Herpes simplex virus glycoprotein D: human monoclonal antibody produced by bone marrow cell line.

Normal bone marrow cells from a donor positive for herpes simplex virus were transformed with Epstein-Barr virus. The resulting lymphoblastoid cell line has secreted immunoglobulin G1 of the kappa type continuously for 2 years. This immunoglobulin, detected both on the cell surface and in the cytoplasm, reacts with cells infected with herpes simplex virus. It defines an antigen that comigrates with the 55-kilodalton glycoprotein D of herpes simplex virus type 1 and neutralizes the infectivity of herpes simplex viruses 1 and 2.

Transgene expression of plasmid DNAs directed by viral or neural promoters in the rat brain.

The use of circular plasmid DNA may be an alternative method for the transfer of genes into the brain and is presumably easier to use than other vectors, such as viruses or genetically engineered cells. The effectiveness and time course of the expression of a reporter gene (LacZ), directed by appropriate promoters, was studied after stereotaxic injection of naked plasmid DNAs into the rat thalamus, cortex or cerebellum. The efficiencies of three different promoters, the human cytomegalovirus (HCMV) promoter and the glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) promoters (specific for astrocytes and neurons, respectively) to drive reporter gene expression were compared. Efficient expression of beta-gal, detected by X-gal histochemistry or immunochemistry, required the use of 50 microg of DNA and was detectable as early as 48 h after injection. Expression increased until day 8, remained stable until day 15, then decreased over 2 months, probably as a result of non-specific degradation of the plasmids within the transfected cells rather than from specific down-regulation of promoters, as the same time course was seen with all three promoters tested. Depending on the promoter used (GFAP or NSE), LacZ was preferentially expressed within astrocytes or neurons, respectively. The GFAP promoter was found to be as efficient as the HCMV promoter, possibly due to the reactive gliosis induced by plasmid injection which is known to up-regulate GFAP expression.

RNA synthesis in cells infected with herpes simplex virus. X. Properties of viral symmetric transcripts and of double-stranded RNA prepared from them.

HEp-2 cells infected with herpes simplex 1 virus contain RNA transcripts capable of forming double-stranded (DS) RNA on annealing. The properties of purified DS RNA were as follows (i) DS RNA is resistant to depolymerization by RNase A or T-1 in 2 times 0.15 M NaCl, plus 0.015 M sodium citrate (SSC) but not 0.1 times SSC or following thermal denaturation. (ii) The Tm of the viral DS RNA was 100 C in 0.1 times SSC. (iii) Undenatured DS RNA does not hybridize with viral DNA: upon denaturation, excess unlabeled RNA drove 50 to 55% of labeled DNA into DNA-RNA hybrid. The kinetics of hybridization indicate that the DS RNA consists of at least two populations of transcripts arising from 29 and 26% of viral DNA and differing 40-fold in molar concentration.

Inhibition of viral RNA methylation in herpes simplex virus type 1-infected cells by 5' S-isobutyl-adenosine.

5' S-isobutyl-adenosine (SIBA), a structural analogue of S-adenosylhomocysteine, reversibly blocks the multiplication of herpes simplex type 1 virus. In the presence of SIBA, viral protein synthesis is inhibited. After removing SIBA the synthesis of proteins starts rapidly again. The new polypeptides are mainly alpha proteins (Honess and Roizman, J. Virol. 14:8-19, 1974,), normally the first to be synthesized after infection. The rapid synthesis of proteins after release of inhibition seems to be directed by mRNA formed in the presence of SIBA as indicated by experiments using actinomycin D but which was undermethylated as shown by analysis of methyl groups on RNA. SIBA inhibits the methylation of mRNA and especially that of the 5' cap. Capping of mRNA thus seems to be essential for efficient translation. The analogue affected various methylations to different extents.

Multiple adjacent or overlapping loci affecting the level of gC and cell fusion mapped by intratypic recombinants of HSV-1.

We have prepared and analyzed 40 HSV-1 intratypic recombinants with regard to plaque morphology and glycoprotein C(gC) phenotypes. Vero cells have been cotransfected with the intact genome of HSV-1(F) and cloned or uncloned DNA fragments from HSV-1(MP) and recombinants inducing the fusion of Vero cells [syncytial (Syn) recombinants] have been selected and purified. Marker transfer of the Syn phenotype has been observed with the cloned BamHI L and B fragments (0.706-0.745 and 0.745-0.810 map units, respectively) as well as with the uncloned HpaI TXO fragment (0.710-0.761) from MP DNA. No marker transfer has been observed with F DNA alone or with the cloned BamHI N fragment (0.863-0.898 map units). When viruses expressing the Syn phenotype in Vero cells were tested in HEp-2 cells, three kinds of recombinants were observed. Members of the first class expressed a wild type, cytoaggregating (Syn+), plaque morphology in these cells. Members of the second class induced the complete fusion (Syn phenotype) of the cells. Members of the third class induced an intermediate plaque morphology, characterized by the formation of groups of polykaryocytes (fused cells) but without formation of a complete syncytium. All recombinants expressing the Syn+ phenotype in HEp-2 cells were also gC+, whereas recombinants expressing the Syn phenotype in these cells were gC- with one exception, in which low levels of gC could be detected (but clearly less than with HSV-1(F]. Concerning polykaryocytic class of recombinants, some of them were gC+ while others expressed only low amounts of gC; no gC- virus was observed within this class of recombinants. The three classes of recombinants were observed with each of the cloned BamHI L and B fragments and also with the HpaI TXO fragment, suggesting the existence of multiple adjacent or overlapping loci affecting plaque morphology and the control of the accumulation or the synthesis of gC at both sides of 0.745 map units.

The pseudorabies virus host-shutoff homolog gene: nucleotide sequence and comparison with alphaherpesvirus protein counterparts.

The virion host shutoff function (VHS) of many herpesviruses is required for inhibition of cellular gene expression in infected cells. This function corresponds to the UL41 open reading frame of herpes simplex virus type 1 (HSV-1) and homolog sequences have been found in other alphaherpesvirus, like HSV-2, Varicella-Zoster virus (VZV) and equine herpesvirus type 1 (EHV-1). In this work, we have cloned and sequenced a pseudorabies virus (PRV) gene which is homologous to the VHS genes of the other alphaherpesvirus. Sequence comparison between all deduced amino acid sequences indicates the presence of several conserved domains, separated by regions containing gaps or low conserved sequences.

Nuclear processing of viral high-molecular-weight RNA in cells infected with herpes simplex virus type 1.

Nuclear RNA isolated from cells infected by herpes simplex virus type 1, strain F, was fractionated on formamide-sucrose gradients into two major classes, greater and less than 45S. These two classes of labeled nuclear RNA were hybridized to viral DNA fragments generated by digestion with the restriction enzymes HindIII and BglII. Early in infection, only a few DNA fragments hybridized to RNA, with slight differences between the two classes. Late in infection, all DNA fragments hybridized, showing that all viral RNA was present in large precursor molecules greater than 14 kilobases. The fragments that correspond to late gene products hybridized more of the small RNA than the large RNA. This suggests that the mRNA corresponding to late genes accumulated after the large precursors have been cleaved. Large (greater than or equal to 45S) and small (< 45S) nuclear RNA and cytoplasmic RNA from cells late in infection were hybridized in excess to in vitro-labeled HindIII M and L fragments. More than 50% of the HIndIII M fragment annealed with the large nuclear RNA, but only 36% of it annealed with the cytoplasmic RNA. The HindIII L fragment hybridized large nuclear RNA and cytoplasmic RNA to the same extent (30% and 26%). These results suggest that RNA complementary to the HindIII M fragment, which is the template for immediate early polypeptides, was regulated in the nuclei at the posttranscriptional level. This seems to suggest that temporal regulation of RNA cleavage occurs in the nucleus.

Herpes simplex type 1 infection of nonpermissive rat XC cells.

Herpes simplex virus type 1 (HSV-1) infection of non permissive XC cells (a rat cell line transformed by Rous sarcoma virus) was studied. Using virus labeled with 3H-thymidine it was shown that adsorption is similar to that in a permissive system. By electron microscopy enveloped particles were observed in cytoplasmic vesicles in XC cells but not in the permissive system. However input viral DNA was degraded both in non permissive cells (XC) and permissive cells (HEp-2) and the degradation products were found incorporated into cellular DNA in the first case or into viral DNA in the second case. In the non permissive XC cells, it was possible to detect a small amount of incorporation of radioactive precursors into the viral DNA, identified by its buoyant density in CsCl of 1.726 g/cm3 and by hybridization with viral DNA. This DNA has the size of the native viral genome and its uptake of radioactive precursors was only partially inhibited by phosphonoacetic acid, a specific inhibitor of HSV-DNA polymerase. With permissive HEp-2 cells in the presence of such inhibitor, the obtained data are roughly the same as with XC cells, both in the presence or in the absence of phosphonoacetic acid. These results suggest that the observed viral DNA synthesis in XC cells is not a true replication but, further, a repair synthesis and, also, that the same events might take place in the permissive system before the onset of viral DNA replication.

Virus polypeptide synthesis induced by herpes simplex virus in non-permissive rat XC cells.

The synthesis of virus polypeptides in rat XC cells infected with herpes simplex virus type 1 (HSV-1; 13VB4tsC75) was studied. At the permissive temperature the virus induced the synthesis, in a cascade fashion, of significant amounts of several early polypeptides (ICP 6, 8 and 39) and those late polypeptides that are relatively resistant to inhibition by phosphonoacetic acid in HEp2 cells (ICP 5, 11, 25, 29, 43 and 44). The infectious cycle appeared to become arrested in XC cells at about 7 to 9 h postinfection, because the relative concentrations of early and latest polypeptides labelled thereafter remained constant and the levels of several of the late virus polypeptides were severely reduced (ICP 2, 10, 24 and 26) or not synthesized at all (ICP 32, 34 and 37). When XC cells were infected at a very high m.o.i., only a small amount of virus DNA synthesis could be detected; the synthesis of cellular DNA was not impaired and the infected XC cells continued to replicate for several weeks at least. When XC cells were infected at the non-permissive temperature, only the immediate-early (IE) ICP 4 could be detected while IE ICP 0 and 22 were not observed. Infection of XC cells with HSV-1 (MP) also resulted in the production of early and late viral polypeptides. On the other hand, in XC cells infected with HSV-1 (F) and HSV-1 (HFEM), the synthesis of virus polypeptides could not be detected.

Genetic and environmental determination of herpes simplex virus type 1 penetration into non-permissive rat XC cells.

We have tested a number of herpes simplex virus type 1 (HSV-1) populations for their ability to enter and express virus polypeptides in non-permissive rat XC cells. The viruses tested included 40 intratypic F(MP) recombinants and different batches of virus, belonging to the same strains, that had been produced in two different permissive systems (HEp-2 or Vero cells). Our results indicated that the ability to infect XC cells was determined in part by genetic elements of the virus genome and in part by phenotypic characteristics conferred by the permissive cell that had produced the virus: a virus strain like HSV-1 MP which, when produced in HEp-2 cells, was able to infect XC cells, lost this ability when produced in Vero cells. Working only with viruses produced in HEp-2 cells we showed that the ability to enter XC cells could be transferred from the MP strain to the F strain (which does not normally infect XC cells efficiently) by transfer of the cloned BamHI B (map units 0.745 to 0.81) or BamHI L (map units 0.706 to 0.745) fragments isolated from MP DNA. The implicated locus or loci seemed to segregate, however, from loci controlling gC synthesis and cell fusion, which have been described as mapping in the same region.

Expression of immediate-early genes in herpes simplex virus type 1-infected XC cells: lack of ICP22 (68K) polypeptide.

The expression of immediate-early (IE) genes of herpes simplex virus type 1 (HSV-1, MP strain) in non-permissive rat XC cells was analysed and compared with the expression of IE genes in permissive HEp-2 cells by the following three methods: analysis of virus polypeptide synthesis in infected cells, Northern blot hybridization between poly(A) nuclear or cytoplasmic RNA and in vitro labelled virus DNA or plasmid-cloned fragments corresponding to IE genes, and ability of poly(A) cytoplasmic RNAs to direct synthesis of virus polypeptides in vitro. ICP4 (175K), ICP0 (110K) and ICP27 (62K) were synthesized in XC cells although in smaller amounts than in HEp-2 cells; ICP4 is functional since early and late polypeptides could be observed. Their corresponding mRNAs were present at low levels in nuclei and in cytoplasm and are functional since the polypeptides were synthesized in a rabbit reticulocyte system. ICP22 (68K) was not detectable in infected XC cells; its mRNA was present in nuclei and in cytoplasm, but it is not functional since the corresponding polypeptide was not synthesized in a rabbit reticulocyte system. This suggests some structural differences in the ICP22 mRNA molecules in infected XC and HEp-2 cells and implicates cellular determinants in the control of the expression of HSV-1 IE genes.

Infection of a restrictive cell line (XC cells) by intratypic recombinants of HSV-1: relationship between penetration of the virus and relative amounts of glycoprotein C.

The ability to induce the synthesis of virus polypeptides in nonpermissive XC cells by several strains, variants, and intratypic recombinants of HSV-1 has been investigated. Our results show that (i) whereas HSV-1 strains mP and MP, and recombinants F(MP)A through D (Ruyechan et al., J. Virol. 29, 677-697, 1979) and HFEMtsB5/MP3 (Manservigi et al., Proc. Nat. Acad. Sci. USA 74, 3913-3917, 1977) induced in XC cells the synthesis of several virus polypeptides (the "positive" viruses), in the case of HSV-1 strain F, variants HFEMtsB5 and mPtsHA1 (both tested at 33 degrees), and recombinants F(MP)E and F(MP)F, no virus specific polypeptides could be detected in these cells (the "negative" viruses). (ii) Failure of the "negative" viruses to synthesize polypeptides in XC cells could be explained by failure of the virions to penetrate the cells since polyethylene glycol, a fusion promotor agent, enabled these viruses to overcome the blockage of their expression. (iii) The ability to modulate penetration into XC cells is genetically determined. A locus affecting this function maps between coordinates 0.70 to 0.82 of HSV-1 DNA and is closely linked to Cr, a locus controlling the synthesis or accumulation of virus glycoprotein C (gC) (Ruyechan et al., 1979). (iv) A decrease in the amount of gC, relative to gB, is associated with an enhanced ability to enter XC cells, suggesting that gC may control penetration into these restrictive cells by negatively modulating the gB promoted fusion between host cell and virion membranes.

Composition and size of Shope fibroma virus deoxyribonucleic acid.

Deoxyribonucleic acid (DNA) extracted from purified virions of Shope fibroma virus (SFV) (by using DNA from Microccocus lysodeikticus as marker) had a buoyant density of 1.6996 +/- 0.0003 g/ml), hence a guanine plus cytosine (G + C) content of 40.4 +/- 0.3%, which is close to the G + C content of the DNA of susceptible rabbit cells (40.9 +/- 0.4%) and different from that of vaccinia virus DNA (35.5 +/- 0.4%). For the determination of the molecular weight of DNA, SFV and vaccinia purified virions, treated with Pronase and detergent, were cosedimented in sucrose density gradients. Results showed that SFV-DNA has a molecular weight of about 153 x 10(6) daltons. By electron microscopy, only one molecule corresponding to this value was observed (its length was 80.3 mum). The others had a median size of 49.8 mum +/- 0.9.

Herpes simplex virus type-1 immediate-early gene expression and shut off of host protein synthesis are inhibited in neomycin-treated human epidermoid carcinoma 2 cells.

Infection of human epidermoid carcinoma-2 (HEp-2) cells by Herpes simplex virus type 1 (HSV-1) leads to significant activation of inositol phospholipid turnover after 15 min. The effect of neomycin, an inhibitor of inositol phospholipid turnover, has been investigated for its effect on HSV-1 multiplication in HEp-2 cells. HSV-1 multiplication is inhibited by neomycin. This inhibition is not due to a block of virus adsorption or penetration. Neomycin inhibits the expression of virus immediate-early genes, as well as expression of early genes and viral DNA synthesis. In neomycin-treated cells, the usual virion-associated shut off of host protein synthesis does not occur. These results indicate that the inositol phospholipid pathway is involved in immediate-early gene expression and shut off of host protein synthesis in HEp-2 cells.

Herpes simplex virus type-1-induced stimulation of ribosomal protein S6 phosphorylation is inhibited in neomycin-treated human epidermoid carcinoma 2 cells and in ras-transformed cells.

Neomycin, an inhibitor of inositol phospholipid turnover, prevents Herpes-simplex-virus-type-1 (HSV-1)-induced stimulation of ribosomal protein S6 phosphorylation, but does not impair the S6 phosphorylation induced by serum. Long-term treatment with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C activity, does not inhibit virus-induced S6 phosphorylation. In ras-transformed cells, S6 phosphorylation is not stimulated after HSV-1 infection. These results suggest that activation of the inositol phospholipid pathway is involved in the HSV-1-induced stimulation of S6 phosphorylation. However, protein kinase C activation does not appear to be necessary for HSV-1-induced S6 phosphorylation.

Ribosome and protein synthesis modifications after infection of human epidermoid carcinoma cells with herpes simplex virus type 1.

Modifications of ribosomes have been investigated in human epidermoid carcinoma-2 cells at different stages of herpes simplex virus type 1 infection. Very early in infection, there is an increase in ribosomal protein S6 phosphorylation even in the absence of serum. The same result is obtained in the presence of actinomycin D. At early infection time, ribosomal proteins S2, S3a and Sa are newly phosphorylated. At early and early-late times, three phosphorylated non-ribosomal proteins (v1, v2 and v3) are differently associated temporally to ribosomes. Analyses of proteins extracted from 40S subunits, 80S ribosomes and polysomes show that v1 and v2 are distributed differently among the different ribosomal populations. S6 phosphopeptides were found to be identical after serum stimulation and after viral infection. In every case phosphoserine and phosphothreonine were identified in S6. Only phosphoserine was found in other phosphorylated proteins. Our results indicate that herpes simplex virus type 1 is able to modify pre-existing ribosomes: (i) by stimulating a pre-existing kinase for S6 phosphorylation even in the absence of serum and of viral genome expression; (ii) by inducing new specific kinase activity(ies); and (iii) by association of new, phosphorylated proteins to ribosomes. These ribosomal modifications are correlated with changes in protein synthesis, as shown by two-dimensional electrophoretic analyses of newly synthesized 35S-labelled proteins.

In vitro divergence of HSV-1 populations propagated in different cell lines.

To investigate how the structure of a virus population is influenced by the particular cell types in which the virus is propagated, laboratory populations of HSV-1 have been serially passaged onto a number of different cell lines, differing either in species or in tissue specificity. After a limited number of in vitro passages, several of the daughter virus populations have diverged in the expression of at least one phenotype, suggesting that different cell types have selected different variants contained in the parental virus population.

Susceptibility to herpes simplex virus type 1 infection of non-permissive rat XC(HPRT-) x permissive mouse L(TK-) hybrid cells.

Somatic cell hybrids between rat XC(HPRT-) cells, non-permissive for herpes simplex virus type 1 (HSV-1) infection, and permissive mouse L(TK-) cells were constructed and karyotyped. Infection of these hybrid cells by HSV-1 strains F and MP revealed that they were susceptible to the virus. The amounts of virus produced by the hybrid cells, as well as the cytopathic effect observed, was very similar to that of the parental L(TK-) cells. Our results suggest that failure of HSV-1 to replicate in XC cells is more likely to be due to the absence of cellular elements required for efficient virus multiplication rather than to the presence of blocking or inhibiting factors.

Cloning, sequencing, and functional characterization of the two subunits of the pseudorabies virus DNA polymerase holoenzyme: evidence for specificity of interaction.

The pseudorabies virus (PRV) genes encoding the two subunits of the DNA polymerase were located on the genome by hybridization to their herpes simplex virus type 1 (HSV-1) homologs, pol and UL42, and subsequently were sequenced. Like the HSV-1 homologs, in vitro translation products of the PRV gene encoding the catalytic subunit (pol) possessed activity in the absence of the Pol accessory protein (PAP). However, the PRV PAP stimulated the activity of Pol fourfold in the presence of 150 mM KCl, using an activated calf thymus DNA template. The stimulation of Pol activity by PAP under high-salt conditions and the inhibition of Pol activity by PAP when assayed in low salt (0 mM KCl) together were used to determine the specificity with which PAP interacted with Pol. Despite functional similarity, HSV-1 UL42 and PRV PAP could neither stimulate the noncognate Pols at high salt nor inhibit them at low salt. Furthermore, a PRV Pol mutant lacking the 30 C-terminal amino acids retained basal Pol activity but could be neither stimulated nor inhibited by the PRV PAP. Sequence comparisons of the Pol proteins of the alphaherpesviruses reveal a conserved domain in the C terminus which terminates immediately before the last 41 residues of both PRV and HSV-1 proteins. These results indicate that the ability and specificity for interaction of the PRV Pol with PAP most likely resides predominantly in the extreme Pol C terminus.

Enhancement of neuronal protection from oxidative stress by glutamic acid decarboxylase delivery with a defective herpes simplex virus vector.

We have developed defective herpes simplex virus 1 (HSV-1) vectors, based on amplicon plasmids with a replication-deficient mutant, as helper for the transfer of the glutamic acid decarboxylase (GAD67) or beta-galactosidase (beta-gal) gene as control directed by HCMV promoter into neuronal-like cells (PC12) and primary neurons. GAD67 protein was detected immunochemically, while GAD67 activity in virus-producing and nonproducing cell lines was detected enzymatically or by GABA release. Infection with GAD67-expressing amplicon vectors enhanced the resistance of PC12 cells to H(2)O(2). This protection was related to increased energy metabolism, as shown by MTT reduction and ATP level, and involved the GABA shunt, as shown by the reduction in ATP level seen in the presence of gamma-vinyl GABA (GVG), a specific GABA transaminase inhibitor. Level of glutathione (GSH), which requires ATP for its synthesis, was increased by the GAD67 transgene. The activity of glucose-6-phosphate dehydrogenase involved in the maintenance of the NADPH that can be used for the regeneration of the GSH pool, was increased by infection with amplicon vectors. Thus, replication-deficient HSV-1 and the GAD67 transgene have complementary neuroprotective effects and infection with GAD67-expressing amplicon vectors was able to protect nondifferentiated cortical neurons from glutamate toxicity mediated by oxidative stress. Such defective GAD67-expressing HSV-1, as neurotropic vector, should be helpful in neurodegenerative diseases implicating alterations of energy metabolism and oxidative stress in neuronal cells expressing GABA transaminase.