Regulatory function of the equine herpesvirus 1 ICP27 gene product.

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.     

The Equine Herpesvirus 1 IR6 Protein That Colocalizes with Nuclear Lamins Is Involved in Nucleocapsid Egress and Migrates from Cell to Cell Independently of Virus Infection

The equine herpesvirus 1 (EHV-1) IR6 protein forms typical rod-like structures in infected cells, influences virus growth at elevated temperatures, and determines the virulence of EHV-1 Rac strains (Osterrieder et al., Virology 226:243–251, 1996). Experiments to further elucidate the functions and properties of the IR6 protein were conducted. It was shown that the IR6 protein of wild-type RacL11 virus colocalizes with nuclear lamins very late in infection as demonstrated by confocal laser scan microscopy and coimmunoprecipitation experiments. In contrast, the mutated IR6 protein encoded by the RacM24 strain did not colocalize with the lamin proteins at any time postinfection (p.i.). Electron microscopical examinations of ultrathin sections were performed on cells infected at 37 and 40°C, the latter being a temperature at which the IR6-negative RacH virus and the RacM24 virus are greatly impaired in virus replication. These analyses revealed that nucleocapsid formation is efficient at 40°C irrespective of the virus strain. However, whereas cytoplasmic virus particles were readily observed at 16 h p.i. in cells infected with the wild-type EHV-1 RacL11 or an IR6-recombinant RacH virus (HIR6-1) at 40°C, virtually no capsid translocation to the cytoplasm was obvious in RacH- or RacM24-infected cells at the elevated temperature, demonstrating that the IR6 protein is involved in nucleocapsid egress. Transient transfection assays using RacL11 or RacM24 IR6 plasmid DNA and COS7 or Rk₁₃ cells, infection studies using a gB-negative RacL11 mutant (L11ΔgB) which is deficient in direct cell-to-cell spread, and studies using lysates of IR6-transfected cells demonstrated that the wild-type IR6 protein is transported from cell to cell in the absence of virus infection and can enter cells by a yet unknown mechanism.     

The EICP22 protein of equine herpesvirus 1 physically interacts with the immediate-early protein and with itself to form dimers and higher-order complexes

The EICP22 protein (EICP22P) of Equine herpesvirus 1 (EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.     

Expression of the full-length form of gp2 of equine herpesvirus 1 (EHV-1) completely restores respiratory virulence to the attenuated EHV-1 strain KyA in CBA mice

Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by EUs4 (gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that EUs4 of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, interferon gamma-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.     

UL31 of herpes simplex virus 1 is necessary for optimal NF-kappaB activation and expression of viral gene products

Previous results suggested that the U(L)31 gene of herpes simplex virus 1 (HSV-1) is required for envelopment of nucleocapsids at the inner nuclear membrane and optimal viral DNA synthesis and DNA packaging. In the current study, viral gene expression and NF-κB and c-Jun N-terminal kinase (JNK) activation of a herpes simplex virus mutant lacking the U(L)31 gene, designated ΔU(L)31, and its genetic repair construct, designated ΔU(L)31-R, were studied in various cell lines. In Hep2 and Vero cells infected with ΔU(L)31, expression of the immediate-early protein ICP4, early protein ICP8, and late protein glycoprotein C (gC) were delayed significantly. In Hep2 cells, expression of these proteins failed to reach levels seen in cells infected with ΔU(L)31-R or wild-type HSV-1(F) even after 18 h. The defect in protein accumulation correlated with poor or no activation of NF-κB and JNK upon infection with ΔU(L)31 compared to wild-type virus infection. The protein expression defects of the U(L)31 deletion mutant were not explainable by a failure to enter nonpermissive cells and were not complemented in an ICP27-expressing cell line. These data suggest that pU(L)31 facilitates initiation of infection and/or accelerates the onset of viral gene expression in a manner that correlates with NF-κB activation and is independent of the transactivator ICP27. The effects on very early events in expression are surprising in light of the fact that U(L)31 is designated a late gene and pU(L)31 is not a virion component. We show herein that while most pUL31 is expressed late in infection, low levels of pU(L)31 are detectable as early as 2 h postinfection, consistent with an early role in HSV-1 infection.     

VP22 of herpes simplex virus 1 promotes protein synthesis at late times in infection and accumulation of a subset of viral mRNAs at early times in infection

VP22, encoded by the U_L49 gene, is one of the most abundant proteins of the herpes simplex virus 1 (HSV-1) tegument. In the present study we show VP22 is required for optimal protein synthesis at late times in infection. Specifically, in the absence of VP22, viral proteins accumulated to wild-type levels until ~6 h postinfection. At that time, ongoing synthesis of most viral proteins dramatically decreased in the absence of VP22, whereas protein stability was not affected. Of the individual proteins we assayed, VP22 was required for optimal synthesis of the late viral proteins gE and gD and the immediate-early protein ICP0 but did not have discernible effects on accumulation of the immediate-early proteins ICP4 or ICP27. In addition, we found VP22 is required for the accumulation of a subset of mRNAs to wild-type levels at early, but not late, times in infection. Specifically, the presence of VP22 enhanced the accumulation of gE and gD mRNAs until ~9 h postinfection, but it had no discernible effect at later times in infection. Also, VP22 did not significantly affect ICP0 mRNA at any time in infection. Thus, the protein synthesis and mRNA phenotypes observed with the U_L49-null virus are separable with regard to both timing during infection and the genes affected and suggest separate roles for VP22 in enhancing the accumulation of viral proteins and mRNAs. Finally, we show that VP22's effects on protein synthesis and mRNA accumulation occur independently of mutations in genes encoding the VP22-interacting partners VP16 and vhs.     

Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other

Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.     

Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection.

The promoter-regulatory regions from the herpes simplex virus type 1 (HSV-1) gene for the immediate-early, 175,000-molecular-weight (175K) protein and the HSV-2 delayed-early gene for a 38K protein were linked to the readily assayable bacterial gene for the enzyme chloramphenicol acetyltransferase (CAT). Unexpectedly, in measurements of the constitutive expression of the recombinant genes 40 to 50 h after transfection of Vero cells, enzyme levels expressed from the delayed-early 38K-promoter-CAT construct (p38KCAT) were at least as high as those from the immediate-early 175K-promoter-CAT construct (p175KCAT). In contrast, enzyme levels expressed after transfection of a similar recombinant gene containing a second delayed-early promoter region, that of the HSV-1 thymidine kinase gene, were ca. 20-fold lower. The amounts of enzyme expressed from both p38KCAT and p175KCAT could be increased by up to 20- to 40-fold after infection of the transfected cells with HSV. In comparison, virus infection had no significant effect on enzyme levels expressed from recombinant CAT genes containing the simian virus 40 early promoter region, with or without the 72-base-pair enhancer element. Experiments with the temperature-sensitive mutants HSV-1 tsB7 and HSV-1 tsK indicate that induction of expression from p175KCAT was mediated by components of the infecting virus particle, whereas that from p38KCAT required de novo expression of virus immediate-early proteins. In addition, we show that functions required to induce expression from both p175KCAT and p38KCAT could also be provided by infection with pseudorabies virus and cytomegalovirus.     

The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation.

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.     

Characterization of the trans-activation properties of equine herpesvirus 1 EICP0 protein

The EICP0 protein of equine herpesvirus 1 (EHV-1) is an early, viral regulatory protein that independently trans-activates EHV-1 immediate-early (IE), early, gamma1 late, and gamma2 late promoters. To assess whether this powerful trans-activator functions in conjunction with three other EHV-1 regulatory proteins to activate expression of the various classes of viral promoters, transient cotransfection assays were performed in which effector plasmids expressing the EICP22, EICP27, and IE proteins were used either singly or in combination with an EICP0 effector construct. These analyses revealed that (i) independently, the EICP0 and IE proteins are powerful trans-activators but do not function synergistically, (ii) the IE protein inhibits the ability of the EICP0 protein to trans-activate the IE, gamma1 late, and gamma2 late promoters, (iii) the EICP22 and EICP0 proteins do not function together to significantly trans-activate any EHV-1 promoter, and (iv) the EICP27 and EICP0 proteins function synergistically to trans-activate the early and gamma1 late promoters. A panel of EICP0 truncation and deletion mutant plasmids was generated and used in experiments to define the domains of the 419-amino-acid (aa) EICP0 protein that are important for the trans-activation of each class of EHV-1 promoters. These studies revealed that (i) carboxy-terminal truncation mutants of the EICP0 protein exhibited a progressive loss of trans-activating ability as increasing portions of the carboxy terminus were removed, (ii) the amino terminus of the EICP0 protein containing the RING finger (aa 8 to 46) and the acidic region (aa 71 to 84) was necessary but not sufficient for activation of all classes of EHV-1 promoters, (iii) the RING finger was absolutely essential for activation of EHV-1 promoters, since deletion of the entire RING finger motif (aa 8 to 46) or a portion of it (aa 19 to 30) completely abrogated the ability of these mutants to activate any promoter tested, (iv) the acidic region contributed to the ability of the EICP0 protein to activate the early and gamma1 late promoters, and deletion of the acidic region enhanced the ability of this mutant to activate the IE promoter, (v) the carboxy terminus (aa 325 to 419), which is rich in glutamine residues, was dispensable for the EICP0 trans-activation function, (vi) a motif resembling a nuclear localization signal (aa 289 to 293) was unnecessary for the EICP0 protein to trans-activate promoters of any temporal class, and (vii) the EICP0 protein was phosphorylated during infection, and deletion of the serine-rich region (aa 210 to 217), a potential site for phosphorylation, reduced by more than 70% the ability of the EICP0 protein to activate the gamma2 late class of promoters.     

Identification and characterization of the ICP22 protein of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.