mRNA decay during herpesvirus infections: interaction between a putative viral nuclease and a cellular translation factor.

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5' end of mRNAs prior to the 3' end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using the Saccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathione S-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

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.

Site-directed mutagenesis of the virion host shutoff gene (UL41) of herpes simplex virus (HSV): analysis of functional differences between HSV type 1 (HSV-1) and HSV-2 alleles.

During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity of KOS when substituted for the corresponding portions of the KOS protein (D. N. Everly, Jr., and G. S. Read, J. Virol. 71:7157-7166, 1997). These results have been extended through the analysis of more than 50 site-directed mutants of UL41 in which selected HSV-2 amino acids were introduced into an HSV-1 background and HSV-1 amino acids were introduced into the HSV-2 allele. The HSV-2 amino acids R22 and E25 were found to contribute dramatically to the greater activity of the HSV-2 allele, as did the HSV-2 amino acids A396 and S423. The substitution of six HSV-2 amino acids between residues 210 and 242 enhanced the HSV-1 activity to a lesser extent. In most cases, individual substitutions or the substitution of combinations of fewer than all six amino acids reduced the UL41 activity to less than that of KOS. The results pinpoint several type-specific amino acids that are largely responsible for the greater activity of the UL41 polypeptide of HSV-2. In addition, several spontaneous mutations that abolish detectable UL41 activity were identified.

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

During lytic herpes simplex virus (HSV) infections, the half-lives of host and viral mRNAs are regulated by the HSV virion host shutoff (Vhs) protein (UL41). The sequences of the UL41 polypeptides of HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical. In spite of this similarity, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. To examine type-specific differences in Vhs function, we compared the Vhs activities of UL41 alleles from HSV-1(KOS) and HSV-2(333) by assaying the ability of a transfected UL41 allele to inhibit expression of a cotransfected reporter gene. Both HSV-1 and HSV-2 alleles inhibited reporter gene expression over a range of vhs DNA concentrations. However, 40-fold less of the HSV-2 allele was required to yield the same level of inhibition as HSV-1, indicating that it is significantly more potent. Examination of chimeric UL41 alleles containing various combinations of HSV-1 and HSV-2 sequences identified three regions of the 333 polypeptide which increase the activity of KOS when substituted for the corresponding amino acids of the KOS protein. These are separated by two regions which have no effect on KOS activity, even though they contain 43 of the 74 amino acid differences between the parental alleles. In addition, alleles encoding a full-length KOS polypeptide with a 32-amino-acid N-terminal extension retain considerable activity. The results begin to identify which amino acid differences are responsible for type-specific differences in Vhs activity.

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.

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 viral 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 transfection 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.

Isolation of a herpes simplex virus type 1 mutant with a deletion in the virion host shutoff gene and identification of multiple forms of the vhs (UL41) polypeptide.

The virion host shutoff (vhs) gene (UL41) of herpes simplex virus type 1 (HSV-1) encodes a virion component that induces degradation of host mRNAs and the shutoff of most host protein synthesis. Subsequently, the vhs protein accelerates the turnover of all kinetic classes of viral mRNA. To identify the vhs (UL41) polypeptide within infected cells and virions, antisera raised against a UL41-lacZ fusion protein were used to characterize the polypeptides encoded by wild-type HSV-1 and two mutants: vhs1, a previously characterized mutant that lacks detectable virion host shutoff activity, and vhs-delta Sma, a newly constructed mutant containing a deletion of 196 codons from UL41. Two forms of the vhs (UL41) polypeptide were identified in cells infected with the wild-type virus or vhs1. Wild-type HSV-1 produced a major 58-kDa polypeptide, as well as a less abundant 59.5-kDa form of the protein, while vhs1 produced 57- and 59-kDa polypeptides that were approximately equally abundant. Although for either virus, both forms of the protein were phosphorylated, they differed in the extent of phosphorylation. While both vhs polypeptides were found in infected cells, only the faster migrating, less phosphorylated form was incorporated into virions. vhs-delta Sma encoded a smaller, 31-kDa polypeptide which, although present in infected cells, was not incorporated into virions. The results identify multiple forms of the vhs (UL41) polypeptide and suggest that posttranslational processing affects its packaging into virions, as well as its ability to induce mRNA degradation.

Herpes simplex virus interferes with monocyte accessory cell function.

HSV is a successful human pathogen that causes severe infections in immunocompromised adults and newborn humans. The possibility that HSV might evade host immune responses by interfering with accessory cell function was investigated in vitro using newborns' monocytes adhered to plastic. These cells lost their ability to present staphylococcal enterotoxin B to resting T cells in a stimulatory form after overnight culture with HSV. The interference with T cell proliferation required live virus and was abolished by heat- or UV inactivation. The T cell proliferative response was restored by the addition of IL-2. Furthermore, HSV-precultured monocytes had a reduced production of IL-1 alpha and TNF-beta after phorbol-ionomycin stimulation. Wild-type HSV and a HSV mutant lacking the primary virion host shutoff protein both interfered with IL-1 synthesis and presentation of staphylococcal enterotoxin B for T cell proliferation. These results suggest that HSV can interfere with the provision by human monocytes of costimulator factors that are essential for T cell stimulation. This effect of HSV may be due to secondary shutoff mechanisms that decrease host protein synthesis or secretion after HSV infection.

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.

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.

Proteins associated with mRNA in cells infected with herpes simplex virus.

The structure of messenger ribonucleoprotein (mRNP) complexes in herpes simplex virus type 1 (HSV-1) infected cells was analyzed by examining the proteins that could be crosslinked to polyadenylated mRNAs by irradiation of intact cells with ultraviolet light. The profiles of crosslinked proteins were qualitatively similar for mRNPs from mock infected and infected cells. However, infection with wild type HSV-1 caused a decrease in the abundance of a major 52 kda protein and an increase in a 49 kda protein. These changes were observed at early times after infection. They occurred following infection with wild type HSV-1 under conditions that blocked viral gene expression, but not following infection with the virion host shutoff mutant vhs 1.

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.

A mutant of herpes simplex virus type 1 exhibits increased stability of immediate-early (alpha) mRNAs.

vhs1 is a herpes simplex virus type 1 mutant defective in the shutoff of both host and alpha polypeptide synthesis. In cycloheximide reversal experiments, alpha mRNAs were significantly more stable in vhs1-infected cells than in cells infected with wild-type virus, whether assayed by in vitro translation or Northern blotting.

In vitro and in vivo transcription initiation sites on the TK-encoding BamHI Q fragment of HSV-1 DNA.

The sites of in vitro transcription initiation on the BamHI Q fragment of herpes simplex virus DNA have been compared with the sites of 5' ends of RNAs made in vivo after virus infection. S1-nuclease protection analysis of these RNAs shows that there are in vivo counterparts for each of the five previously identified in vitro transcripts. The whole-cell-extract RNA polymerase II transcription system faithfully initiates RNAs predominantly at bona fide in vivo start sites and gives few, if any, false positive start sites. Further, antiparallel, self-complementary transcripts from the BamHI Q fragment were observed in the coding region of the HSV thymidine kinase gene.

Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides.

Six mutants isolated from herpes simplex virus type 1 were judged to be defective with respect to the virion-associated function acting to rapidly shut off host polypeptide synthesis in herpes simplex virus-infected cells. The mutants were capable of proper entry into the cells, but, unlike the parent wild-type virus, they failed to shut off host polypeptide syntehsis in the presence of actinomycin D. They were consequently designated as virion-associated host shutoff (vhs) mutants. In the presence of actinomycin D, three of the mutants, vhs1, -2, and -3, failed to shut off the host at both 34 and 39 degrees C, whereas vhs4, -5, and -6 exhibited a temperature-dependent vhs phenotype. Since the mutants were capable of growth at 34 degrees C, it appeared that the vhs function was not essential for virus replication in cultured cells. Temperature-shift experiments performed with the vhs4 mutant showed that an active vhs function was required throughout the shutoff process and that, once established, the translational shutoff could not be reversed. In the absence of actinomycin D, the mutants induced a generalized, secondary shutoff of host translation, which required the synthesis of beta (early) or gamma (late) viral polypeptide(s). The vhs mutants appeared to be defective also with respect to post-transcriptional shutoff of alpha (immediate early) viral gene expression, since (i) cells infected with mutant viruses overproduced alpha viral polypeptides, (ii) there was an increased functional stability of alpha mRNA in the vhs1 mutant virus-infected cells, and (iii) superinfection of vhs1-infected cells with wild-type virus, in the presence of actinomycin D, resulted in a more pronounced shutoff of alpha polypeptide synthesis from preformed alpha mRNA than equivalent superinfection with vhs1 virus. The data suggest that the synthesis of alpha polypeptides in wild-type virus infections is subject to a negative post-transcriptional control involving viral gene product(s) present in infected cell lysates constituting virus stocks. The vhs1 mutant and possibly other vhs mutants contain a mutation in the gene encoding this function.

In vitro transcription of the thymidine kinase gene of herpes simplex virus.

We transcribed in vitro a cloned 3.5-kilobase fragment of herpes simplex virus type 1 DNA that contains the gene for the viral thymidine kinase. Extracts from uninfected HeLa cells produced five in vitro transcripts, one of which initiated at the in vivo start site for the thymidine kinase mRNA (an early viral message). A second in vitro transcript initiated at or near the start site for a major late in vivo viral mRNA. The remaining three in vitro transcripts may correspond to minor in vivo mRNA species. Sequences similar to the "T-A-T-A" and "C-A-A-T" boxes, which may be involved in the control of transcription of a variety of viral and cellular genes, were found to precede the initiation site of each of the five in vitro transcripts. Considerable overlap of transcription units was observed.

Genetic studies of cell fusion induced by herpes simplex virus type 1.

Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infections (complementation test). In single infections, fusion began 4 to 6 h after infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was a significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild-type infected cells. Fusion was decreased in mixed infections with the mutants and wild-type virus, but the mutants displayed a codominant fusion phenotype. Fusion was not decreased in mixed infection with pairs of mutants, indicating that the mutants, with one possible exception, are members of the same complementation group. A linkage map was established for six of the mutants by analysis of recombination frequencies.

Kinetics of cell fusion induced by a syncytia-producing mutant of herpes simplex virus type I.

We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 X 10(4) cells/cm2 and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a smiple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay.