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Previous work has shown that the 68-kDa immediate-early protein of herpes simplex virus type 1 (HSV-1), also known as ICP22, is involved in the control of viral gene expression, although the precise mechanism remains to be elucidated. In order to study the function(s) of this protein, we constructed expression vectors containing the coding sequence of the ICP22 gene placed under the control of the SV40 or HCMV promoter. After cell transfection, ICP22 synthesis was studied by immunoblotting, using a specific antiserum. In transient expression experiments in COS cells in which the ICP22 vector was under the control of the SV40 promoter, we found that ICP22 was able to inhibit chloramphenicol acetyltransferase (CAT) expression under the control of either the alpha 22 (IE4) promoter or other immediate-early promoters, such as alpha 4 (IE3), alpha 0 (IE1), and alpha 27 (IE2). CAT expression under the control of the alpha 4 (IE3) promoter was inhibited in these cells by expression of ICP22 under the control of the HCMV promoter; it was also inhibited in RAT-1 cells by ICP22 expressed under the control of the SV40 or HCMV promoter. In contrast, CAT expression directed by the SV40 or HCMV promoters was only weakly or not inhibited by the ICP22 vectors. We also constructed an expression vector for UL13, a gene whose product is implicated in the phosphorylation of ICP22. Although CAT expression under the control of the alpha 4 (IE3) promoter was also negatively regulated by the UL13 gene product, the effects of the ICP22 (directed by the SV40 or HCMV promoter) and UL13 vectors were not synergistic; furthermore, at a particular molar ratio of the two vectors, inhibition of CAT activity was partially reversed. The results in the present work suggest that ICP22 can negatively regulate the expression of immediate-early viral genes and that its phosphorylation by UL13 protein kinase might be involved in the modulation of its function.

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.

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.

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.

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.

In order to examine cellular gene involvement in HSV-1 expression, we constructed different rat embryo fibroblast cell lines immortalized by adenovirus E1A or c-myc, with or without the human EJ bladder carcinoma transforming oncogene EJ-ras. HSV-1 multiplication was strongly inhibited in cells expressing EJ-ras genes compared to immortalized control cells. Virus adsorption and penetration were not quantitatively modified, but HSV-1 DNA replication was inhibited. The expression of viral thymidine kinase (TK) activity after infection by recombinant virus with the TK coding sequence under immediate-early (IE) promoter control showed that IE gene expression is inhibited in cells expressing EJ-ras. Analysis of IE gene transcription by Northern-blot hybridization and by nuclear run-off transcription assay indicates that this inhibition takes place at the transcriptional level.

SUMMARY 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.

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.

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.