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

Summary 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.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis.

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

Herpes simplex virus immediate-early promoters are responsive to virus and cell trans-acting factors.

The promoters for each of the immediate-early genes from herpes simplex virus type 1 were cloned and fused to a chloramphenicol acetyltransferase cassette. These chimeric genes were used as targets in a transient expression assay to determine how the immediate-early gene products ICP4 and ICP0 and the virion-associated stimulatory protein Vmw65 affected their expression in HeLa and Vero cells. The basal level of expression from these cassettes differed significantly depending on the extent of 5'-flanking sequence and the cell line that served as host. The promoters from IE-4 and IE-0 behaved in a qualitatively similar fashion independent of the host cell. However, the promoter for ICP27 had a unique response pattern: in Vero cells it acted as an alpha gene promoter, whereas in HeLa cells its response was more like that of a beta gene promoter. The promoter sequences for ICP22 and ICP47 behaved as the IE-4 and IE-0 promoters did in HeLa cells, but their response to the effector molecules in Vero cells was unlike that of other alpha gene promoters we have studied. Evidence is also presented for a role for ICP27 in autoregulation.     

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize immediate-early protein ICP27.

The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H-2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino-acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity.     

Herpes simplex virus types 1 and 2 modulate autophagy in SIRC corneal cells

Autophagy and apoptosis function as important early cellular defense mechanisms in infections and other diseases. The outcome of an infection is determined by a complex interplay between the pathogenic microorganism and these intracellular pathways. To better understand the cytopathogenicity of Herpes simplex virus types 1 and 2 (HSV-1 and -2), we studied the effect of these viruses on the autophagic and apoptotic processes in the SIRC corneal cell line. Infection with the KOS strain of HSV-1 and a wild-type strain of HSV-2 enhanced autophagosome formation, triggered cytoplasmic acidification, increased LC3B lipidation and elevated the ratio of apoptotic cells. The autophagy inhibitor bafilomycin A1 triggered a significant increase in the apoptotic responses of HSV-1- and HSV-2-infected cells. Thus, both HSV types affect autophagy and apoptosis in a coordinated fashion, and autophagy plays cytoprotective role in HSV-infected cells via antagonizing apoptosis. Together these data implicate autophagy in the pathogenic mechanism of herpetic keratitis.     

A post-alpha gene function turns off the capacity of a host protein to bind DNA in cells infected with herpes simplex virus 1.

HEp-2 cell proteins electrophoretically separated in denaturing polyacrylamide gels and electrically transferred to nitrocellulose sheets contain a polypeptide which efficiently binds linear native DNA end labeled with 32P but not denatured DNA. The polypeptide has an apparent molecular weight of ca. 130,000. The activity of the protein was stable, and no appreciable turnover was observed after exposure of uninfected cells to inhibitory concentrations of cycloheximide for intervals of up to 24 h. However, the activity was absent from lysates of cells harvested 6 h or later postinfection with wild-type viruses. To identify the viral function involved in the loss of DNA-binding activity, we tested the lysates of cells infected with several mutants. Thus, the DNA-binding activity was unaffected in cells infected with a temperature-sensitive mutant (herpes simplex virus 1 tsLB2) in the alpha 4 gene and was maintained at a nonpermissive temperature (39 degrees C). Experiments involving (i) temperature shift-down of cells infected with tsLB2 in the presence of cycloheximide, (ii) withdrawal of cycloheximide in the presence and absence of actinomycin D from cells infected with wild-type virus, (iii) infection of cells at 33 and 39 degrees C with herpes simplex virus 1 tsHA1 carrying a temperature-sensitive lesion in the beta 8 gene, and (iv) infection of cells in the presence of inhibitory concentrations of phosphonoacetate led to the conclusion that the viral functions responsible for the loss of DNA-binding capacity were specified by either beta or gamma genes not dependent on viral DNA synthesis for their expression.     

Herpes simplex virus US3 protein kinase regulates host responses and determines neurovirulence

The US3 of HSV encodes a serine/threonine protein kinase that is highly conserved among members of the alphaherpesviruses. It is an accessory gene that is not required for viral replication in cultured cells but appears essential for viral survival in humans. Although accumulating in vitro evidence suggested that the viral protein kinase is multifunctional, little information is available about its functions in vivo. Several reports point out that, upon invasion into the peripheral nervous system, HSV blocks virus-induced neuronal apoptosis, while presumably subverting host immune responses, largely through actions of the US3 protein kinase. In addition, the US3 protein kinase confers the viral neurovirulence. In the present article, functions of the HSV US3 protein kinase are briefly reviewed, with special attention given to its role in regulating host responses and neurovirulence.