Detailed structural analysis of two spliced HSV-1 immediate-early mRNAs.

The structures of two HSV-1 immediate-early mRNAs have been determined by nuclease-digestion procedures using 5' and 3' end-labelled DNA probes. These mRNAs, which map across the junctions between the short unique (US) and short repeat (IRS and TRS) genome regions, have common 5' portions located in IRS and TRS. The 3' portions, which extend into opposite ends of US, and unique. The DNA sequence encoding the common 5' portions largely comprises a 247 base pair (bp) leader region and a single intron of variable size. The variation in intron length is due to different copy numbers of a 22 bp tandem reiteration. A small proportion of the mRNA population is unspliced, but otherwise is identical to the more abundant spliced species.     

Comparison of the host immune response to herpes simplex virus 1 (HSV-1) and HSV-2 at two different mucosal sites

A study was undertaken to compare the host immune responses to herpes simplex virus 1 (HSV-1) and HSV-2 infection by the ocular or genital route in mice. Titers of HSV-2 from tissue samples were elevated regardless of the route of infection. The elevation in titers of HSV-2, including cell infiltration and cytokine/chemokine levels in the central nervous system relative to those found following HSV-1 infection, was correlative with inflammation. These results underscore a dichotomy between the host immune responses to closely related alphaherpesviruses.     

Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 X HSV-2 recombinants.

In an earlier paper (Morse et al., J. Virol 24:231--248, 1977) we reported on the provenance of the DNA sequences in 26 herpes simplex virus type 1 (HSV-1) X HSV-2 recombinants as determined from analyses of their DNAs with at least five restriction endonucleases. This report deals with the polypeptides specified by the recombinants and by their HSV-1 and HSV-2 parents. We have identified (i) the corresponding HSV-1 and HSV-2 polypeptides with molecular weights ranging from 20,000 to more than 200,000, (ii) the polypeptides that undergo rapid post-translational processing, and (iii) polypeptides that vary intratypically in apparent molecular weight. By comparing the segregation patterns of the polypeptides with those of the DNA sequence of the recombinants, we have mapped the templates specifying 26 polypeptides and several viral functions on the physical map of HSV DNA. The data show the following: (i) alpha polypeptides map at the termini of the L and S components of the HSV DNA. Although alpha ICP 27 maps entirely within the reiterated region of the L component, the template for alpha ICP 4 may lie only in part within the reiterated sequences of the S component. Of note is the finding that cells infected with a recombinant that contains both HSV-1 and HSV-2 DNA sequences in the S component produced alpha ICP 4 of both HSV-1 and HSV-2. (ii) Templates specifying beta and gamma polypeptides map in the L component and appear to be randomly distributed. (iii) Thymidine kinase and resistance to phosphonoacetic acid mapped in the L component. In addition, we have taken advantage of the rapid inhibition of host protein synthesis characteristic of HSV-2 infections and syncytial plaque morphology to also map the template(s) responsible for these functions in the L component. The implications of the template arrangement in HSV DNA are discussed.     

The differential requirement for cyclin-dependent kinase activities distinguishes two functions of herpes simplex virus type 1 ICP0

Herpes simplex virus type 1 (HSV-1) ICP0 directs the degradation of cellular proteins associated with nuclear structures called ND10, a function thought to be closely associated with its broad transactivating activity. Roscovitine (Rosco), an inhibitor of cyclin-dependent kinases (cdks), inhibits the replication of HSV-1, HSV-2, human cytomegalovirus, varicella-zoster virus, and human immunodeficiency virus type 1 by inhibiting specific steps or activities of viral regulatory proteins, indicating the broad and pleiotropic effects that cdks have on the replication of these viruses. We previously demonstrated that Rosco inhibits the transactivating activity of ICP0. In the present study, we asked whether Rosco also affects the ability of ICP0 to direct the degradation of ND10-associated proteins. For this purpose, WI-38 cells treated with cycloheximide (CHX) were mock infected or infected with wild-type HSV-1 or an ICP0(-) mutant (7134). After release from the CHX block, the infections were allowed to proceed for 2 h in the presence or absence of Rosco at a concentration known to inhibit ICP0's transactivating activity. The cells were then examined for the presence of ICP0 and selected ND10-associated antigens (promyelocytic leukemia antigen [PML], sp100, hDaxx, and NDP55) by immunofluorescence. Staining for the ND10-associated antigens was detected in 90% of 7134- and mock-infected cells stained positive for all ND10-associated antigens in the presence or absence of Rosco. Similar results were obtained with a non-ND10-associated antigen, DNA-PK(cs), a known target of ICP0-directed degradation. The results of the PML and DNA-PK(cs) immunofluorescence studies correlated with a decrease in the levels of these proteins as determined by Western blotting. Thus, the differential requirement for Rosco-sensitive cdk activities distinguishes ICP0's ability to direct the dispersal or degradation of cellular proteins from its transactivating activity.     

Induction of cellular stress overcomes the requirement of herpes simplex virus type 1 for immediate-early protein ICP0 and reactivates expression from quiescent viral genomes

Herpes simplex virus type 1 (HSV-1) mutants impaired in the activities of the structural protein VP16 and the immediate-early (IE) proteins ICP0 and ICP4 establish a quiescent infection in human fibroblasts, with most cells retaining an inactive, repressed viral genome for sustained periods in culture. To date, the quiescent state has been considered stable, since it has been reversed only by provision of herpesviral proteins, such as ICP0, not by alteration of the cell physiological state. We report that the interaction of HSV-1 with human fibroblasts can be altered significantly by transient treatment of cultures with sodium arsenite, an inducer of heat shock and oxidative stress, or gramicidin D, a toxin that selectively permeabilizes cell membranes, prior to infection. These regimens stimulated gene expression from IE-deficient HSV-1 mutants in a promoter sequence-independent manner and also overcame the replication defect of ICP0-null mutants. Reactivation of gene expression from quiescent HSV-1 genomes and the resumption of virus replication were observed following addition of arsenite or gramicidin D to cultures. Both agents induced reorganization of nuclear domain 10 structures, the sites of quiescent genomes, but appeared to do so through different mechanisms. The results demonstrate that the physiological state of the cell is important in determining the outcome of infection with IE-deficient HSV-1 and show novel methods for reactivating quiescent HSV-1 in fibroblasts with a high efficiency.     

In situ detection of alkaline nuclease activity in cells infected with herpes simplex virus type 1 (HSV-1)

An in situ assay for detection of alkaline nuclease activities has been adapted to the herpes simplex virus type 1 (HSV-1) system. Six major nuclease activities which migrate with molecular weights of 90,000, 85,000, 80,000, 76,000, 71,000 and 65,000, and six minor species of molecular weights 87,000, 81,000, 57,000, 18,500, 17,500 and 16,500 were detected in lysates of HSV-1 infected cells following SDS-polyacrylamide gel electrophoresis and enzyme activation in situ. An ELISA assay and an immunoprecipitation study indicated that the six major HSV-induced nuclease species are virus-specific. Moreover, a reconstruction experiment in which 14C-labelled protein markers were incubated with mock- and HSV-infected cell lysates demonstrates that the nuclease fractions detected in situ were not due to endogenous proteolytic activity. The 80,000, 76,000, 71,000 and 65,000 species were first detected at 4 h post-infection, whereas all others were detectable by 6 h post-infection. The activities of the major cellular nucleases of molecular weights 50,000. 48,000 and 45,000 decreased as a function of time post-infection. The level of expression of each of the virus-induced species was dependent upon the multiplicity of infection, and all virus-induced activities exhibited biochemical properties characteristic of purified HSV-1 alkaline nuclease, including activation and inhibition by specifications. The 76,000 HSV-induced alkaline nuclease species was also demonstrated to possess endonucleolytic activity.     

The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation.

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.     

Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection.

The promoter-regulatory regions from the herpes simplex virus type 1 (HSV-1) gene for the immediate-early, 175,000-molecular-weight (175K) protein and the HSV-2 delayed-early gene for a 38K protein were linked to the readily assayable bacterial gene for the enzyme chloramphenicol acetyltransferase (CAT). Unexpectedly, in measurements of the constitutive expression of the recombinant genes 40 to 50 h after transfection of Vero cells, enzyme levels expressed from the delayed-early 38K-promoter-CAT construct (p38KCAT) were at least as high as those from the immediate-early 175K-promoter-CAT construct (p175KCAT). In contrast, enzyme levels expressed after transfection of a similar recombinant gene containing a second delayed-early promoter region, that of the HSV-1 thymidine kinase gene, were ca. 20-fold lower. The amounts of enzyme expressed from both p38KCAT and p175KCAT could be increased by up to 20- to 40-fold after infection of the transfected cells with HSV. In comparison, virus infection had no significant effect on enzyme levels expressed from recombinant CAT genes containing the simian virus 40 early promoter region, with or without the 72-base-pair enhancer element. Experiments with the temperature-sensitive mutants HSV-1 tsB7 and HSV-1 tsK indicate that induction of expression from p175KCAT was mediated by components of the infecting virus particle, whereas that from p38KCAT required de novo expression of virus immediate-early proteins. In addition, we show that functions required to induce expression from both p175KCAT and p38KCAT could also be provided by infection with pseudorabies virus and cytomegalovirus.     

Mechanism of immunologic resistance to herpes simplex virus 1 (HSV-1) infection.

Susceptibility of adult mice to i.p. infection with HSV-1 was greatly increased by administration of a single dose of cyclophosphamide. Mortality of cyclophosphamide-treated virus-infected mice was associated with increased virus replication and pathologic changes in brain and liver. The development of a fatal infection in immunosuppressed mice could be curtailed after transfer of specifically immune spleen cells. Passively transferred antibody had no such effect. Protective activity of spleen cells was significantly reduced after pretreatment with anti-theta serum. Significant protection was also achieved when normal spleen cells plus immune serum were administered simultaneously. Our results indicate that protection against this virus infection is predominantly T cell dependent, and suggests that antibody-dependent cell-mediated protection may also be operative in vivo.