The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5′ cap or a 3′ poly(A) tail in the RNA substrate, requires Mg2+, and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5′ quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system.

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To identify pharmacokinetic (PK) drug-drug interactions between tipranavir-ritonavir (TPVTipranavir coadministered with low-dose ritonavir (TPV/r) is an effective treatment option in treatment-experienced human immunodeficiency virus (HIV)-infected patients with resistance to more than one protease inhibitor (PI) (9). TPV/r is associated with adverse effects (AEs) that include increased triglycerides and cholesterol. NIAID Division of AIDS (DAIDS) grade 3 to 4 cholesterol elevation (Ͼ400 mg/dl) and grade 3 to 4 triglyceride elevation (Ͼ750 mg/dl) were higher in the TPV/r-treated patients than in the comparator-boosted PI-treated patients in phase III studies (9). Grade 3 to 4 cholesterol elevation was 4.3 versus 0.6/100 patient exposure years, and triglyceride elevation was 27.8 versus 21.6/100 patient exposure years in the TPV/r versus comparator-boosted PI- (1); however, their use in HIV-infected patients may be limited by clinically significant drug-drug interactions with PIs (5). Atorvastatin is metabolized extensively by cytochrome P450 3A4 (CYP3A4) to metabolites that have in vitro inhibitory activity for HMG-CoA reductase similar to that of atorvastatin. Approximately 70% of the circulating inhibitory activity for HMG-CoA reductase has been attributed to these active metabolites (15). Since TPV/r has a net inhibitory effect on CYP3A4

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The herpes simplex virus (HSV) virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. vhs displays weak amino acid sequence similarity to the fen-1 family of nucleases and suffices to induce accelerated RNA turnover through endoribonucleolytic cleavage events when it is expressed as the only HSV protein in a rabbit reticulocyte in vitro translation system. Although vhs selectively targets mRNAs in vivo, the basis for this selectivity remains obscure, since in vitro activity is not influenced by the presence of a 5′ cap or 3′ poly(A) tail. Here we show that vhs activity is greatly altered by placing an internal ribosome entry site (IRES) from encephalomyocarditis virus or poliovirus in the RNA substrate. Transcripts bearing the IRES were preferentially cleaved by the vhs-dependent endoribonuclease at multiple sites clustered in a narrow zone located immediately downstream of the element in a reaction that did not require ribosomes. Targeting was observed when the IRES was located at the 5′ end or placed at internal sites in the substrate, indicating that it is independent of position or sequence context. These data indicate that the vhs-dependent nuclease can be selectively targeted by specific cis-acting elements in the RNA substrate, possibly through secondary structure or a component of the translational machinery.