attenuates Herpes Simplex Virus 2 in Mice

“…A second possible explanation for the slower replication and spread of the RNase-deficient virus in IFN-␣/␤R Ϫ/Ϫ mice is that dominant negative effects of enzymatically inactive vhs may be exaggerated within the nervous system. Indeed, cell-type-specific differences in sensitivity to the shutoff activity of vhs within various neuronal cell populations have been observed (2,43,56) and may be related to variations in the levels of individual translation factors in different cells (24) as well as to MOIs (56). Of interest, the vhs-1 mutant of HSV-1 is RNase deficient yet reduces protein synthesis in cultured cerebellar granule neurons infected at a high MOI (2), suggesting a negative effect of enzymatically inactive vhs bound to translation initiation complexes.…”

“…vhs mutants show restricted replication in vivo and have significantly reduced capacities to cause disease, establish latency, and reactivate. HSV-1 vhs also delays apoptosis of cultured infected neurons (2). What role each of these vhs functions may play in promoting HSV virulence is not known.…”

Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the U L 47 tegument protein

“…HSV-1 blocks the responses in two fundamentally different ways, by bringing into cells at the time of entry both tegument proteins and prepackaged mRNAs and by encoding proteins made immediately after infection with numerous functions designed to block host innate immune responses (1)(2)(3)(4)(5). Among the best-known and highly effective tegument proteins actively blocking host responses is the virion host shutoff RNase encoded by the U L 41 ORF (6)(7)(8). This RNase is an endoribonuclease with the specificity of RNase A (9), and it is active immediately after entry of the virus into cells and during the initial stages of infection.…”

The Nuclear-Cytoplasmic Shuttling of Virion Host Shutoff RNase Is Enabled by pU L 47 and an Embedded Nuclear Export Signal and Defines the Sites of Degradation of AU-Rich and Stable Cellular mRNAs

“…One of the main components of the armamentarium to defeat the early innate immune responses to infection is the VHS-RNase, a tegument protein encoded by U L 41, a late (␥2) viral protein that is introduced into the infected cells along with the capsid containing viral DNA and approximately another 19 tegument proteins (4,32,33).…”

“…It plays a key role in blocking host responses to infection capable of curtailing viral replication (1)(2)(3)(4). It functions as an endoribonuclease with the specificity of RNase A (5).…”