Classes Of Viral Genes Research Articles

Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications.

ABSTRACTLike their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016) contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation) associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes.

Virus‐encoded endonucleases: expected and novel functions

Endonucleases catalyze critical steps in the processing, function, and turnover of many cellular RNAs. It is, therefore, not surprising that a number of viruses encode endonucleases that play important roles in viral gene expression. The virion host shutoff (Vhs) endonuclease of herpes simplex virus, the SOX protein of Kaposi Sarcoma Herpesvirus (KSHV), and the influenza virus PB1 endonuclease have well-characterized functions that stem from their abilities to cleave RNA. Vhs accelerates turnover of many cellular and viral mRNAs, redirecting the cell from host to viral gene expression, counteracting key elements of the innate immune response, and facilitating sequential expression of different classes of viral genes. SOX reduces synthesis of many host proteins during the lytic phase of KSHV infections. PB1 is a component of the influenza RNA polymerase that snatches capped oligonucleotides from cellular pre-mRNAs to serve as primers during viral mRNA synthesis. However, all three proteins have important second functions. Vhs stimulates translation of the 3' cistron of bicistronic mRNAs that have selected cellular internal ribosome entry sites, and stimulates polysome loading and translation of selected viral mRNAs at late times during productive infections. SOX has an alkaline exonuclease activity that is important for processing and maturation of newly synthesized copies of the KSHV genome. The influenza RNA polymerase binds the cap and 5' region of viral mRNAs and recruits eIF4G and other factors to viral mRNAs, allowing them to be translated under conditions of reduced eIF4E functionality. This review will discuss the novel and expected functions of these viral endonucleases.

Imaging immediate-early and strict-late promoter activity during oncolytic herpes simplex virus type 1 infection and replication in tumors

An increasing number of oncolytic viruses have been developed and studied for cancer therapy. In response to needs for non-invasive monitoring and imaging of oncolytic virotherapy, several different approaches, including a positron emission tomography-based method, a method using secreted marker peptides, and optical imaging-based methods, have been reported. Among these modalities, we utilized the luciferase-based bioluminescent assay/imaging systems to determine the kinetics and dynamics of a productive viral infection. The replication cycle of herpes simplex virus type 1 (HSV-1) is punctuated by a temporal cascade of three classes of viral genes: immediate-early (IE), early (E) and late (L) genes. U(L)39- and gamma(1)34.5-deleted, replication-conditional HSV-1 mutants that express firefly luciferase under the control of the IE4/5 or strict-late gC promoters were generated. These oncolytic viruses were examined in cultured cells and a mouse tumor model. IE promoter- and strict-late promoter-mediated luciferase expression was confirmed to indicate viral infection and replication, respectively. Incorporation of a strict-late promoter-driven luciferase cassette into oncolytic HSV-1 vectors would be useful for assessing tumor oncolysis in preclinical tumor treatment studies.

Prolonged activation of NF-κB by human cytomegalovirus promotes efficient viral replication and late gene expression

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-κB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-κB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498–4507). Because a second, sustained increase in NF-κB activity following the initial phase of NF-κB activation was also observed, we investigated the role that this prolonged NF-κB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-κB activation was blocked by pretreatment with NF-κB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-κB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-κB in the regulation of the later class of viral genes.

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.

Replacement of the herpes simplex virus type 1 Vmw175 DNA binding domain with its varicella-zoster virus counterpart results in a protein with novel regulatory properties that can support virus growth.

The alphaherpesviruses encode major immediate early transactivator proteins that are essential for the expression of later classes of viral genes. We have previously shown that the extensive sequence similarity between the herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) members of the family (proteins Vmw175 and VZV140k) extends to function, since a virus which expresses VZV140k in place of Vmw175 is able to grow, albeit at reduced efficiency. We have also shown that the DNA binding characteristics of the isolated DNA binding domains of Vmw175 and VZV140k are related but distinct. In order to assess whether the different DNA binding properties of the two proteins are responsible for the differences in their individual transcriptional regulatory functions, we constructed a plasmid and an HSV-1 virus in which the VZV140k DNA binding domain coding sequences replace those of Vmw175. The characteristics of the resultant hybrid protein in transfection assays and during virus infection suggest that the nature of the DNA binding domain plays a significant role in the transactivation and repression properties of the Vmw1 75 family of proteins.

Expression of a Murine Cytomegalovirus Early-Late Protein in "Latently" Infected Mice

Monoclonal antibodies were isolated from the spleen of a latently infected mouse to identify possible antigenic markers for latent murine cytomegalovirus infection. One antibody, AM3, was selected for further study. Characterization of the antigen recognized by AM3 confirmed that it is the early-late phosphoprotein pp50. The antigen was readily detected by immunoautoradiography in the spleens of acutely and latently infected mice. Low levels of the AM3/pp50 transcript were also detected in latently infected tissues by in situ hybridization. Presence of AM3/pp50 mRNA, as well as IE-1 transcripts, was confirmed by reverse transcription-polymerase chain reaction in 3 of 5 latently infected spleens. The expression of both immediate-early and early-late classes of viral genes suggests that persistent infection may be a component of the MCMV life cycle operationally defined as latency and that this gene is not specific for latency.

Regulation of the Epstein-Barr virus DNA polymerase gene.

The gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.

Activities of heterodimers composed of DNA-binding- and transactivation-deficient subunits of the herpes simplex virus regulatory protein ICP4

Two mutant strains (vi12 and vi13) of herpes simplex virus that contain insertion mutations in the sequences that encode the DNA-binding domain of viral regulatory protein ICP4 were generated. Both mutations disrupted specific DNA binding and resulted in transcriptionally inactive molecules; however, the ability of the mutant proteins to form dimers was retained. The mutant proteins formed heterodimers with an ICP4 deletion mutant (X25) that is nonfunctional but retains the ability to bind to consensus sites. Significantly elevated levels of early (E or beta) and "leaky late" (beta gamma or gamma 1) gene expression were observed upon coexpression of the insertion mutant and X25 ICP4 polypeptides. While the heterodimers composed of the vi13 and X25 peptides possessed DNA-binding activity, those composed of vi12 and X25 did not, indicating that DNA binding by the heterodimers may not be required for restored activity. Despite significant levels of early gene expression and viral DNA synthesis in vi12-infected X25 cells, true late (gamma 2) mRNA was not synthesized. This indicates that the structural requirements for ICP4 induction of different classes of viral genes may be different.

The major transcriptional regulatory protein of herpes simplex virus type 1 includes a protease resistant DNA binding domain

Herpes simplex virus type 1 expresses five immediate-early (IE) polypeptides. In the absence of functional Vmw175 (the product of IE gene 3) activation of transcription of later classes of viral genes and repression of IE gene expression does not occur. The recognition of specific DNA sequences by Vmw175 requires, as determined by sensitivity to mutation, a part of the protein highly conserved in the corresponding proteins of related herpes viruses. However, mutations in other parts of the protein can also disrupt specific DNA binding. This paper shows that the DNA binding domain of Vmw175 can be liberated as a functional unit by digestion with proteinase K. Analysis of mutant Vmw175 proteins showed that the proteinase K resistant domain has an amino terminus between amino acid residues 229 and 292, while its carboxy terminus is between residues 495 and 518. Mutations outside this region which affect DNA binding by the intact protein do not eliminate binding of the proteinase K resistant domain. This implies that direct DNA binding by Vmw175 involves a linear subsection of the polypeptide, and that mutations in other parts of the polypeptide which affect DNA binding of the whole protein do so by indirect means.

A Prominent Serine-rich Region in Vmw175, the Major Transcriptional Regulator Protein of Herpes Simplex Virus Type 1, is not Essential for Virus Growth in Tissue Culture

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) genes of which at least three are involved in the transcriptional regulation of later classes of viral genes. Perhaps the most important of these regulatory proteins is Vmw175, a nuclear phosphoprotein of 1298 predicted amino acid residues. In the absence of functional Vmw175 the virus fails to activate early or late genes or to repress IE gene expression. All viruses of the sub-family alphaherpes-virinae encode polypeptides that are closely related to Vmw175. Mutational studies have shown that regions of homology within this family of gene regulators are generally of functional importance. One of the most striking conserved stretches of amino acid sequence is a run of serine residues followed by a highly acidic region in the amino-terminal fifth of the polypeptide. We have constructed an HSV-1 virus which lacks this serine-rich run within Vmw175. Surprisingly, the virus was viable in tissue culture cells and expressed apparently normal amounts of viral polypeptides. In plaque assays it was very slightly temperature-sensitive and, depending on the state of the host cells, could generate plaques with a syncytial morphology. The mutant protein was able to bind to DNA in a manner indistinguishable from that of the wild-type polypeptide. We conclude that despite its conservation in all of the alphaherpes-virinae so far sequenced, the serine-rich homology is not important for virus growth in tissue culture.

Delineation of regulatory domains of early (beta) and late (gamma 2) genes by construction of chimeric genes expressed in herpes simplex virus 1 genomes.

The expression of the gamma 2 class of viral genes in cells infected with herpes simplex virus 1 requires viral DNA synthesis and functional viral products made earlier in infection. To identify the sequences required for gamma 2 gene expression, we constructed recombinant viruses in which regions of the thymidine kinase gene (tk), a beta gene normally expressed early in infection, were replaced by specific domains of a gamma 2 gene. The phenotypic attributes examined were (i) sensitivity or resistance of expression in cells exposed to sufficient phosphonoacetate to block viral DNA synthesis, properties of gamma 2 and beta genes, respectively; (ii) expression early in infection, a property of beta genes; and (iii) expression late in infection, a property of gamma 2 genes. We report that replacement of nucleotides -200 to +51 of tk with nucleotides -77 to +104 of the gamma 2 gene conferred upon tk all of the tested attributes of gamma 2 genes. The tk sequence in the 5' transcribed noncoding domain downstream of nucleotide +51 played no apparent role in the expression of the chimeric genes. Similarly, tk sequence downstream of -16 and gamma 2 sequence upstream of -12, when juxtaposed in correct orientations, yielded a chimeric gene that was poorly expressed. In contrast, the chimeric gene consisting of tk sequence upstream of -16 fused to gamma 2 sequence downstream of -12 had the attributes of both beta and gamma 2 genes in that it was expressed both early and late in infection and was partially resistant to phosphonoacetate. The capacity for expression late in infection encoded in the gamma 2 5' transcribed noncoding domain was observed in cells infected with a recombinant virus in which gamma 2 nucleotides +17 to +104 were inserted into the 5' transcribed noncoding domain of the tk gene. We conclude that whereas in the beta genes exemplified by the tk gene the regulatory domains are mainly upstream from nucleotide +51, the sequence(s) that confers gamma 2 regulation is downstream from the TATAA box.

Analysis of the functional domains of herpes simplex virus type 1 immediate-early polypeptide Vmw110

At the onset of infection by herpes simplex virus type 1 (HSV-1) in tissue culture, five immediate-early (IE) genes are expressed. At least four of the IE gene products have some role in the transcriptional regulation of later classes of viral genes. This study concerns the product of IE gene 1, Vmw110. Several laboratories have shown that a cloned copy of IE gene 1 can stimulate gene expression in trans in transfection experiments; this transactivation can occur either with Vmw110 by itself or in a strongly synergistic fashion with the product of IE gene 3, Vmw175. As an approach to study the mechanism of this effect, a large number of in-frame insertion mutations in the Vmw110 coding region have been constructed. The effect of the insertions on the ability of Vmw110 to activate transcription in the presence of Vmw175 has been described. This report concerns the effect of the insertions on the ability of Vmw110 to activate gene expression in the absence of Vmw175. In addition, a family of in-frame deletion mutations has been constructed and tested in transfection and immunofluorescence assays. The results show (1) that mutations in different domains of the Vmw110 polypeptide can have different effects on function in the presence or absence of Vmw175; (2) that mutations can affect the pattern of immunofluorescent intranuclear staining of Vmw110; and (3) that deletions in the region of a particular highly basic region of the polypeptide result in failure of Vmw110 to be transported into the nucleus.

The regions of the herpes simplex virus type 1 immediate early protein Vmwl75 required for site specific DNA binding closely correspond to those involved in transcriptional regulation

The immediate-early (IE) protein Vmw175 (ICP4) of HSV-1 is required for the transcription of later classes of viral genes and the repression of IE gene expression. We have previously constructed a panel of plasmid-borne insertion and deletion mutants of the gene encoding Vmw175 and assayed their ability to regulate transcription in transient transfection assays. By this approach we have mapped the regions of the Vmw175 amino acid sequence that are required for transcriptional activation and repression of herpes virus promoters. This paper describes the use of nuclear extracts, made from cells transfected with these mutant plasmids, in gel retardation DNA binding assays in order to define the regions of Vmw175 involved in binding to a specific Vmw175 DNA binding site. The results show that amino acid residues 275-495 (a region which is highly conserved between Vmw175 and the varicella-zoster virus "IE" 140K protein) include structures which are critically required for specific DNA binding, transactivation and repression. This raises the interesting paradox that although the specific DNA sequence recognized by Vmw175 is not commonly found in its target promoters, the protein domain required for recognition of this sequence is required for promoter activation.

Sp1 binds to promoter sequences and activates herpes simplex virus 'immediate-early' gene transcription in vitro.

During a herpes simplex virus (HSV-1) lytic infection, three classes of viral genes are transcribed in a temporally regulated manner. The HSV-1 'immediate-early' (IE) promoter sequences contain multiple copies of a hexanucleotide sequence, GGGCGG, known as a GC box, and one or more copies of an 11-base pair (bp) conserved A + T-rich element, designated TAATGARAT. The TAATGARAT elements are thought to mediate the trans-activation of IE RNA synthesis by a virion-associated protein(s), and the flanking G + C-rich sequences appear both to potentiate this induction and to direct IE promoter activity in vivo. The similarity of the herpesvirus GC box repeats to those of the simian virus 40 (SV40) early promoter prompted the in vitro analysis of HSV IE transcription reported here. We show that the mammalian gene-specific transcription factor Sp1 binds to eight distinct regions of the HSV short terminal repeat and stimulates transcription 25-fold from the divergent IE-3 (ICP-4) and IE-4/5 promoters.