Vhs Activity Research Articles

Generation and characterization of UL41 null pseudorabies virus variant in vitro and in vivo

BackgroundThe alphaherpesvirus virion host shutoff (vhs) gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis. The roles of vhs in HSV-1 and HSV-2 have been studied extensively in previous studies, however, relatively little is known about the vhs protein of PRV.MethodsA novel method combining CRISPR/Cas9 and Gibson assembly was developed to generate UL41 null PRV variant. The properties of UL41 null PRV in vitro and in vivo were further characterized. And the vhs activity of UL41 protein of PRV variant was evaluated by luciferase assay, Western-blot and RT-qPCR.ResultsGibson assembly based on homologous recombination can accomplish one-step insertion of viral DNA fragments into donor plasmids efficiently (> 80%). Cas9/gRNA further largely enhanced the efficiency of homologous recombination. Using this method we were able to rapidly generate the UL41 null and revertant viruses of PRV variant. Compared to wild type (JS-2012), the UL41 null virus showed significantly smaller plaques and lower titers in Vero cells and impaired lethality and neuroinvasion in mice. Further the UL41 protein from different PRV strains exhibited unequal vhs activity in vitro, which of JS-2012 showed significantly weaker vhs activity than that of European-American strains. In addition UL41 null virus can also significantly decrease the expression of host genes during the early period of infection, which suggests other viral factors may be also involved in host shutoff.ConclusionsCRISPR/Cas9 combined with Gibson assembly efficiently generated UL41 null PRV. Compared to wild type, UL41 null PRV showed impaired both replication capability in vitro and neuroinvasion in vivo. Further UL41 protein of PRV variant showed significantly weaker vhs activity than that of PRV SC (European-American-like strain), suggesting the deficiency of vhs activity by the PRV variant UL41 protein.

Role of the UL41 Protein of Pseudorabies Virus in Host Shutoff, Pathogenesis and Induction of TNF-.ALPHA. Expression

The vhs (virion host shutoff) is highly conserved in alphaherpesvirus, including pseudorabies virus (PRV). In an attempt to explore the function of vhs of PRV, we constructed and characterized a mutant virus (Δ41). In the absence of vhs activity, Δ41 mutant is highly attenuated in mice model and the lethality is correlated with the virus dissemination in neural tissues. As with herpes simplex virus type 1 (HSV-1), the prototype virus of alphaherpesvirus, the pronounced decrease in cellular protein synthesis triggered by wild type PRV was largely restored in cells infected with Δ41 virus. Furthermore, tumor necrosis factor-α (TNF-α) protein expression was elevated significantly in spleen of mice infected with vhs mutant virus. Since TNF-α has been indicated to be an important cytokine in the innate immune response against various infections, our results implicate vhs may contribute to the protection against PRV lethality via the action of TNF-α. Overall, we confirm the shutoff function of vhs protein in PRV, and demonstrate the role that vhs protein plays in virulence, and regulation of cytokine production.

Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay

Marek's disease, a T cell lymphoma, is an economically important disease of poultry caused by the Marek's disease virus (MDV), a highly cell-associated alphaherpesvirus. A greater understanding of viral gene function and the contribution of sequence variation to virulence should facilitate efforts to control Marek's disease in chickens. To characterize a naturally occurring single nucleotide polymorphism (SNP; AY510475:g.108,206C > T) in the MDV UL41 gene that results in a missense mutation ( AAS01683:p.Arg377Cys), bacterial artificial chromosome (BAC)-derived MDVs that differed only in the UL41 SNP were evaluated using a head-to-head competition assay in vitro. Monitoring the frequency of each SNP by pyrosequencing during virus passage determined the ratio of each viral genome in a single monolayer, which is a very sensitive method to monitor viral fitness. MDV with the UL41*Cys allele showed enhanced fitness in vitro. To evaluate the mechanism of altered viral fitness caused by this SNP, the virion-associated host shutoff (vhs) activity of both UL41 alleles was determined. The UL41*Cys allele had no vhs activity, which suggests that enhanced fitness in vitro for MDV with inactive vhs was due to reduced degradation of viral transcripts. The in vitro competition assay should be applicable to other MDV genes and mutations.

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

We have previously shown that herpes simplex virus type 1 (HSV-1) infection is associated with early destabilization/degradation of infected cell mRNAs and consequent shutoff of host protein synthesis by the activity of the virion-associated host shutoff (vhs) UL41 protein. Wild-type (wt) virus destabilized/degraded the housekeeping beta-actin and alpha-tubulin mRNAs as well host stress functions, like the heat shock 70 protein induced postinfection. vhs mutants did not degrade the mRNAs. Elaborate studies by others have been concerned with the mode of mRNA degradation and the mRNAs affected. We now describe vhs activity in primary cultures of mouse cerebellar granule neurons (CGNs). Specifically, (i) upon infection in the presence of actinomycin D to test activity of input viral particles, there was a generalized inhibition of protein synthesis, which depended on the input multiplicity of infection (MOI). (ii) Low-MOI infection with vhs-1 mutant virus was associated with increased synthesis of all apparent proteins. Higher MOIs caused some shutoff, albeit significantly lower than that of wt virus. This pattern could reflect an interaction(s) of vhs-1 protein with host machinery involved in cellular mRNA destabilization/degradation, sequestering this activity. (iii) wt virus infection was associated with cell survival, at least for a while, whereas mutant virus induced apoptotic cell death at earlier times. (iv) wt virus replicated well in the CGNs, whereas there was no apparent replication of the vhs-1 mutant virus. (v) The vhs-1 mutant could serve as helper virus for composite amplicon vectors carrying marker genes and the human p53 gene. Ongoing studies test the use of vhs-1-based composite oncolytic vectors towards cancer gene therapy.

Role of the VP16-Binding Domain of vhs in Viral Growth, Host Shutoff Activity, and Pathogenesis

The virion host shutoff (vhs) protein of herpes simplex virus type 1 causes the degradation of host and viral mRNA immediately upon infection of permissive cells. vhs can interact with VP16 through a 20-amino-acid binding domain, and viruses containing a deletion of this VP16-binding domain of vhs (Delta20) and a corresponding marker rescue (Delta20R) were constructed and characterized. Transient-transfection assays showed that this domain was dispensable for vhs activity. The Delta20 recombinant virus, however, was unable to induce mRNA degradation in the presence of actinomycin D, while degradation induced by Delta20R was equivalent to that for wild-type virus. Delta20, Delta20R, and KOS caused comparable RNA degradation in the absence of actinomycin D. Western blot analysis of infected cells indicated that comparable levels of vhs were expressed by Delta20, Delta20R, and KOS, and there was only a modest reduction of vhs packaging in Delta20. Immunoprecipitation of protein from cells infected with Delta20 and Delta20R showed equivalent coprecipitation of vhs and VP16. Pathogenesis studies with Delta20 showed a significant decrease in replication in the corneas, trigeminal ganglia, and brains, as well as a significant reduction in clinical disease and lethality, but no significant difference in the establishment of, or reactivation from, latency compared to results with KOS and Delta20R. These results suggest that the previously described VP16-binding domain is not required for vhs packaging or for binding to VP16. It is required, however, for RNA degradation activity of tegument-derived vhs and wild-type replication and virulence in mice.

Inhibition of TAP by pseudorabies virus is independent of its vhs activity

Previously we have shown that pseudorabies virus (PrV) down-regulates the expression of porcine MHC class I molecules by interfering with the transporter associated with antigen processing (TAP). During lytic PrV infection, the half-lives of both host and viral mRNA are regulated by the product of virion host shut-off (vhs) gene, UL41. PrV vhs protein induces degradation of cellular mRNA including those encoding class I and TAP. Therefore, further elucidation of specific mechanisms of down-regulation of class I molecules by PrV necessitates construction of a vhs deletion mutant. Two such mutants (vhsΔ1 and vhsΔ2) were generated by homologous recombination between the wild type (wt) PrV Indiana-F strain, and plasmids containing truncated UL41 gene of PrV into which the enhanced green fluorescent protein (EGFP) cassette was inserted. Compared with the wt virus, both the vhs mutants exhibited slower in vitro growth kinetics. The mutants, like the wt virus, inhibited the peptide transport activity of TAP and down-regulated cell surface expression of class I molecules. These findings suggest that, inhibition of TAP activity in PrV-infected cells is due to mechanism(s) specifically directed at class I pathway and not due to the non-specific vhs activity of the virus.

Effects of virion host shut-off activity of bovine herpesvirus 1 on MHC class I expression.

Previously, we have shown that bovine herpesvirus 1 (BHV-1) down-regulates the expression of major histocompatibility complex class I molecules by interfering with transport of peptides by the transporter associated with antigen processing (TAP). Further studies revealed that BHV-1 down-regulates the expression of mRNA for class I molecules and other cellular proteins. To further elucidate the mechanisms of down-regulation of class I molecules, a virion host shut-off (vhs) deletion mutant was generated. The mutant, like the wildtype (wt) virus, interfered with transport of peptides by the TAP, and down-regulated cell surface expression of class I molecules. However, unlike the wt virus, the mutant did not impair the synthesis of class I molecules. These results indicate that down-regulation of class I molecules by BHV-1 is mediated by vhs activity of the virus, as well as mechanisms specifically directed at the class I pathway. Absence of vhs activity should result in decreased pathogenicity and enhanced immunogenicity of BHV-1 vhs deletion mutant, making it a better vaccine candidate.

Herpesvirus Papio 2 Encodes a Virion Host Shutoff Function

Infection of baboons with herpesvirus papio 2 (HVP-2) produces a disease that is similar to human infection with herpes simplex viruses (HSV). Molecular characterization of HVP-2 has demonstrated that the virion contains a factor which rapidly shuts off host cell protein synthesis after infection. Reduction of host cell protein synthesis occurs in parallel with the degradation of mRNA species. A homolog of the HSV virion host shutoff (vhs) gene was identified by Southern and DNA sequence analysis. The sequence of the HVP-2 vhs gene homolog had greater than 70% identity with the vhs genes of HSV 1 and 2. Disruption of the HVP-2 vhs open reading frame diminished the ability of the virus to shut off protein synthesis and degrade cellular mRNA, indicating that this gene was responsible for the vhs activity. The HVP-2 model system provides the opportunity to study the biological role of vhs in the context of a natural primate host. Further development of this system will provide a platform for proof-of-concept studies that will test the efficacy of vaccines that utilize vhs-deficient viruses.

MRNA degradation by the virion host shutoff (Vhs) protein of herpes simplex virus: genetic and biochemical evidence that Vhs is a nuclease.

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating the decay of all mRNAs, it helps redirect the cell from host to viral gene expression and facilitates the sequential expression of different classes of viral genes. While it is clear that Vhs induces mRNA degradation, it is uncertain whether it is itself an RNase or somehow activates a cellular enzyme. This question was addressed by using a combination of genetic and biochemical approaches. The Vhs homologues of alphaherpesviruses share sequence similarities with a family of mammalian, yeast, bacterial, and phage nucleases. To test the functional significance of these similarities, Vhs was mutated to alter residues corresponding to amino acids known to be critical to the nuclease activity of cellular homologues. In every instance, mutations that inactivated the nuclease activity of cellular homologues also abolished Vhs activity. Recent experiments showed that Vhs interacts with the cellular translation initiation factor eIF4H. In this study, the coexpression of Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein in bacteria resulted in the formation of a complex of the proteins. The wild-type Vhs/GST-eIF4H complex was isolated and shown to have RNase activity. In contrast, Vhs mutations that altered key residues in the nuclease motif abolished the nuclease activity of the recombinant Vhs/GST-eIF4H complex. The results provide genetic and biochemical evidence that Vhs is an RNase, either alone or as a complex with eIF4H.

The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity.

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214-->Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.

36] Herpes simplex virus vhs protein

Publisher Summary The virion host shutoff (vhs) protein encoded by herpes simplex virus (HSV) gene UL41 is responsible for the rapid shutoff of host protein synthesis that occurs during the earliest stages of HSV infection. This chapter summarizes the current understanding of the mechanism of action and regulation of vhs activity, and provides a detailed description of a simple and convenient In Vitro assay for vhs-dependent ribonuclease activity. vhs is a structural component of the HSV virion that is synthesized late in infection and packaged into the tegument of the mature virus particle (the space between the envelope and the nucleocapsid). It is then delivered into the cytoplasm of newly infected cells after fusion of the virion envelope with the host plasma membrane, where it triggers host shutoff before the onset of de novo viral gene expression, vhs-induced shutoff is characterized by strong inhibition of host protein synthesis, disruption of preexisting polyribosomes, and accelerated turnover of host mRNAs. The vhs-dependent shutoff system exhibits little specificity, destabilizing most, if not all, cellular and viral mRNAs in the infected cell. The rapid decline in host mRNA levels presumably helps viral mRNAs gain access to the cellular translational apparatus. The vhs system provides a striking and readily dissected example of gene regulation at the level of mRNA stability in mammalian cells, and may therefore help illuminate the host mRNA turnover pathways that regulate cell growth, differentiation, and oncogenesis.

A vhs-like activity of bovine herpesvirus-1

Bovine herpesvirus 1 (BHV-1) is a major pathogen of cattle, causing significant disease including immunosuppression in infected animals. In vitro, the surface expression of major histocompatibility complex (MHC) class I molecules, crucial for an appropriate anti-viral immune response of the host, is down-regulated by BHV-1 infection. Northern blot analyses revealed that the mRNAs for MHC class I and class II molecules were significantly down-regulated in BHV-1 infected cells, starting as early as 2 h after infection. Furthermore, mRNA expression of the two house keeping genes actin and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) was also repressed after infection. This BHV-1 induced effect on cellular metabolism resembled the virion host shutoff (vhs) activity of herpes simplex virus (HSV). Similar to the HSV vhs activity, the putative BHV-1 vhs activity was not abrogated in cells infected in the presence of actinomycin D (ActD) which suggested that no viral gene expression is required for the vhs function and the putative vhs protein is associated with the virion. Sequence comparison indicated a BHV-1 open reading frame having a 60% similarity to the HSV vhs sequence. This putative BHV-1 open reading frame contained the four conserved regions of the alphaherpesvirus vhs protein. Since an HSV vhs-mutant exhibited less virulence and good immunogenicity, we suggest that a BHV-1 vhs- mutant may hold promising potential as a candidate vaccine.

Evidence that herpes simplex virus VP16 is required for viral egress downstream of the initial envelopment event.

During infection with herpes simplex virus type 1 (HSV-1), VP16 serves multiple functions, including transcriptional activation of viral immediate early genes and downregulation of the virion host shutoff protein vhs. Furthermore, VP16 has been shown to be involved in some aspect of virus assembly and/or maturation. Experiments with a VP16 null virus, 8MA, suggested that VP16 plays a direct role in virion assembly, since removal of VP16 from the HSV-1 genome results in reduced levels of encapsidated DNA and a failure to produce extracellular enveloped particles. However, VP16 null mutants display a severe translational arrest due to unrestrained vhs activity, thus complicating interpretation of these data. We examine here the role of VP16 in virion assembly and egress in the context of a vhs null background, using the virus 8MA/DeltaSma (VP16(-) vhs(-)). Comparison of 8MA and 8MA/DeltaSma with respect to viral DNA accumulation and encapsidation and accumulation of the major capsid protein, VP5, revealed that the 8MA lethal phenotype is only partially due to uncontrolled vhs activity, indicating that VP16 is required in HSV-1 virion formation. Electron microscopy confirmed these results and further showed that VP16 is required for HSV-1 egress beyond the perinuclear space. In addition, we describe the isolation and characterization of an 8MA derivative capable of propagation on Vero cells, due to second site mutations in the vhs and UL53 (gK) genes. Taken together, these results show that VP16 is required for viral egress downstream of the initial envelopment step and further underscore the importance of VP16 in controlling vhs activity within an infected cell.

Picornavirus internal ribosome entry site elements target RNA cleavage events induced by the herpes simplex virus virion host shutoff protein.

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.

The Virion Host Shutoff Function of Herpes Simplex Virus Degrades the 5′ End of a Target mRNA before the 3′ End

During lytic infections, the virion host shutoff (vhs) function of herpes simplex virus (HSV) disaggregates host polysomes and induces rapid turnover of both cellular and viral mRNAs. To examine the steps in vhs-induced mRNA degradation, an RNase protection assay was used to compare the relative decay rates of sequences from the 5′ and 3′ ends of a selected target mRNA. In cells infected with wild-type HSV-1, sequences at the 5′ end of the HSV-1 thymidine kinase mRNA were degraded more rapidly than those at the 3′ end of the transcript. In contrast, in cells infected with a vhs mutant, the decay rates of sequences at the 5′ and 3′ termini of the transcript were much slower and were essentially indistinguishable from each other. Vhs-induced degradation of the transcribed portion of the mRNA was not preceded by detectable shortening of the poly(A) tail in vivo; nor was a poly(A) tail required to make an RNA a target for the vhs activity in vitro. The results suggest that degradation of sequences at or near the 5′ end of an mRNA is an early step in vhs-induced decay.

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

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 virion-associated 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-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). 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).

Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function.

Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.

The Virion Host Shutoff Protein of Herpes Simplex Virus Inhibits Reporter Gene Expression in the Absence of Other Viral Gene Products

The virion host shutoff (vhs) function of herpes simplex virus induces degradation of host mRNAs at early times and rapid turnover of vital mRNAs throughout infection. Previous studies have shown that disruption of the UL41 gene abrogates vhs activity, but have not determined whether the UL41 polypeptide is the direct inducer of mRNA degradation or whether it is the only virion component required for this activity. In this paper we report that transfaction of cells with UL41 inhibits expression of a cotransfected CAT reporter gene and that the inhibition is not dependent upon other viral genes. Inhibition of CAT expression was due to UL41-dependent reduction of CAT mRNA levels. UL41 alleles encoding polypeptides that lacked vhs activity during virus infections exhibited a similar lack of activity in transfected cells. The results indicate that the UL41 polypeptide is the direct inducer of host mRNA degradation following virus infection and that it is the only virion component directly required for this activity. A 382-amino-acid nonsense polypeptide missing the last 107 residues of UL41 lacked inhibitory activity, but was packaged into virions, while a 343-amino-acid nonsense polypeptide lacked both inhibitory activity and the ability to be packaged.

In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus

The virion host shutoff (vhs) gene of herpes simplex virus encodes a virion polypeptide that induces degradation of host mRNAs at early times and rapid turnover of viral mRNAs throughout infection. To better investigate the vhs function, an in vitro mRNA degradation system was developed, consisting of cytoplasmic extracts from HeLa cells infected with wild-type herpes simplex virus type 1 or a mutant encoding a defective vhs polypeptide. Host and viral mRNAs were degraded rapidly in extracts from cells productively infected with wild-type herpes simplex virus type 1 but not in extracts from mock-infected cells or cells infected with the mutant vhs1. In contrast, 28S rRNA was stable in all three kinds of extract. Accelerated turnover of host mRNAs was also observed in extracts from cells infected with wild-type virus in the presence of dactinomycin, indicating that the activity was induced by a structural component of the infecting virions. The in vitro vhs activity was inactivated by heat or proteinase K digestion but was insensitive to brief treatment of the extracts with micrococcal nuclease. It was not inhibited by placental RNase inhibitor, it exhibited a strong dependence upon added Mg2+, it was active at concentrations of K+ up to 200 mM, and it did not require the components of an energy-generating system. In summary, the in vitro mRNA degradation system appears to accurately reproduce the vhs-mediated decay of host and viral mRNAs and should be useful for studies of the mechanism of vhs action.