Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize immediate-early protein ICP27.

The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H-2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino-acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity.     

Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities.

The HSV-1 alpha (immediate-early) protein ICP27 expressed in transfected cells can activate the expression of certain HSV-1 promoters as well as inhibit the transactivated expression of others. We constructed a set of plasmids encoding mutant ICP27 molecules truncated at their carboxyl termini and used transfection assays to determine the functional properties of the mutant proteins. A polypeptide containing the amino-terminal 263 amino acid residues of ICP27 retained partial ability to activate gene expression but was unable to inhibit transactivation. Mutant proteins possessing 406 or 504 amino acids of ICP27 were unable to activate gene expression but retained full ability to inhibit transactivation. These results define two separable regulatory activities of ICP27, one positive and one negative, which can modulate gene expression in transfected cells. Immunoblot and immunofluorescence experiments were used to study the immunological reactivities and intracellular localizations of the mutant proteins. All proteins possessing the amino-terminal 263 amino acids of ICP27 reacted with an ICP27-specific monoclonal antibody and were localized to the cell nucleus. The mutant proteins, however, exhibited a number of phenotypes with regard to intranuclear localization. A mutant possessing 504 residues of ICP27 was similar to the wild-type protein in apparently localizing to all regions of the nucleus. A mutant containing 406 residues of ICP27, on the other hand, was mostly excluded from the nucleolar regions, while a 263-residue mutant was localized predominantly in the nucleoli. Thus, some aspect of ICP27 structure or function can dramatically affect its intranuclear distribution.     

The regions important for the activator and repressor functions of herpes simplex virus type 1 alpha protein ICP27 map to the C-terminal half of the molecule.

The herpes simplex virus type 1 (HSV-1) alpha or immediate-early proteins ICP4 (IE175), ICP0 (IE110), and ICP27 (IE63) are trans-acting proteins which affect HSV-1 gene expression. We previously showed that ICP27 in combination with ICP4 and ICP0 could act as a repressor or an activator in transfection assays, depending on the target gene (R. E. Sekulovich, K. Leary, and R. M. Sandri-Goldin, J. Virol. 62:4510-4522, 1988). To investigate the regions of the ICP27 protein which specify these functions, we constructed a series of in-frame insertion and deletion mutants in the ICP27 gene. These mutants were analyzed in transient expression assays for the ability to repress or to activate two different target genes. The target plasmids used consisted of the promoter regions from the HSV-1 beta or early gene which encodes thymidine kinase and from the beta-gamma or leaky late gene. VP5, which encodes the major capsid protein, each fused to the chloramphenicol acetyltransferase gene. Our previous studies showed that induction of pTK-CAT expression by ICP4 and ICP0 was repressed by ICP27, whereas the stimulation of pVP5-CAT expression seen with ICP4 and ICP0 was significantly increased when ICP27 was also added. In this study, a series of transfection assays was performed with each of the ICP27 mutant plasmids in combination with plasmids containing the ICP4 and ICP0 genes with each target. The results of these experiments showed that mutants containing insertions or deletions in the region from amino acids 262 to 406 in the carboxy-terminal half of the protein were unable to stimulate expression of pVP5-CAT but were able to repress induction of pTK-CAT activity by ICP4 and ICP0. Mutants in the carboxy-terminal 78 amino acids lost both activities; that is, these mutants did not show repression of pTK-CAT activity or stimulation of pVP5-CAT activity, whereas mutants in the hydrophilic amino-terminal half of ICP27 were able to perform both functions. These results show that the carboxy-terminal half of ICP27 is important for the activation and repression functions. Furthermore, the carboxy-terminal 62 amino acids are required for the repressor activity, because mutants with this region intact were able to repress. Analysis of the DNA sequence showed that there are a number of cysteine and histidine residues encoded by this region which have some similarity to zinc finger metal-binding regions found in other eucaryotic regulatory proteins. These results suggest that the structural integrity of this region is important for the function of ICP27.     

Induction in vitro of primary cytotoxic T-lymphocyte responses with DNA encoding herpes simplex virus proteins.

Vaccines which successfully protect against virus infections usually need to induce a broadly reactive immune response which includes the induction of cytotoxic T lymphocytes (CTL). In this study, we have used a convenient in vitro approach to investigate if plasmid DNAs encoding proteins of herpes simplex virus (HSV) are capable of inducing primary CD8+ CTL. Dendritic cells or macrophages were transfected with either plasmid DNA encoding glycoprotein B or DNA encoding the immediate-early protein ICP27. These antigen-presenting cells (APC) were then used to stimulate enriched populations of naive T cells in microcultures for 5 days in vitro. Antigen-specific CD8+ CTL which reacted both with specific protein-expressing targets and with syngeneic targets infected with HSV could be demonstrated. Dendritic cells, as APC, generated the maximal responses, but such cells needed to be transfected with DNA in the presence of a cationic lipid. However, macrophages could act as APC when they were exposed to purified DNA. HSV-primed splenocytes were also shown to generate specific CTL responses when they were stimulated with purified DNA encoding ICP27. The novel approach described in this paper promises to be extremely useful, since defining immunogenicity profiles and identifying epitopes on viral proteins should be easier and more convenient when working with DNA and investigating variables in vitro. This is particularly the case with complex viruses such as HSV, most of whose encoded proteins have yet to be isolated in sufficient quantity or purity to perform in vivo immunological studies.     

Mutations in the activation region of herpes simplex virus regulatory protein ICP27 can be trans dominant.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein which is required for virus replication. Transfection experiments have demonstrated that ICP27 along with the HSV-1 transactivators ICP4 and ICP0 can positively regulate the expression of some late HSV-1 target plasmids and can negatively regulate the expression of some immediate-early and early target plasmids. We previously showed that mutants defective in the activation of a late target plasmid mapped to the carboxy-terminal half of the protein, whereas mutants defective in the repression of an early target plasmid mapped within the C-terminal 78 amino acids of ICP27 (M. A. Hardwicke, P. J. Vaughan, R. E. Sekulovich, R. O'Conner, and R. M. Sandri-Goldin, J. Virol. 63:4590-4602, 1989). In this study, we cotransfected ICP27 activator and repressor mutants along with wild-type ICP27 plasmid to determine whether these mutants could interfere with the wild-type activities. Mutants which were defective only in the activation function were dominant to the wild-type protein and inhibited the activation of the late target plasmid pVP5-CAT, whereas mutants defective in the repressor function did not inhibit either the activation of pVP5-CAT or the repression of the early target plasmid pTK-CAT. Furthermore, cell lines which stably carried three different activator mutants were impaired in their ability to support the growth of wild-type HSV-1 strain KOS, resulting in virus yields 5- to 40-fold lower than in control cells. The defect in virus replication appeared to stem from a decrease in the expression of HSV-1 late gene products during infection as measured by steady-state mRNA levels and by immunoprecipitation analysis of specific polypeptides. These results indicate that ICP27 activator mutations specifically interfere with the activation function of the protein both in transfection and during infection. Moreover, these results suggest that the repressor region may be important for binding of the polypeptide, since mutations in this region did not interfere with the activities of wild-type ICP27 and therefore presumably could not compete for binding.     

Six epitopes reacting with human cytotoxic CD8+ T cells in the central region of the HIV-1 NEF protein.

In order to identify the target epitopes recognized by specific CTL in the NEF protein of HIV-1, 33 peptides derived from the HIV-BRU sequence were tested with NEF-specific CTL generated from HIV-seropositive donors. Six different epitopes were identified and several points were remarkable: 1) They were all located in two regions of the central part of the NEF protein corresponding to residues 73 to 94 and 113 to 147, respectively. 2) The CTL issued from a single donor could recognize several peptides of the NEF protein. 3) Some of these peptides could be recognized in association with at least two or three different HLA class I molecules. 4) Two different overlapping epitopes were present in a relatively short sequence of 15 amino acids. These results suggest that multiple epitopes corresponding to different HLA restrictions could coexist in a relatively small region of the NEF protein. The implications of these results in vaccine strategies using synthetic peptides bearing CTL epitopes are discussed.     

Specific cytotoxic T lymphocytes recognize the immediate-early transactivator Zta of Epstein-Barr virus.

We identified the immediate-early transactivator Zta of Epstein-Barr virus as a target for specific cytotoxic T lymphocytes (CTL). Cells pulsed with overlapping synthetic peptides representing the entire amino acid sequence of Zta proved to be efficient for the in vitro stimulation of Zta-specific CTL in several donors. With peptide-pulsed target cells, we found that CTL from several donors recognize a peptide comprising 15 amino acids. The immune response against this peptide exerted by CTL lines from different donors was found to be restricted by two different molecules of the major histocompatibility complex: HLA-B8 and HLA-Cw6. The latter molecule could for the first time be identified as a restricting element for a CTL response. The epitope of the HLA-B8-restricted CTL could be mapped to an octameric sequence between amino acid positions 190 and 197 of the Zta protein, whereas the minimal epitope of HLA-Cw6-restricted CTL consists of 11 to 15 residues between positions 187 and 201. Thus, the HLA-B8 and HLA-Cw6 epitopes widely overlap but are not completely identical. In vitro stimulation of blood lymphocytes from a panel of HLA-B8-positive or HLA-Cw6-positive virus carriers, using autologous cells pulsed with the Zta peptides comprising the HLA-B8 or HLA-Cw6 epitope, respectively, revealed in both cases that most of these donors developed a Zta-specific cytotoxic activity. These data, as well as the high spread of the major histocompatibility complex molecules HLA-B8 and HLA-Cw6 in most populations, suggest that an efficient CTL response directed against gene products of the immediate-early group of the lytic cycle exists in vivo in a considerable portion of virus carriers. A CTL response against proteins expressed immediately after the switch into the lytic cycle could eliminate lytically activated cells at an early stage and would thus efficiently prevent the production and release of progeny virions.     

Herpes simplex virus 1 VP22 regulates translocation of multiple viral and cellular proteins and promotes neurovirulence

Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD(50)) for neurovirulence in mice following intracerebral inoculation about 10(3)-fold lower than the LD(50) of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo.     

Analysis of the phosphorylation sites of herpes simplex virus type 1 regulatory protein ICP27

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167-182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infection. In this study, we mapped several of the phosphorylation sites of ICP27 following in vivo radiolabeling. Phosphoamino acid analysis showed that serine is the only amino acid that is phosphorylated during infection. Two-dimensional phosphopeptide mapping showed a complex tryptic phosphopeptide pattern with at least four major peptides and several minor peptides. In addition, ICP27 purified from transfected cells yielded a similar phosphopeptide pattern, suggesting that cellular kinases phosphorylate ICP27 during viral infection. In vitro labeling showed that protein kinase A (PKA), PKC, and casein kinase II (CKII) were able to differentially phosphorylate ICP27, resulting in distinct phosphopeptide patterns. The major phosphorylation sites of ICP27 appeared to cluster in the N-terminal portion of the protein, such that a frameshift mutant that encodes amino acids 1 to 163 yielded a phosphopeptide pattern very similar to that seen with the wild-type protein. Further, using small deletion and point mutations in kinase consensus sites, we have elucidated individual serine residues that are phosphorylated in vivo. Specifically, the serine at residue 114 was highly phosphorylated by PKA and the serine residues at positions 16 and 18 serve as targets for CKII phosphorylation in vivo. These kinase consensus site mutants were still capable of complementing the growth of an ICP27-null mutant virus. Interestingly, phosphorylation of the serine at residue 114, which lies within the major nuclear localization signal, appeared to modulate the efficiency of nuclear import of ICP27.     

ICP27 interacts with SRPK1 to mediate HSV splicing inhibition by altering SR protein phosphorylation

Infection with some viruses can alter cellular mRNA processing to favor viral gene expression. We present evidence that herpes simplex virus 1 (HSV-1) protein ICP27, which contributes to host shut-off by inhibiting pre-mRNA splicing, interacts with essential splicing factors termed SR proteins and affects their phosphorylation. During HSV-1 infection, phosphorylation of several SR proteins was reduced and this correlated with a subnuclear redistribution. Exogenous SR proteins restored splicing in ICP27-inhibited nuclear extracts and SR proteins isolated from HSV-1-infected cells activated splicing in uninfected S100 extracts, indicating that inhibition occurs by a reversible mechanism. Spliceosome assembly was blocked at the pre-spliceosomal complex A stage. Furthermore, we show that ICP27 interacts with SRPK1 and relocalizes it to the nucleus; moreover, SRPK1 activity was altered in the presence of ICP27 in vitro. We propose that ICP27 modifies SRPK1 activity resulting in hypophosphorylation of SR proteins impairing their ability to function in spliceosome assembly.     

Immediate-Early Transactivator Rta of Epstein-Barr Virus (EBV) Shows Multiple Epitopes Recognized by EBV-Specific Cytotoxic T Lymphocytes

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells.     

Varicella-zoster virus open reading frame 4 protein is functionally distinct from and does not complement its herpes simplex virus type 1 homolog, ICP27.

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a putative immediate-early protein which is homologous to herpes simplex virus type 1 (HSV-1) ICP27 on the basis of gene location and similarity in amino acid sequence. In transient expression assays, however, ORF4 and ICP27 exhibit different properties. ICP27 alone has little activity on target plasmids, but it acts as a transactivator or a transrepressor in the presence of other HSV-1 transactivators. In contrast, ORF4 directly transactivates plasmids containing homologous or heterologous promoters and has no apparent transrepressing activity. To further illuminate the functional similarities and differences between ORF4 and ICP27, Vero cell lines which express ORF4 under the inducible metallothionein promoter were constructed. Cell lines expressing functionally active ORF4 protein upregulated the expression of transfected VZV target plasmids but were unable to efficiently complement HSV-1 ICP27 mutants. These results indicate that, despite structural similarities, VZV ORF4 and HSV-1 ICP27 behave differently in transient expression assays and may play different roles in virus replication.     

Mutational analysis of ICP0R, a transrepressor protein created by alternative splicing of the ICP0 gene of herpes simplex virus type 1.

The immediate-early protein ICP0 (infected-cell polypeptide 0) of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator of both viral and nonviral promoters in transient expression assays. Failure to splice the second of two introns in the ICP0 gene results in the utilization of an alternate stop codon that generates a truncated form of ICP0 called ICP0R. This protein exists in low levels in HSV-1-infected cells and functions as a dominant negative repressor of ICP0-mediated transactivation in transient expression assays. To conduct a detailed structure-function analysis of ICP0R, a series of insertion and deletion mutants of this protein were generated and analyzed in transfection assays. These studies indicated that segments of ICP0R that were rich in acidic amino acid residues (amino acids 9 to 76 and 233 to 241) or glycine residues (amino acids 242 to 262) were dispensable for the dominant negative phenotype. In contrast, the RING finger domain (amino acids 116 to 156) and surprisingly the sequences carboxy terminal to it (amino acids 157 to 232) were absolutely essential for transdominant repression. Consistent with these findings, the amino acid sequences of these two regions were conserved among other alphaherpesvirus ICP0 homologs. A construct containing only amino acids 76 to 232 inhibited ICP0-mediated transactivation almost as efficiently as wild-type ICP0R and represented the minimal sequences necessary for the dominant negative phenotype. These results demonstrated that the critical functional domain shared by both ICP0R and ICP0 is much more complex than a simple RING finger motif. Western blot (immunoblot) analyses of transfected cell lysates revealed that nearly all of the mutant constructs directed the expression of stable ICP0R proteins of the predicted molecular weight. However, there was a striking inverse correlation between the ability of a mutant construct to mediate transrepression and the amount of protein that it synthesized, indicating that dominant negative inhibition is achieved through the action of very little ICP0R protein.     

Infection and inhibition of human cytotoxic T lymphocytes by herpes simplex virus.

The effect of herpes simplex virus type 1 (HSV-1) infection on human cytotoxic T-lymphocyte (CTL) lytic function was assessed. All HSV-infected CTL populations tested were significantly inhibited in lysing target cells. The inhibition of CTL lytic function by infection with HSV-1 was independent of T-cell receptor-mediated antigen recognition and did not involve virus-induced shutoff of host protein synthesis, the expression of the HSV-1 transactivation protein, ICP4, or replicating virus. Understanding the functional impairment of CTL following infection with HSV may have important implications for HSV-induced immunosuppression and the mechanism of HSV persistence in immunocompetent hosts.     

The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection.

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.     

HLA-A*0201-restricted CD8+ cytotoxic T lymphocyte epitopes identified from herpes simplex virus glycoprotein D

Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited high-affinity binding in vitro to purified human HLA-A*0201 molecules. Three of these four peptide epitopes, gD53-61, gD70-78, and gD278-286, significantly stabilized HLA-A*0201 molecules on T2 cell lines and are highly conserved among and between HSV-1 and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A*0201-positive, HSV-1-seropositive, and/or HSV-2-seropositive healthy individuals, the most frequent and robust CD8+ T cell responses, assessed by IFN-gamma ELISPOT, CD107a/b cytotoxic degranulation, and tetramer assays, were directed mainly against gD53-61, gD70-78, and gD278-286 epitopes. In addition, CD8+ T cell lines generated by gD53-61, gD70-78, and gD278-286 peptides recognized infected target cells expressing native gD. Lastly, CD8+ T cell responses specific to gD53-61, gD70-78, and gD278-286 epitopes were induced in HLA-A*0201 transgenic mice following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.     

Herpes simplex virus ICP27 activation of stress kinases JNK and p38

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0-/4-) and d103 (4-/22-/47-) mutants activated p38 and JNK, while the d106 (4-/22-/27-/47-) and d107 (4-/27-) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.     

Development and preparation of recombinant gD antigen of the herpes simplex type 1 (HSV-1) virus]

The most potent antigen among HSV-1 proteins are glycoproteins gB(UL27) and gD(US6). Multiple amino acid sequence alignment of these proteins shows that gD protein is the most specific for HSV-1. Analysis of gD protein epitopes detected the main antigenic determinants not cross-reactive with antigens of other viruses. Virus was isolated and genome DNA was prepared from morphological elements of a patient with herpes simplex infection. US6 gene fragment was cloned in pUC19 vector. Cloning in bacterial expression vectors helped obtain beta-galactosidase-fused recombinant HSV-1 gD protein with 6-histidines affine target for high-performance chromatography purification. ELISA with a set of HSV-1-positive and negative donor sera and a commercial panel of HSV-1 sera (Vektor-Best) showed that recombinant gD can be used as an antigen to HSV-1-specific IgG.