Mutational analysis of the promoter region of the alpha 27 gene of herpes simplex virus 1 within the context of the viral genome.

In herpes simplex virus 1-infected cells, the first set of genes to be expressed (alpha genes) is induced by the alpha gene trans-inducing factor (alpha TIF), a virion structural protein. The cis-acting site in the 5' untranscribed domain of alpha genes was previously reported to be the sequence 5'-GYATGNTAATGARATTCYTTGNGGG noncoding (where Y is a pyrimidine, R is a purine, N is any base), which binds a host protein designated alpha H1 (also termed the octamer binding protein, OTF-1, Oct-1, etc.) and which, together with this and possibly other proteins, forms complexes with alpha TIF. To determine the role of the various components of this cis-acting site and of other sequences shared by the alpha genes, we constructed 17 mutants spanning 110 nucleotides of the promoter domain of the alpha 27 gene and made a series of chimeric genes. Each chimeric gene embodying one set of these mutations was inserted into the viral genome and measurements were made of (i) accumulated mRNA under conditions in which only alpha genes were expressed and (ii) the capacity of the mutated sequence to form complexes containing alpha TIF and alpha H1 proteins. We report that (i) transversions in the "TAAT" sequence abolished both complex formation and induction of the chimeric alpha gene, (ii) mutations in the octamer binding site sequence upstream from TAAT or of the downstream GARAT abolished alpha TIF complex formation and also reduced alpha mRNA accumulation, (iii) mutations in a "CAAT" box also reduced expression of mRNA without affecting the formation of DNA-protein complexes containing alpha TIF, and (iv) mutations in sequences immediately downstream from TAATGARAT and in a pair of GA-rich elements reduced alpha mRNA expression whereas mutations between these elements had no effect on the accumulation of the mRNA. The results are consistent with the conclusion that both the alpha H1 octamer binding site ATGNTAAT and the GARAT sequence play a significant role in the induction of alpha genes. For optimal gene expression, however, additional elements downstream from the GARAT sequence and in other regions of the alpha promoter must be present.     

Host cell proteins bind to the cis-acting site required for virion-mediated induction of herpes simplex virus 1 alpha genes.

The herpes simplex virus 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. In productively infected cells, the alpha genes are expressed first, and a virion protein, the alpha-trans-inducing factor (alpha-TIF), acts in trans to enhance their expression. Induction of the alpha genes by alpha-TIF requires the presence of a trans-induction cis-acting site (alpha-TIC), and one to three homologs of the alpha-TIC sequence are contained in the regulatory domains of all alpha genes. We report that small DNA fragments from regulatory domains of alpha 0, alpha 4, and alpha 27 genes containing alpha-TIC homologs formed complexes with host but not viral proteins. DNase protection studies indicated that the major host protein complex alpha-H1 detected in DNA gel retardation assays bound asymmetrically across the alpha-TIC site. All DNA fragments containing alpha-TIC homologs, but not those lacking the homolog, competed for the binding of this complex. The location of the binding site of the other host proteins is not yet known. Simian virus 40 DNA fragments containing a homolog of the alpha-TIC sequence also competed with herpes simplex virus DNA fragments carrying authentic alpha-TIC homologs for the alpha-H1 protein complex.     

Differentiation between alpha promoter and regulator regions of herpes simplex virus 1: the functional domains and sequence of a movable alpha regulator.

The herpes simplex virus genome consists of at least three groups of genes--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. We have established that the elements involved in regulation of alpha genes are a sequence that promotes gene expression and a sequence that confers alpha regulation on the gene by responding to trans-acting regulatory signals. The domains of these sequences were mapped by determining the regulation of thymidine kinase (TK) in L cells converted to TK+ phenotype by chimeric TK indicator genes. The chimeric genes were constructed from appropriate portions of the TK gene fused to donor sequences derived from the 5' nontranscribed and nontranslated leader portions of the viral alpha gene 4. The results were as follows. (i) The natural beta TK indicator extending 5' up to -80 and the chimeric alpha TK extending 5' up to -110 both converted cells to TK+ phenotype but were not regulated. (ii) A segment of the regulator region of the alpha gene 4, extending 5' from position -110, confers inducible alpha-type regulation when fused to the nonregulated but expressible beta TK indicator described above. (iii) The extent of gene induction appears to hinge on the size of the regulatory region inserted into the chimeric gene and correlates with the presence of repeated consensus sequences and G+C-rich inverted repeats in the regulatory region of the alpha gene 4 and other alpha genes.     

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.     

Separation of sequences defining basal expression from those conferring alpha gene recognition within the regulatory domains of herpes simplex virus 1 alpha genes.

The genes of herpes simplex virus 1 form three major groups--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. To determine how the infected cell differentiates between these gene groups, alpha-regulated chimeric genes were constructed in earlier studies by fusing the structural sequences of the thymidine kinase (TK) gene, a beta gene, to the 5' noncoding sequences of alpha genes. These studies showed that (i) one or more structural components of the virion act in trans to increase alpha gene expression and (ii) the 5' noncoding sequences of alpha genes contain cis-acting domains that promote gene expression and confer alpha-gene regulation. These two domains could be moved independently, but the regulatory domain required a promoter for its function. We report here the properties of three sequences containing features common to the regulatory regions of all alpha genes. Sequence 1, containing (G + C)-rich inverted repeats, increased the basal level of TK expression when fused 5' to either the alpha gene 4 promoter or the truncated beta TK promoter. The effect was to some extent orientation dependent. Moreover, sequence 1 restored beta regulation to the truncated beta TK promoter but did not confer alpha-specific regulation on any of the chimeric genes tested. Sequences 2 (49 base pairs) and 3 (29 base pairs), containing an (A + T)-rich homolog from alpha gene 27 and alpha gene 0, respectively, restored alpha-specific regulation to the alpha promoter gene but only sequence 2 conferred alpha regulation on the truncated beta promoter gene. Our results indicate that (i) in natural beta TK the promoter and regulatory domains overlap, (ii) sequence 1 determines basal level of expression and substitutes for a promoter component that is essential for beta but not alpha regulation, and (iii) conversion of a gene with a promoter into an alpha gene requires two elements. Sequence 2 may contain both whereas sequence 3 contains only one.     

Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.

Transfection of Syrian hamster embryo cells with limit digests of Bgl II-, Hpa I-, or Bgl II/Hpa I-cleaved DNA from herpes simplex virus type 2 (strain S-1) but not with salmon sperm DNA resulted in the appearance of refractile, morphologically altered cells at a frequency of 10(-5)/0.005 microgram of viral DNA within two to four passages. Transformed lines manifested reduced serum requirement and anchorage-independent growth and were tumorigenic in newborn hamsters. They expressed ICP10, a viral protein immunologically identical to the cervical-tumor-associated AG-4 antigen. Transforming activity was localized in the 16.5 x 10(6)-dalton Bgl II/Hpa I double-digest fragment CDs-1, which exhibited sequence homology to the Bgl II/Hpa I fragment CD of DNA from herpes simplex virus type 2 strain 333, mapping between coordinates 0.43 and 0.58 on the physical map of strain 333 DNA. This fragment, CD333, was also shown to induce neoplastic transformation.     

Alpha 4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter-regulatory domains of alpha genes and of selected other viral genes

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. However, the mechanism by which alpha 4 regulates gene expression remained unknown. We report that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from infected-cell lysates as detected by a gel electrophoresis binding assay. The protein(s) exhibits sequence specificity since autologous DNA fragments but not heterologous DNA fragments, synthetic polydeoxynucleotide chains, or salmon sperm DNA competitively displace the DNA probe from the complexes. Murine monoclonal antibody to alpha 4 protein added before or after DNA-protein complex formation further retarded the electrophoretic mobility of the complexes whereas monoclonal antibody to alpha 0, alpha 27, or to a viral glycoprotein had no effect. Complexes consisting of the promoter-regulatory domain of the beta-class thymidine kinase gene and infected cell proteins were low in abundance and could be detected only in the presence of antibody to alpha 4 protein. The alpha 4 protein, therefore, forms stable complexes with promoter-regulatory domains of alpha genes and of selected other herpes simplex virus type 1 genes either alone or in combination with other proteins.     

Casein kinase II specifically nucleotidylylates in vitro the amino acid sequence of the protein encoded by the alpha 22 gene of herpes simplex virus 1.

An earlier report has shown that eight viral proteins with a common amino acid sequence (R/P)RA(P/S)R are nucleotidylyated in vitro by nuclear extracts from cells infected with herpes simplex virus 1. One, the product of the alpha 22 gene, is nucleotidylylated in the absence of viral proteins made late in infection. A chimeric protein (GST22P) consisting of amino acids 50-200 of the alpha 22 coding sequence fused to the C terminus of the glutathione S-transferase was nucleotidylylated by enzymes in nuclear extracts of infected or mock-infected cells and also by a casein kinase II enzyme purified from the sea star. The enzyme did not nucleotidylylate common casein kinase II substrates (casein, phosvitin) and the reaction was inhibited by heparin. The results are consistent with the hypothesis that nucleotidylylation of the eight viral proteins involves casein kinase II.     

Activation of NF-kappaB in cells productively infected with HSV-1 depends on activated protein kinase R and plays no apparent role in blocking apoptosis

Microarray data reported elsewhere indicated that herpes simplex virus 1 induces the up-regulation of nuclear factor kappaB (NF-kappaB)-regulated genes, including that of its inhibitor, IkappaBalpha, consistent with the reports that wild-type virus induces the activation of NF-kappaB. In this report we show that activation of NF-kappaB in infected cells is linked to the activation of protein kinase R (PKR). Specifically: (i) PKR is activated in infected cells although the effects of the activated enzyme on protein synthesis are negated by the viral gene gamma134.5, which encodes a protein phosphatase 1alpha accessory factor that enables the dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. NF-kappaB is activated in wild-type murine embryonic fibroblasts but not in related PKR-null cells. (ii) In cells infected with a replication-competent Deltagamma134.5 mutant (R5104), but carrying a US11 gene expressed early in infection, eukaryotic translation initiation factor 2alpha is not phosphorylated, and in in vitro assays, PKR bound to the US11 protein is not phosphorylated on subsequent addition of double-stranded RNA. Here we report that this mutant does not activate PKR, has no effect on the accumulation of IkappaBalpha, and does not cause the translocation of NF-kappaB in infected cells. (iii) One hypothesis advanced for the activation of NF-kappaB is that it blocks apoptosis induced by viral gene products. The replication-competent R5104 mutant does not induce the programmed cell's death. We conclude that in herpes simplex virus 1-infected cells, activation of NF-kappaB depends on activation of PKR and that NF-kappaB is not required to block apoptosis in productively infected cells.     

Replication-incompetent herpesvirus vector delivery of an interferon alpha gene inhibits human immunodeficiency virus replication in human monocytes.

Human monocytes and macrophages are nondividing cells that serve as a major reservoir for human immunodeficiency virus (HIV) at all stages of infection. To investigate viral-mediated gene delivery as a means of inhibiting HIV replication in human monocytes, a replication-incompetent herpes simplex virus vector was developed that expressed human interferon alpha. Monocytes infected with this herpes simplex virus vector and then challenged with HIV showed dramatically reduced cytopathic effects and HIV replication compared to control treated monocytes. Similar effects on HIV replication were observed if monocytes were first infected with HIV and then treated with the recombinant vectors. These results demonstrate that replication-incompetent herpes simplex virus gene delivery of interferon alpha directly to human monocytes can greatly decrease HIV replication and suggest that such a vector might deliver therapeutically important genes directly to sites of HIV infection.     

The U(L)41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins

The herpes simplex virus 1 ORF U(L)41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or beta-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3' domain disappears rapidly, a large 5' domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3' untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.     

Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1.

The protein encoded by the gamma 134.5 gene of herpes simplex virus precludes premature shutoff of protein synthesis in human cells triggered by stress associated with onset of viral DNA synthesis. The carboxyl terminus of the protein is essential for this function. This report indicates that the shutoff of protein synthesis is not due to mRNA degration because mRNA from wild-type or gamma 134.5- virus-infected cells directs protein synthesis. Analyses of the posttranslational modifications of translation initiation factor eIF-2 showed the following: (i) eIF-2 alpha was selectively phosphorylated by a kinase present in ribosome-enriched fraction of cells infected with gamma 134.5- virus. (ii) Endogenous eIF-2 alpha was totally phosphorylated in cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene but was not phosphorylated in mock-infected or wild-type virus-infected cells. (iii) Immune precipitates of the PKR kinase that is responsible for regulation of protein synthesis of some cells by phosphorylation of eIF-2 alpha yielded several phosphorylated polypeptides. Of particular significance were two observations. First, phosphorylation of PKR kinase was elevated in all infected cells relative to the levels in mock-infected cells. Second, the precipitates from lysates of cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene contained an additional labeled phosphoprotein of M(r) 90,000 (p90). This phosphoprotein was present in only trace amounts in the immunoprecipitate from cells infected with wild-type virus or mutants lacking a portion of the 5' domain of gamma 134.5. We conclude that in the absence of gamma 134.5 protein, PKR kinase complexes with the p90 phosphoprotein and shuts off protein synthesis by phosphorylation of the alpha subunit of translation initiation factor eIF-2.     

Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products.

In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.     

Possible peptide chain termination mutants in thymide kinase gene of a mammalian virus, herpes simplex virus.

Mutations in the viral gene coding for the thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) induced by herpes simplex virus have been obtained by selection of virus resistant to bromodeoxyuridine when grown in thymidine-kinase-deficient LMTK- mouse cells. Proteins labeled after infection of Vero (monkey) cells with herpes simplex virus were analyzed by gel electrophoresis and one protein of about 40,000 daltons was consistently altered in a number of thymidine-kinase-deficient mutants. Many viral mutants lacked this peptide and one class of these mutants induced the synthesis of new shorter peptides. Revertant virus could be selected which simultaneously regained the ability to induce thymidine kinase activity, regained the intact thymidine kinase peptide, and lost the ability to synthesize the shorter peptide fragment. These mutants comprise a class of animal virus mutants which have the properties expected of peptide chain termination mutants.