Expression of an early, nonstructural antigen of herpes simplex virus in cell transformed in vitro by herpes simplex virus.

Hyperimmune rabbit antiserum to an early, nonstructural herpes simplex virus type 2 (HSV-2)-induced polypeptide (VP143) reacted in immunofluorescence tests with a variety of cell lines transformed by HSV-2. Cytoplasmic fluorescence was observed in 10 to 50% of HSV-2-transformed cells, whereas no fluorescence was observed in cells transformed by other oncogenic DNA viruses or by a chemical carcinogen. VP143-specific reactivity could be absorbed from anti-VP143 serum with HSV-2-transformed cells but not with cells transformed by other agents. When HSV-2-transformed cells were synchronized in mitosis and examined at various times postmitosis for VP143-specific fluorescence, the expression of VP143 was shown to be cell cycle dependent.     

High level expression and purification of herpes simplex virus type 1 immediate early polypeptide Vmw110.

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) polypeptides. This paper reports the construction of a baculovirus vector which expresses large amounts of Vmw110, the product of IE gene 1. The expressed protein has been purified to near homogeneity and has a mobility on SDS polyacrylamide gels identical to that of Vmw110 produced during HSV-1 infection. Characterisation of its properties indicated that it forms dimers and perhaps higher order oligomers in solution and that the purified protein binds to both single stranded and double stranded calf thymus DNA cellulose columns. However, filter binding experiments were unable to detect any stable association of Vmw110 with DNA in solution.     

Comprehensive characterization of extracellular herpes simplex virus type 1 virions

The herpes simplex virus type 1 (HSV-1) genome is contained in a capsid wrapped by a complex tegument layer and an external envelope. The poorly defined tegument plays a critical role throughout the viral life cycle, including delivery of capsids to the nucleus, viral gene expression, capsid egress, and acquisition of the viral envelope. Current data suggest tegumentation is a dynamic and sequential process that starts in the nucleus and continues in the cytoplasm. Over two dozen proteins are assumed to be or are known to ultimately be added to virions as tegument, but its precise composition is currently unknown. Moreover, a comprehensive analysis of all proteins found in HSV-1 virions is still lacking. To better understand the implication of the tegument and host proteins incorporated into the virions, highly purified mature extracellular viruses were analyzed by mass spectrometry. The method proved accurate (95%) and sensitive and hinted at 8 different viral capsid proteins, 13 viral glycoproteins, and 23 potential viral teguments. Interestingly, four novel virion components were identified (U_L7, U_L23, U_L50, and U_L55), and two teguments were confirmed (ICP0 and ICP4). In contrast, U_L4, U_L24, the U_L31/U_L34 complex, and the viral U_L15/U_L28/U_L33 terminase were undetected, as was most of the viral replication machinery, with the notable exception of U_L23. Surprisingly, the viral glycoproteins gJ, gK, gN, and U_L43 were absent. Analyses of virions produced by two unrelated cell lines suggest their protein compositions are largely cell type independent. Finally, but not least, up to 49 distinct host proteins were identified in the virions.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Isolation by high-performance liquid chromatography and partial characterization of a 57,000-dalton herpes simplex virus type 1 polypeptide.

A Nonidet P-40 extract of HSV-1-purified virions was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). The first peak fraction eluted at 25% organic solvent. Polyacrylamide gel electrophoresis showed that it contained a 57,000-dalton polypeptide. The polypeptide was characterized by determination of the amino acid composition and the N-terminal amino acid sequence. Adsorption of the detergent extract before RP-HPLC showed that the polypeptide reacted with monoclonal antibodies LP1 directed against herpes simplex virus polypeptide VP-16.     

A 3'' co-terminus of two early herpes simplex virus type 1 mRNAs.

A 3' co-terminus of two early herpes simplex virus type 1 mRNAs has been identified using the nuclease -S1 mapping procedure with cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region 0.56-0.60, are unspliced and are transcribed rightwards on the prototype genome orientation. The position of their 3' ends has been located on the virus DNA sequence and lies downstream from the polyadenylation signal 5'-AATAAA-3'. This hexanucleotide sequence also was present in the complementary DNA strand and was shown to be the polyadenylation signal for a leftwards-transcribed late mRNA. The abundance within the cytoplasm of the 5.0 kb and 1.2 kb mRNAs was investigated. Results indicated that these mRNAs were regulated in concert. It is suggested that sequences at the 3' co-terminus may be involved in their regulation.     

Purification and characterization of UL9, the herpes simplex virus type 1 origin-binding protein.

UL9, the origin-binding protein of herpes simplex virus type 1 (HSV-1), has been overexpressed in an insect cell overexpression system and purified to homogeneity. In this report, we confirm and extend recent findings on the physical properties, enzymatic activities, and binding properties of UL9. We demonstrate that UL9 exists primarily as a homodimer in solution and that these dimers associate to form a complex nucleoprotein structure when bound to the HSV origin of replication. We also show that UL9 is an ATP-dependent helicase, capable of unwinding partially duplex DNA in a sequence-independent manner. Although the helicase activity of UL9 is demonstrable on short duplex substrates in the absence of single-stranded DNA-binding proteins, the HSV single-stranded DNA-binding protein ICP8 (but not heterologous binding proteins) stimulates UL9 to unwind long DNA sequences of over 500 bases. We were not able to demonstrate unwinding of fully duplex DNA sequences containing the HSV origin of replication. However, in experiments designed to detect origin-dependent unwinding, we did find that UL9 wraps supercoiled DNA independent of sequence or ATP hydrolysis.     

Virion component of herpes simplex virus type 1 KOS interferes with early shutoff of host protein synthesis induced by herpes simplex virus type 2 186.

Herpes simplex virus (HSV) strains HSV type 1 (HSV-1) KOS and HSV-2 186 are representative of delayed and early shutoff strains, respectively, with regard to their ability to inhibit protein synthesis in Friend erythroleukemia cells. When these cells were simultaneously infected with HSV-1 KOS and HSV-2 186, HSV-1 KOS interfered with the rapid suppression of globin synthesis induced by HSV-2 186. The observed interference was competitive and not due to exclusion of HSV-2 by HSV-1 at the level of adsorption. Furthermore, UV-irradiated HSV-1 KOS was also effective at interfering with the early shutoff function of HSV-2 186, indicating that a virion component is responsible for the observed interference.     

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene.     

Hand-to-hand transmission of herpes simplex virus type 1

Droplets of tissue culture fluid containing herpes simplex virus type 1 were placed on the palm of the hand. Each 0.01 ml droplet was taken from a stock virus suspension with a titre of 10(7.5) TCID50/0.1 ml. At 0, 15, 30, 60 and 120 min a droplet was firmly touched with the middle finger of the right hand, after which, attempts were made to recover virus from the finger. At 0 min, when the virus-containing droplet was in a liquid state, there was a 100% rate of virus recovery. By 15 min the droplets had dried out, and after touching dried out droplets there was a 40% virus recovery rate, even though experimental procedures demonstrated that infectious virus was present in the dried out droplets at all test times. If the finger was moistened with tap water or saliva, there was a 100% recovery rate of virus after touching dried out droplets, as well as after touching droplets in a liquid state.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize virus nonstructural proteins.

The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.     

Immunological characterization of herpes simplex virus type 1 and 2 polypeptide(s) involved in viral ribonucleotide reductase activity

Mammalian cells infected with herpes simplex virus (HSV) express a novel ribonucleotide reductase which is biochemically and immunologically distinct from the uninfected-cell enzyme. Using polyvalent rabbit antiserum raised against partially purified HSV type 2 reductase as well as monoclonal antibodies to HSV type 1 and HSV type 2 early antigens, we have been able to show that in both serotypes reductase activity is associated with phosphoproteins of molecular weights 144,000 and 38,000 encoded between map units 0.566 and 0.602 in the viral genomes. The major antigenic species (144,000) have been tentatively identified as HSV type 1 ICP6 and HSV type 2 ICP10.     

gA and gB glycoproteins of herpes simplex virus type 1: two forms of a single polypeptide.

Utilizing a combination of preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-hydroxylapatite column chromatography, we have separated and purified the gA and gB glycoproteins of the major virus-specific glycoprotein region from herpes simplex virus type 1-infected cells. By using purified antigen preparations, antisera specific to each of these glycoproteins were produced. Immunoprecipitation from detergent extracts of infected cells and radioimmune precipitation of the purified antigens have shown that the anti-gA and anti-gB sera each recognize both the gA and the gB glycoproteins. The anti-gA serum was also shown to neutralize virus despite the presence of only minute quantities of the gA glycoprotein in virions. Pulse-chase studies have indicated that the gA and gB glycoproteins are synthesized from a common precursor polypeptide. Together, these data demonstrate that the gA and gB glycoproteins of herpes simplex virus type 1 are antigenically similar but not identical and probably represent two different forms of the same polypeptide which differ in their degree of glycosylation.     

Characterization of a herpes simplex virus type 2 75,000-molecular-weight glycoprotein antigenically related to herpes simplex virus type 1 glycoprotein C.

Evidence is presented that the herpes simplex virus type 2 glycoprotein previously designated gF is antigenically related to herpes simplex virus type 1 gC (gC-1). An antiserum prepared against type 1 virion envelope proteins immunoprecipitated gF of type 2 (gF-2), and competition experiments revealed that the anti-gC-1 component of the antiserum was responsible for the anti-gF-2 cross-reactivity. An antiserum prepared against fully denatured purified gF-2, however, and three anti-gF-2 monoclonal antibodies failed to precipitate any type 1 antigen, indicating that the extent of cross-reactivity between gC-1 and gF-2 may be limited. Several aspects of gF-2 synthesis and processing were investigated. Use of the enzymes endo-beta-N-acetylglucosaminidase H and alpha-D-N-acetylgalactosaminyl oligosaccharidase revealed that the fully processed form of gF-2 (about 75,000 [75K] apparent molecular weight) had both complex-type N-linked and O-linked oligosaccharides, whereas newly synthesized forms (67K and 69K) had only high-mannose N-linked oligosaccharides. These last two forms were both reduced in size to 54K by treatment with endo-beta-N-acetylglucosaminidase H and therefore appear to differ only in the number of N-linked chains. Neutralization tests and radioiodination experiments revealed that gF-2 is exposed on the surfaces of virions and that the 75K form of gF-2 is exposed on cell surfaces. The similarities and differences of gF-2 and gC-1 are discussed in light of recent mapping results which suggest collinearity of their respective genes.