The herpes simplex virus virion host shutoff function.

The virion host shutoff (vhs) function of herpes simplex virus (HSV) limits the expression of genes in the infected cells by destabilizing both host and viral mRNAs. vhs function mutants have been isolated which are defective in their ability to degrade host mRNA. Furthermore, the half-life of viral mRNAs is significantly longer in cells infected with the vhs-1 mutant virus than in cells infected with the wild-type (wt) virus. Recent data have shown that the vhs-1 mutation resides within the open reading frame UL41. We have analyzed the shutoff of host protein synthesis in cells infected with a mixture of the wt HSV-1 (KOS) and the vhs-1 mutant virus. The results of these experiments revealed that (i) the wt virus shutoff activity requires a threshold level of input virions per cell and (ii) the mutant vhs-1 virus protein can irreversibly block the wt virus shutoff activity. These results are consistent with a stoichiometric model in which the wt vhs protein interacts with a cellular factor which controls the half-life of cell mRNA. This wt virus interaction results in the destabilization of both host and viral mRNAs. In contrast, the mutant vhs function interacts with the cellular factor irreversibly, resulting in the increased half-life of both host and viral mRNAs.     

Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.     

Herpes simplex virus tegument protein VP22 contains an internal VP16 interaction domain and a C-terminal domain that are both required for VP22 assembly into the virus particle

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N- and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22-a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly.     

Virion component of herpes simplex virus type 1 KOS interferes with early shutoff of host protein synthesis induced by herpes simplex virus type 2 186.

Herpes simplex virus (HSV) strains HSV type 1 (HSV-1) KOS and HSV-2 186 are representative of delayed and early shutoff strains, respectively, with regard to their ability to inhibit protein synthesis in Friend erythroleukemia cells. When these cells were simultaneously infected with HSV-1 KOS and HSV-2 186, HSV-1 KOS interfered with the rapid suppression of globin synthesis induced by HSV-2 186. The observed interference was competitive and not due to exclusion of HSV-2 by HSV-1 at the level of adsorption. Furthermore, UV-irradiated HSV-1 KOS was also effective at interfering with the early shutoff function of HSV-2 186, indicating that a virion component is responsible for the observed interference.     

The virion host shutoff protein of herpes simplex virus type 1: messenger ribonucleolytic activity in vitro.

Shortly after tissue culture cells are infected with herpes simplex virus (HSV) type 1 or 2, the rate of host protein synthesis decreases 5- to 10-fold and most host mRNAs are degraded. mRNA destabilization is triggered by the virion host shutoff (vhs) protein, a virus encoded, 58-kDa protein located in the virion tegument. To determine whether it can function as a messenger RNase (mRNase), the capacity of vhs protein to degrade RNA in vitro in absence of host cell components was assessed. Two sources of vhs protein were used in these assays: crude extract from virions or protein translated in a reticulocyte-free system. In each case, wild-type but not mutant vhs protein degraded various RNA substrates. Preincubation with anti-vhs antibody blocked RNase activity. These studies do not prove that vhs protein on its own is an mRNase but do demonstrate that the protein, either on its own or in conjunction with another factor(s), has the biochemical property of an mRNase, consistent with its role in infected cells.     

Selective ablation of virion host shutoff protein RNase activity attenuates herpes simplex virus 2 in mice

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.     

Identification of a region in the herpes simplex virus scaffolding protein required for interaction with the portal

The herpes simplex virus type 1 capsid is a protective shell that acts as a container for the genetic material of the virus. After assembly of the capsid, the viral DNA is translocated into the capsid interior through a channel formed by the portal. The portal is composed of a dodecamer of UL6 molecules which form a ring-like structure found at a single vertex within the icosahedron. Formation of portal-containing capsids minimally requires the four structural proteins (VP5, VP19C, VP23, and UL6) and a scaffolding protein (UL26.5). Recently, an interaction between UL26.5 and the portal has been identified, suggesting the scaffold functions by delivering the portal to the growing capsid shell. The aim of this study was to identify regions within UL26.5 required for its interaction with the portal. A specific region was identified by mutational analysis. Deletion of scaffold amino acids (aa) 143 to 151 was found to be sufficient to inhibit formation of the scaffold-portal complex as assayed in vitro. The aa 143 to 151 contain the sequence YYPGE, which is highly conserved among alpha herpesviruses. Although it did not bind to the portal, the Delta143-151 mutant was found to retain the ability to support assembly of morphologically normal capsids in vitro. Such capsids, however, did not contain the portal. The results suggest assembly of portal-containing capsids requires formation of a scaffold-portal complex in which intermolecular contact is dependent on scaffold aa 143 to 151.     

Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein.

Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fusion proteins were then incubated with 35S-labeled herpes simplex virus type 1-infected cell lysates containing VP5. The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads. Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C-terminal domain alone is sufficient for interaction with VP5. Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif. Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one. A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong alpha-helix structure. Most interestingly, the alpha-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the alpha-helix. Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction. Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5. In addition, a cyclic analog of the minimal peptide which is designed to stabilize an alpha-helical structure competed more efficiently than the minimal peptide. The evidence suggests that the C-terminal end of ICP35 forms an alpha-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5.     

Analysis of the interaction between the essential herpes simplex virus 1 tegument proteins VP16 and VP1/2

The incorporation of tegument proteins into the herpes simplex virus 1 (HSV-1) virion during virion assembly is thought to be a complex, multistage process occurring via numerous interactions between the tegument and the capsid, within the tegument, and between the tegument and the envelope. Here, we set out to examine if the direct interaction between two essential tegument proteins VP1/2 and VP16 is required for connecting the inner tegument with the outer tegument. By using glutathione S-transferase (GST) pulldowns, we identified an essential role of lysine 343 in VP16, mutation of which to a neutral amino acid abrogated the interaction between VP1/2 and VP16. When the K343A substitution was inserted into the gene encoding VP16 (UL48) of the viral genome, HSV-1 replicated successfully although its growth was delayed, and final titers were reduced compared to titers of wild-type virus. Surprisingly, the mutated VP16 was incorporated into virions at levels similar to those of wild-type VP16. However, the analysis of VP16 on cytoplasmic capsids by fluorescence microscopy showed that VP16 associated with cytoplasmic capsids less efficiently when the VP16-VP1/2 interaction was inhibited. This implies that the direct interaction between VP1/2 and VP16 is important for the efficiency/timing of viral assembly but is not essential for HSV-1 replication in cell culture. These data also support the notion that the incorporation of tegument proteins into the herpesviruses is a very complex process with significant redundancy.     

Control of mRNA stability by the virion host shutoff function of herpes simplex virus

vhs1 is a mutant of herpes simplex virus type 1 that is defective in the virion host shutoff function responsible for the degradation of cellular mRNAs and the concomitant shutoff of host protein synthesis. In this study, the effect of the vhs1 mutation on the metabolism of viral mRNAs was examined by measuring the half-lives and patterns of accumulation of 10 different viral mRNAs representing all kinetic classes. The vhs1 mutation had the effect of dramatically lengthening the cytoplasmic half-lives of all 10 mRNAs. In wild-type virus infections, the 10 mRNAs had similar half-lives, suggesting that little, if any, target mRNA selectivity was exhibited by the vhs function. The vhs1 mutation caused overaccumulation of a number of mRNAs. The effect was most dramatic for the alpha (immediate-early) mRNA for ICP27 and the beta (early) mRNAs encoding thymidine kinase, ICP8, and DNA polymerase. Whereas in wild-type infections these mRNAs increased to peak levels and subsequently declined in abundance, in vhs1 infections they continued to accumulate until late times. A significant but less dramatic overaccumulation was observed for several beta-gamma (delayed-early) and gamma (late) mRNAs. The results suggest that the vhs protein plays an important role in determining the half-lives of viral mRNAs belonging to all kinetic classes and in so doing is important in the normal downregulation at late times of alpha and beta gene expression.     

Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein

Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo, (ii) necessary for the colocalization of UL31 and UL34 in infected cells, and (iii) essential for normal viral replication.     

The virion host shutoff protein of herpes simplex virus type 1 has RNA degradation activity in primary neurons

The virion host shutoff protein (Vhs) of herpes simplex virus type 1 induces destabilization of mRNA following infection. Our study of primary neurons from CD-1 mice demonstrates that vhs is functional in neurons but that more Vhs is required to mediate RNA degradation in neurons than in other susceptible cells.     

Particle formation by a conserved domain of the herpes simplex virus protein VP22 facilitating protein and nucleic acid delivery

VP22, a structural protein of herpes simplex virus, exhibits unusual trafficking properties which we proposed might be exploited in gene and protein delivery applications. To pursue the use of the protein itself for cargo delivery into cells, we developed an expression system for the C-terminal half of VP22, residues 159-301 (VP22.C1), and purified the protein in high yields. Addition of short oligonucleotides (ODNs) induced the assembly of novel particles, which were regular spheres with a size range of 0.3 to 1.0 microm in diameter, incorporating both protein and ODN. Following the particles in living cells using fluorescently tagged ODNs, we show that they enter efficiently within 2-4 h, and reside stably in the cell cytoplasm for up to several days. Remarkably, however, light activation induced particle disruption and release of the protein and ODN to the nucleus and cytoplasm within seconds, a process that we have captured by time lapse microscopy. In addition to delivering antisense ODNs, ribozymes, and RNA/DNA hybrids, the VP22.C1 protein could also be modified to include peptides or proteins. These particles have the potential for delivery of a wide range of therapeutic agents in gene therapy and vaccine development.