Herpes simplex virus type 1 HindIII fragment L encodes spliced and complementary mRNA species.

We have used DNA bound to cellulose to isolate and translate in vitro herpes simplex virus type 1 (HSV-1) mRNA's encoded by HindIII fragment L (mapping between 0.592 and 0.647), and 8.450-base-pair (8.45-kb) portion of the long unique region of the viral genome. Readily detectable, late mRNA's 2.7 and 1.9 kb in size encoding 69,000- and 58,000-dalton polypeptides, respectively, were isolated. A very minor late mRNA family composed of two colinear forms, one 2.6 kb and one 2.8 kb, was isolated and found to encode only an 85,000-dalton polypeptide. A major early mRNA, 1.8 kb in size encoding a 64,000-dalton polypeptide, was also isolated. High-resolution mapping of these mRNA's by using S1 nuclease and exonuclease VII digestion of hybrids between them and 5' and 3' end-labeled DNA fragments from the region indicated that the major early mRNA contained no detectable splices, and about half of its 3' end was complementary to the 3' region of the very minor 2.6- to 2.8-kb mRNA's encoded on the opposite strand. These mRNA's also contained no detectable splices. The major late 2.7-kb mRNA was found to be a family made up of members with no detectable splices and members with variable-length (100 to 300 bases) segments spliced out very near (ca. 50 to 100 bases) the 5' end.     

Herpes simplex virus latency

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are ubiquitous human pathogens. They share with other herpesviruses the ability to establish lifelong latent infection of the host. Periodic reactivation from latency is responsible for most of the clinical disease burden of HSV infection. This review focuses on what we have learned from molecular studies in model systems of HSV latency, and the implications these findings have for treating recurrent HSV disease.     

Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.     

Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection.

We showed that the expression of a single protein, glycoprotein D (gD-1), specified by herpes simplex virus type 1 (HSV-1) renders cells resistant to infection by HSV but not to infection by other viruses. Mouse (LMtk-) and human (HEp-2) cell lines containing the gene for gD-1 under control of the human metallothionein promoter II expressed various levels of gD-1 constitutively and could be induced to express higher levels with heavy metal ions. Radiolabeled viruses bound equally well to gD-1-expressing and control cell lines. Adsorbed viruses were unable to penetrate cells expressing sufficient levels of gD-1, based on lack of any cytopathic effects of the challenge virus and on failure to detect either the induction of viral protein synthesis or the shutoff of host protein synthesis normally mediated by a virion-associated factor. The resistance to HSV infection conferred by gD-1 expression was not absolute and depended on several variables, including the amount of gD-1 expressed, the dosage of the challenge virus, the serotype of the challenge virus, and the properties of the cells themselves. The interference activity of gD-1 is discussed in relation to the role of gD-1 in virion infectivity and its possible role in permitting escape of progeny HSV from infected cells.     

Herpes simplex virus (HSV)

Herpes simplex virus (HSV), the prototype of the herpesvirus family, causes a variety of diseases in human. In this review, I focus on the molecular mechanism of HSV infection including recent advance on this research field.     

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.     

Herpes simplex virus type 1-induced hydrocephalus in mice.

Adult ICR/Slc or BALB/c mice developed hydrocephalus when attenuated herpes simplex virus type 1 (HSV-1) (strain Ska) was injected intracerebrally 2 to 4 weeks earlier and then after mice were challenged with the same virus or virulent HSV-1. Initial inoculation of the Ska strain elicited acute meningitis and ependymitis with transient mild hydrocephalus. Viral antigen was seen in the meninges and subependymal areas, and the virus was titrated during the acute phase of infection. After the second virus inoculation, more prominent inflammation was evoked in the same area, and the animals developed hydrocephalus, although viral antigen and infectious virus were hardly detected. When the mice were immunosuppressed with cyclophosphamide, they ceased to develop hydrocephalus. BALB/c nude mice did not show the same pathology, even though they were treated in the same way. When irradiated mice, which had been infected with the Ska strain intracerebrally 2 weeks earlier, received syngeneic immune spleen cells, they developed hydrocephalus. The T-cell nature of the effector cells was confirmed by the elimination of the pathology after treatment of the donor cells with anti-Thy-1.2 plus complement. No hydrocephalic mice were observed after treatment of the donor cells with anti-Lyt-1.2 plus complement, which gave further evidence of the T-cell nature of the effector cells as the Lyt-1+.2+ antigen-bearing subsets. Intervals between priming and challenge virus inoculation could be more than 18 months. The presence of purified HSV-1 envelope protein was feasible for the development of the hydrocephalic animals.     

Temperature sensitive mutant of Herpes simplex virus type 1. I. Pathogenicity and immunogenicity]

The aim of the study was to characterize biological features of the sensitive mutant of HSV-1, derived from McIntyre strain by numerous virus passages at lowered replication temperature (28 degrees C). Pathogenicity of obtained ts mutant for inbred mice lines, CFW/Pzh and BALB/cPzh, was determined. Statistically significant decrease in virulence of the mutant for these mouse lines was demonstrated, as compared with the native virus strain, propagated at 37 degrees C. Immunogenic activity of ts mutant of HSV-1 defined by the possibility of mouse protection against infection with high virulent was determined. Mice, which at the time of immunization with ts mutant received Depo-Medrol--an immunosuppressive agent--were also found to be capable of inducing defense mechanisms to infection with the native strain.     

Herpes simplex virus eliminates host mitochondrial DNA

Mitochondria have crucial roles in the life and death of mammalian cells, and help to orchestrate host antiviral defences. Here, we show that the ubiquitous human pathogen herpes simplex virus (HSV) induces rapid and complete degradation of host mitochondrial DNA during productive infection of cultured mammalian cells. The depletion of mitochondrial DNA requires the viral UL12 gene, which encodes a conserved nuclease with orthologues in all herpesviruses. We show that an amino-terminally truncated UL12 isoform-UL12.5-localizes to mitochondria and triggers mitochondrial DNA depletion in the absence of other HSV gene products. By contrast, full-length UL12, a nuclear protein, has little or no effect on mitochondrial DNA levels. Our data document that HSV inflicts massive genetic damage to a crucial host organelle and show a novel mechanism of virus-induced shutoff of host functions, which is likely to contribute to the cell death and tissue damage caused by this widespread human pathogen.     

Herpes simplex virus vectors for gene therapy

Herpes simplex virus (HSV) has a number of advantages as a vector for delivering specific genes to the nervous system. These include its large size, wide host range, and its ability to establish long-lived asymptomatic infections in neuronal cells in which a specific region of the viral genome continues to be expressed. Unfortunately, the large size of this virus and difficulty in manipulating it has led to its use as a vector lagging behind that of other, smaller viruses such as the retroviruses. In addition, the virus's ability to replicate lytically in the brain, under some circumstances, causing encephalitis, has led to fears about its potential safety for ultimate use in humans. This review will discuss a number of new approaches that are aimed at rendering simpler the insertion of foreign genes into the virus and making it as safe as possible. Ultimately, these advances offer real hope for the use of HSV vectors in gene therapy procedures.