Expression of herpes simplex virus type 1 glycoprotein B in insect cells. Initial analysis of its biochemical and immunological properties.

A recombinant baculovirus (vAc-gB1) was constructed which expresses the glycoprotein B (gB) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with these recombinant viruses, a protein that was close in size to authentic HSV-1 gB was detected by gB polyclonal antibody. The recombinant gB was found on the membrane of Sf9 cells and was susceptible to tunicamycin, glycosidase F (PNGase F) and partially susceptible to Endo-H. Antibodies raised in mice to this recombinant recognized viral gB and neutralized the infectivity of HSV-1 in vitro. Mice inoculated with the recombinant gB were protected from lethal challenge with HSV-1.     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。     

Incorporation of green fluorescent protein into the essential envelope glycoprotein B of herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major virion component, essential for various steps of virus replication in cells, such as entry and maturation, and cell fusion. In addition, gB is a strong inducer of the immune response in humans and has been involved in neuropathogenesis. To analyze gB during infection, a recombinant HSV-1 was generated containing gB fused to the green fluorescent protein (GFP). The GFP-gB fusion protein was incorporated into fully infectious viral particles. In cells infected with the recombinant KGFP-gB, the spontaneous fluorescence emitted by the fusion protein was observed as early as 5 h post infection, and its transport through cell compartments was followed during an entire viral replication cycle. The results show that GFP can be inserted into an essential viral envelope component of HSV-1 such as gB while preserving the infectivity of the resulting recombinant. This virus allows the investigation of several events of the viral life cycle involving gB, and provides the basis for the development of new diagnostic assays.     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗…

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白的重组痘苗病毒能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株，首先将经聚合酶链反应修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７．５Ｋ早／晚期启动子控制下，将同位重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＾＋痘苗病毒天     

A vaccinia virus — herpes simplex virus (HSV) glycoprotein B1 recombinant or an HSV vaccine overcome the HSV type 2 induced humoral immunosuppression and protect against vaginal challenge in BALB/c mice

Primary infections with herpes simplex virus type 2 (HSV-2) suppress the antibody response to secondary HSV-1 and -2 infections in the BALB/c mouse. In contrast, a challenge by the i.p. route using a vaccinia virus-HSV-1 glycoprotein B (VV gB1) recombinant induces a significant enhancement of the antibod response. This booster reaction is also observed if a challenge with a formalin-inactivated HSV-1 vaccine is performed. Although no or low humoral and vaginal antibodies are detectable after a single i.p. infection with the VV gB1 recombinant or the HSV-1 vaccine, protection against vaginal challenge with HSV-2 is induced. This points to the important role of cellular immunity for vaginal infections.     

Transactivation by herpes simplex virus proteins ICP4 and ICP0 in vaccinia virus infected cells.

Vaccinia virus recombinants containing the sequences from herpes simplex virus type 1 (HSV-1) encoding the immediate early (IE)(alpha) proteins ICP4 and ICP0, under the control of a mutated vaccinia virus 11K late promoter, were constructed. A cDNA copy of the gene encoding ICPO and an ICP4-encoding genomic segment were each inserted into the vaccinia virus genome at the thymidine kinase (TK) locus by homologous recombination. Steady-state analyses revealed that RNAs homologous to the IE-0 and IE-4 sequences accumulated in cells infected by recombinants with the kinetics of a typical vaccinia late mRNA. Western blot analyses demonstrated that the expression level of both ICPO and ICP4, produced by the recombinant viruses, was comparable to that in HSV-1-infected cells at late times postinfection. Both proteins synthesized in cells infected by the recombinants were located in the nucleus as revealed by immunofluorescence. Although in vitro studies reveal that extracts from vaccinia-virus-infected cells lose the ability to transcribe genes that contain RNA polymerase II promoters (Puckett and Moss (1983), Cell 35, 441-448) both ICPO and ICP4 expressed by the recombinant viruses can transactivate plasmids containing a reporter gene driven by the promoters for the HSV-1 TK and glycoprotein C genes. Nuclear extracts prepared from cells infected with the vaccinia virus vector expressing ICP4 exhibited sequence-specific DNA-binding activity.     

Expression Analysis of Recombinant Herpes Simplex Virus Type 1 DNase

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)¹. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503–626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein re aching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear loca lization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparison of T cell responses to glycoprotein B (gB-1) of herpes simplex virus type 1 and its non-glycosylated precursor protein, pgB-1.

The ability of non-glycosylated precursor glycoprotein B (pgB) to induce T cell responses in herpes simplex virus (HSV) infected mice was compared with fully glycosylated glycoprotein B (gB) and with whole virus. pgB was as effective as gB in priming for virus- and glycoprotein-specific T cells. pgB could also re-stimulate virus or glycoprotein primed cells in vitro as efficiently as gB. In addition, priming with pgB protected mice against a lethal challenge with HSV type 1 (HSV-1) and could induce the early in vivo production of IL-2 and IL-3 in infected mice. In all of these responses, pgB was as effective as gB. Thus, the carbohydrate side chains on gB do not appear to be necessary for T cell recognition of this protein.     

Common epitopes of glycoprotein B map within the major DNA-binding proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

Bovine herpesvirus 2 (BHV-2) specifies a glycoprotein of 130 kDa (gB BHV-2) which shows extensive homology to glycoprotein B (gB-1) of herpes simplex virus 1 (HSV-1). The BHV-2-specific 130-kDa glycoprotein is able to induce cross-reacting antibodies, some of which even cross-neutralize HSV-1. In order to determine the genome localization of gB BHV-2 and in order to identify conserved antigenic domains in both glycoproteins, we established libraries of subgenic fragments of BHV-2 and HSV-1 DNA in the prokaryotic expression vector lambda gt11 and screened them with cross-reacting monoclonal antibodies which allowed us to identify recombinant lambda gt11 clones expressing gB fusion protein. Nucleotide sequencing of inserted DNA fragments within these recombinant lambda gt11 clones revealed that they originated from the carboxy-terminal part of the major DNA-binding proteins (dbp) of BHV-2 (dbp BHV-2) and its counterpart ICP8 in HSV-1. Antisera raised against the beta-galactosidase fusion protein of recombinant phage lambda-113/2 coding for an 84 amino acid (aa) polypeptide originating from dbp BHV-2 neutralized infectivity of BHV-2 and HSV-1 in the presence of complement and precipitated [3H] glucosamine-labeled gB BHV-2 and gB-1. This antiserum also reacts with ICP8 and presumably with dbp BHV-2. Two hypotheses are discussed to explain this unexpected result: (i) epitopes in the carboxy-terminal part of gB BHV-2 and gB-1 are similar to antigenic determinants in the amino-terminal region of the gBs, thus providing cross-reacting antibody-binding sites; (iii) during gene expression a carboxy-terminal part of dbp BHV-2 and ICP8 genes might be spliced to the amino-terminal region of the glycoproteins gB BHV-2 and gB-1.     

Characterisation and physical mapping of an HSV-1 glycoprotein of approximately 115 X 10(3) molecular weight

A type-specific monoclonal antibody that efficiently neutralises HSV-1 immunoprecipitated a glycoprotein of slightly greater electrophoretic mobility than gB from HSV-1 infected cells. Pulse and pulse chase experiments indicate that this glycoprotein is distinct from HSV-1 glycoproteins gB, gC, gD, and gE. This was confirmed by the reactions of LP11 with a series of intertypic recombinants the results of which indicate that the LP11 target gene is located close to the HSV-1 thymidine kinase gene between map positions 0.28 and 0.31. In accordance with the presently agreed convention this glycoprotein should be designated gH-1, and it may correspond to the 110K glycoprotein described by S. D. Showalter, M. Zweig, and B. Hampar (1981), Infect. Immun. 34, 684-692. Antibody LP11 inhibits plaque formation when added to cell monolayers after infection suggesting that gH-1 may play a role in cell-to-cell spread of infectious virus.     

The glycoprotein B gene and its syn3 locus of herpes simplex virus type 1 are involved in the synthesis of virus-associated growth factor (HSGF-1).

A putative growth factor (HSGF-1) associated with herpes simplex virus type 1 (HSV-1), which is similar to PRGF associated with pseudorabies virus, and/or HSGF-2 associated with HSV-2, was described. Experiments with four syncytial (syn) and four nonsyncytial (syn+) HSV-1 strains showed that the ability of this virus to produce HSGF-1 in infected cells is associated with the syn+ phenotype. Double infection of cells with syn+ and syn strain resulted either in enhancement or complete inhibition of HSGF-1 production, depending on the chosen pair of syn+ and syn strains. The studies with the recombinants between the syn+ strain KOS and syn strain ANGpath in the gene for glycoprotein B (gB) and syn3 locus revealed that the gB gene and its syn3 locus play a role in the HSGF-1 synthesis.     

Activation of HIV-1 enhancer sequence by vaccinia virus

To investigate whether vaccinia virus (VV) can augment gene expression of human immunodeficiency virus type 1 (HIV-1), co-transfection experiments were carried out in which recombinant plasmids containing various portions of the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into cultured cells. A high level of enhancement in CAT activity directed by the HIV-1 LTRs containing the enhancer sequence was observed in cells infected with VV, as in the cells infected with type 1 herpes simplex virus (HSV-1). The sequence responsible for this augmentation of CAT activity was different from that recognized by HIV-1 Tat. These data clearly demonstrated that VV transactivates HIV-1 LTR through a mechanism distinct from that of activation by HIV-1 Tat.     

Analysis of the intracellular maturation of the herpes simplex virus type 1 glycoprotein gH in infected and transfected cells.

We have expressed the HSV-1 glycoprotein, gH, in transiently transfected COS-1 cells. The expressed protein was retained intracellularly, contained unprocessed carbohydrate, and was unrecognized by the monoclonal antibody, LP11. In addition, the protein was aggregated. These properties suggest that unlike other HSV glycoproteins, gH is misfolded in transfected cells. Pulse-chase studies of HSV-1-infected cells indicate that the kinetics of processing of gH are comparable to those of gB, gC, and gD. Rescue studies suggest that gH may interact with another protein during maturation in infected cells. However, we were unable to detect any stable interaction, although analysis of gH on neutral sucrose gradients shortly after synthesis indicated a possible transient association with a high molecular weight molecule or complex. The processing and cell surface expression of gH were also analyzed in HSV-1 virus mutants lacking gB, gC, or gD. Our results indicate that the maturation and cell surface transport of gH did not require the presence of these HSV-1 glycoproteins. In addition, three truncation mutants were constructed by linker insertion mutagenesis. Each of the three truncated proteins was synthesized, but the proteins were aggregated, contained only endo H-sensitive carbohydrate, and none were secreted.     

Requirements for transport of HSV-1 glycoproteins to the cell surface membrane of human fibroblasts and Vero cells

The intracellular transport of the HSV-1 glycoproteins gA/gB, gC and gD has been followed by the indirect immunofluorescence technique (IIF). Infected tissue culture cells were stained with monoclonal antibodies made to the individual glycoproteins and with fluorochrome-coupled wheat germ agglutinin reacting specifically with Golgi apparatus of the cells. Staining of either infected, human fibroblasts or of VERO cells at 9 hours p.i. with antibodies to gA/gB showed a prominent ring-like nuclear fluorescence and distinct staining of the Golgi apparatus in the cells. Antibodies to gC and gD stained mainly the Golgi apparatus and areas close to or at the surface of the cells. By immunocytolysis of HSV-1-infected VERO cells the viral glycoproteins were demonstrable at the surface of cells but growth of infected cells in the presence of either TM or monensin inhibited the expression of most of the viral glycoproteins at the cell surface. Blocking of the glycosylation of the viral glycoproteins with tunicamycin (TM) was followed by accumulation of the core of the glycoproteins gA/gB and gD in granular structures close to the nucleus as seen by immunofluorescence microscopy. Antibodies to gC did also stain granules close to the nucleus but in addition the periphery of the cells were stained. Inhibition of intracellular transport from the Golgi apparatus by the carboxylic ionophore monensin was followed by accumulation of all the HSV-1 glycoproteins in vesicles derived from the Golgi apparatus in both human fibroblasts and VERO cells. Our data thus support the hypothesis that the HSV-1 glycoproteins are processed in the Golgi apparatus before the transport to and incorporation into the cell surface membrane of infected cells and into virion envelopes.     

Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope glycoproteins of Lassa virus

A recombinant vaccinia virus that expresses the nucleoprotein gene of Lassa virus (Josiah strain) under the control of the P7.5 promoter was constructed using the lacZ coexpression transfer vector pSC11. Southern blot analysis demonstrated that recombination of the sequences inserted within the thymidine kinase gene of the transfer vector into the HindIII J fragment of vaccina virus genomic DNA occurred properly. A 63-kDa protein identical in electrophoretic mobility to authentic Lassa nucleoprotein was observed in recombinant virus-infected cell lysates. The reactivity of vaccinia-expressed Lassa proteins to a panel of monoclonal antibodies representing multiple epitopes on each of the N, G1, and G2 proteins was determined by indirect immunofluorescence. Lassa proteins expressed in recombinant vaccinia virus-infected cells reacted in a manner indistinguishable from that of the proteins expressed in Lassa virus-infected cells, indicating that there are no significant differences between authentic and recombinant virus-expressed proteins. Vaccine efficacy trials in guinea pigs indicated that both the nucleoprotein and the envelope glycoproteins are capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN, 79% vaccinated with V-LSGPC, and 58% vaccinated with both recombinant viruses survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived. The protection resulting from vaccination with the recombinant virus vaccines did not correlate with the levels of prechallenge serum antibodies, suggesting that a cell-mediated immune response is a critical component of protective immunity to Lassa fever.     

Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus

Summary.  The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. Accepted November 20, 1996 Received October 10, 1996     

Expression and characterization of baculovirus expressed herpes simplex virus type 1 glycoprotein L

Summary We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein L (gL) in Sf9 cells. Sf9 cells infected with this recombinant virus synthesized three polypeptides of 26–27 kDa 28 kDa, and 31 kDa. The 28 and 31 kDa species were sensitive to tunicamycin and N-glycosidase F (PNGase F) treatment, suggesting that they were glycosylated. As shown by both indirect immunofluorescence and Western blot analysis, using polyclonal antibodies to synthetic gL peptides indicated that the baculovirus expressed gL was abundant on the surface of baculovirus gL infected Sf9 cells. A small fraction of the 31 kDa polypeptide was secreted into the extracellular medium as judged by Western blot analysis. The secreted form of gL was completely resistant to Endoglycosidase H (Endo-H), while the membrane associated form of gL was only partially resistant to Endo-H treatment, suggesting that the secreted gL represented a subpopulation of the membrane bound gL. Mice vaccinated with baculovirus expressed gL produced serum antibodies that reacted with authentic HSV-1 gL. However, these mice produced no HSV-1 neutralizing antibody (titer <1: 10) and they were not protected from lethal intraperitoneal or lethal ocular challenge with HSV-1. Thus, when used as a vaccine in the mouse model, gL, similar to our findings with HSV-1 gH, but unlike our results with the other 6 HSV-1 glycoproteins that we have expressed in this baculovirus system, did not provide any protection against HSV-1 challenge.     

Conserved cytoplasmic domain sequences mediate the ER export of VZV, HSV-1, and HCMV gB

Glycoprotein B (gB) is conserved among the herpesviruses and participates in both virus entry and cell-cell spread. The ER export of VZV gB is mediated by two cytoplasmic domain regions, aa 818-826, which contains a YXXphi motif, and the C-terminal 17 aa. The current study examines whether related sequences in the cytoplasmic domains of HSV-1 and HCMV gB similarly influence the ER export of their gB homologs. Directed mutations were introduced into the cytoplasmic domains of HSV-1 and HCMV gB, and the efficiencies with which the mutated proteins acquired Golgi-dependent modifications were determined. Sequences homologous to VZV gB aa 818-826 were required for normal ER export of both HSV-1 gB and HCMV gB. However, the C-terminal regions of HSV-1 and HCMV gB had no impact on ER export. Therefore, alpha- and betaherpesvirus gB homologs share conserved ER export signals, but species-specific differences in the ER export of gB also exist.     

Conservation of a gene cluster including glycoprotein B in bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

A library of subgenomic fragments of bovine herpesvirus type 2 (BHV-2) DNA was constructed in the expression cloning vector lambda gt11 and screened with monoclonal antibodies to the glycoprotein gb BHV-2, which is homologous to glycoprotein gB (gB-1) of herpes simplex virus type 1 (HSV-1). Lambda gt11 clones containing gB BHV-2-specific sequences were used to identify lambda EMBL3 vectors with DNA inserts which contained the complete gB BHV-2 gene. Nucleotide sequencing revealed that the gB BHV-2 gene is highly conserved compared to gB-1. The amino acid sequences and the predicted secondary structures of both glycoproteins are very similar. Two further open reading frames (ORF) in close vicinity to the gene encoding gB BHV-2 showed considerable homology to HSV-1 genes. They code for the major DNA-binding protein (dbp) of BHV-2 and a putative 72-kDa polypeptide. The gene of the latter protein corresponding to ICP18.5 of HSV-1 is interspersed between the ORFs of gB BHV-2 and the dbp of BHV-2. All three genes map in the unique long region of the genome. Their homology and the colinear arrangement compared to HSV-1 indicate a close relationship between the two viruses.