Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production.

The antibiotic tunicamycin, which blocks the synthesis of glycoproteins, inhibited the production of infectious herpes simplex virus. In the presence of this drug, [14C]glucosamine and [3H]mannose incorporation was reduced in infected cells, whereas total protein synthesis was not affected. Gel electrophoresis of [2-3H]mannose-labeled polypeptides failed to detect glycoprotein D or any of the other herpes simplex virus glycoproteins. By use of specific antisera we demonstrated that in the presence of tunicamycin the normal precursors to viral glycoproteins failed to appear. Instead, lower-molecular-weight polypeptides were found which were antigenically and structurally related to the glycosylated proteins. Evidence is presented to show that blocking the addition of carbohydrate to glycoprotein precursors with tunicamycin results in the disappearance of molecules, possibly due to degradation of the unglycosylated polypeptides. We infer that the added carbohydrate either stabilizes the envelope proteins or provides the proper structure for correct processing of the molecules needed for infectivity.     

Guanylylation and adenylylation of the alpha regulatory proteins of herpes simplex virus require a viral beta or gamma function.

Herpes simplex virus genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Most of the products of the first group, the alpha genes, appear to have regulatory functions. We report that the alpha proteins, infected cell proteins 4, 0, 22, and 27 of herpes simplex virus 1 and 4, 0, and 27 of herpes simplex virus 2, were labeled in the isolated nuclei of infected HeLa cells with [alpha-32P]GTP or [alpha-32P]ATP late in infection and that these proteins represent the largest group of virus-specific proteins labeled in this fashion. Studies with [2-3H]ATP, in which the label is in the purine ring, showed that a portion of the label in alpha proteins and in at least one other infected cell protein is due to nucleotidylylation. Analyses of the labeling reactions in nuclei of (i) cells infected with temperature-sensitive mutants at nonpermissive temperatures, (ii) cells infected with wild-type virus and harvested at different times postinfection, and (iii) cells treated with inhibitors of protein synthesis or of synthesis of viral DNA led to the conclusion that viral gene functions expressed after the synthesis of alpha proteins are required for the labeling of the alpha proteins with [alpha-32P]GTP. We conclude that several of the alpha proteins are extensively posttranslationally modified and that these modifications include nucleotidylylation.     

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.     

Identification of viral polypeptides involved in pseudorabies virus ribonucleotide reductase activity.

We studied pseudorabies virus-induced ribonucleotide reductase and found that it exhibited biochemical properties very similar to those of herpes simplex virus reductase. A polyclonal rabbit antiserum (P9) directed against the carboxy terminus of subunit H2 polypeptide (38,000 daltons) of herpes simplex virus reductase neutralized the pseudorabies virus reductase, as well as the herpes simplex virus isozyme. This serum recognized two pseudorabies virus-specified polypeptides of 34,000 and 110,000 daltons, which may represent the two subunits of the enzyme. Furthermore, as already shown for herpes simplex virus reductase (E. A. Cohen, P. Gaudreau, P. Brazeau, and Y. Langelier, Nature [London] 321:441-443, 1986), we show that the nonapeptide itself specifically inhibited pseudorabies reductase activity.     

Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. We carried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared our sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells.     

Characterization of a polypeptide present in herpes simplex virus type 2-transformed and -infected hamster embryo cells

Transformation of hamster embryo cells by herpes simplex virus stimulated the production of a 35-kilodalton (35K) protein that was specifically immunoprecipitated, along with other polypeptides, by rabbit hyperimmune serum. This 35K polypeptide was further analyzed by partial digestion with Staphylococcus aureus V8 protease in parallel with a 35K polypeptide from herpes simplex virus type 2-infected cells. These polypeptides had almost identical partial-proteolytic cleavage maps, indicating that they are probably the same or that they are very similar polypeptides.     

Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA

RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.     

Characterization of mutants of herpes simplex viruses, types 1 and 2, that produce fragments of the thymidine kinase polypeptide

Seven tk- mutants of herpes simplex virus, type 2 (HSV-2), and three tk- mutants of herpes simplex virus, type 1 (HSV-1), were isolated which did not produce the thymidine kinase (TK) polypeptides but formed smaller polypeptides not seen in wild-type infected cells. Positive TK mRNA selection by hybridization to the cloned tk genes followed by in vitro translation identified the TK polypeptides. Comparisons of the products of partial proteolysis of the polypeptides of four HSV-2 and two HSV-1 tk- mutants to those of the parental TK polypeptides indicated that, in each case, the novel polypeptide was a fragment of the TK polypeptide, showing that these mutants have defects in the structural gene for tk. HSV-2 mutants of this sort have not been previously described. They and the HSV-1 mutants are similar to HSV-1 mutants reported previously. In addition, it was found that TK mRNA was present early in infection but was absent late in infection, suggesting that the shutoff of TK synthesis is due to message degradation. Also, HSV-2 TK mRNA did not hybridize to the cloned HSV-1 tk gene indicating that these genes have extensively diverged.     

Structure and origin of defective genomes contained in serially passaged herpes simplex virus type 1 (Justin).

Restriction enzyme and hybridization analyses have revealed that high-density DNA prepared from passage 15 of serially passaged herpes simplex virus type 1 (Justin) contains three major classes of modified viral DNA molecules, each composed of distinct but closely related types of repeate units. The DNA sequences within the three types of repeat units are colinear with the DNA sequences located at the right end (between coordinates 0.94 and 1.0) of the parental herpes simplex virus type 1 genome. Thus, the three types of repeat units each contain the entire repeat sequence (ac) (which brackets the unique sequences of the small [S] component of herpes simplex virus type 1 DNA) and differ only with respect to the amount of unique S sequences which they contain. The three classes of high-density DNA molecules were found to be stably propagated between passages 6 and 15 of this series.     

Gene expression of herpes simplex virus. III. Effect of arabinosyladenine on viral polypeptide synthesis

Arabinosyladenine, an established antiherpetic drug, was used to block herpes simplex virus type 1 DNA synthesis quantitatively in infected xeroderma pigmentosum cells. Kinetic analyses of viral polypeptides synthesized in the presence and absence of this drug revealed that there were at least six distinct kinetic classes of polypeptides. These differed in time of appearance after infection, time of maximum rate of synthesis, kinetics of turnoff, and sensitivity to arabinosyladenine. This study showed that arabinosyladenine had the following three main effects on herpes simplex virus type 1 gene expression. (i) The turnon of immediate early and delayed early polypeptides (kinetic classes 1 and 2) was retarded. (ii) The turnoff of early (immediate early and delayed early) polypeptides (classes 1 through 3) was delayed. (iii) The synthesis of late polypeptides (class 4 through 6) was inhibited by arabinosyladenine, with class 6 severely (80 to 90%) inhibited. The kinetic data presented here, along with the findings of other workers on the effects of inhibition of viral DNA synthesis, suggest that viral DNA replication is required for optimum synthesis of late viral polypeptides.     

DNA-binding protein associated with herpes simplex virus DNA polymerase.

Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified. The purified protein has been used to prepare antisera (both polyclonal rabbit serum and monoclonal antibodies). These reagents have been used to characterize the protein, to demonstrate its quite distinct map location from that of the DNA polymerase on the herpes simplex virus genome, and to demonstrate the close association between the two polypeptides.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

Deoxypyrimidine kinase (deoxythymidine [TdR] kinase and deoxycytidine kinase) activity was induced in human embryonic lung cells after infection with varicella-zoster virus (VZ virus). Increased enzyme activity was also produced by using cell-associated virus as inoculum instead of cell-free virus. Anti-VZ virus serum inhibited both the appearance of cytopathic effect and the induction of enzyme activity. The induced TdR kinase activity was more thermostable than that induced by herpes simplex virus type 1. Also, the TdR kinase activity of VZ virus-infected cells was inhibited by dTTP less than in mock-infected cells and more than in herpes simplex virus type 1-infected cells.     

Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion.

The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component.     

Identification and genetic mapping of a herpes simplex virus capsid protein that binds DNA

Herpes simplex virus virion protein 19C (VP19C) is a constituent of both unenveloped (nuclear) and enveloped (cytoplasmic) capsids. In this paper we report that 32P-labeled DNA, either supercoiled or linear double stranded, efficiently bound to VP19C electrically transferred from denaturing polyacrylamide gels containing electrophoretically separated proteins from purified capsids. Analyses of the polypeptides specified by herpes simplex virus type 1 X herpes simplex type 2 recombinants with respect to electrophoretic mobility and binding of 32P-labeled DNA indicate that VP19C maps at the same location as infected cell polypeptide 32 and is derived from it.     

Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells.

HEp-2 cells were infected with herpes simplex virus type 1 and labeled with [3H]thymidine and 14C-amino acids. Infected cells or nuclei prepared from them were extracted with Triton X-100 and NaCl, utilizing a method recently described, and the low-speed supernatant (extract) was partially purified by sedimentation on sucrose gradients. A nucleoprotein complex which sedimented as a wide peak around 200S was identified. The nucleoprotein complex contained viral DNA, which banded at the expected density in CsCl isopycnic gradients and was intact after measurements taken on electron microscopic photographic enlargements. The autoradiographic pattern of 14C-labeled proteins after electrophoresis showed that only a few of the virus-specific polypeptides were present in the nucleoprotein complexes, in particular, VP5, VP12, VP15.2, VP19, and VP24. Cellular histones were absent. The extracts and the nucleoprotein complexes were centrifuged to equilibrium in metrizamide density gradients without prefixation. Electron microscopic direct visualization of the nucleoprotein complexes after sucrose or metrizamide purification revealed that the proteins were preferentially associated with one end of the DNA molecule and formed large irregular terminal thickenings or capsid-like transparent shells enclosing polyglobular cores. No nucleosomes were observed on herpes simplex virus nucleoprotein complexes. The same type of complex was detected after phosphonoacetic acid addition, and grossly altered nucleocapsids were formed.     

Characteristics of chromatin preparations from herpes simplex virus infected cells

Chromatin isolated from herpes simplex virus type 1-infected baby hamster kidney cells contains a number of tightly associated virus-induced polypeptides. A subset of these proteins bind to immobilized DNA in vitro (Vmw 175, 155, 130, 63, 43, 38/39). Virus-induced polypeptides extractable with acid from infected cell chromatin include Vmw 155, the major capsid protein of herpes simplex virus type 1 virions, and Vmw 63 and 38/39 which are heterogeneous with respect to charge and are phosphorylated. These chromatin preparations, in the presence of deoxynucleoside triphosphates and MgCl2 were capable of synthesizing viral and cell DNA in a reaction which was stimulated by the addition of ATP, riboNTPs and potassium acetate. In vitro synthesized viral DNA co-sedimented with prelabelled parental DNA but the single-stranded product was smaller than parental DNA. Density labelling indicated that extensive synthesis was taking place and all BamHI fragments of viral DNA were represented by the DNA synthesized in vitro.