Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Recombinogenic properties of herpes simplex virus type 1 DNA sequences resident in simian virus 40 minichromosomes.

In a previous work, it was demonstrated that the bacterial transposon Tn5 is capable of undergoing sequence inversion via recombination between its duplicated IS50 elements when replicated by the herpes simplex virus type 1 (HSV-1) origin oris but not by the simian virus 40 (SV40) origin orisv. Further analysis of the latter phenomenon indicated that this lack of recombination was the result of topological constraints imposed by the SV40 minichromosome, such that recombination events could be readily detected in Tn5 derivatives in which the IS50 elements were arranged in a direct rather than inverted orientation. With this information, a second set of experiments were carried out to examine how the highly recombinogenic sequences which mediate the inversion of the long (L) and short (S) components of the HSV-1 genome behave in an SV40 minichromosome. Tandem copies of the L-S junction of the HSV-1 genome were observed to promote deletions in an SV40 shuttle plasmid at a frequency that was considerably greater than that of duplicated bacterial plasmid vector DNA. However, the presence of superinfecting HSV-1 did not enhance the frequency of these recombination events. These results support our previous findings that HSV-1 genome isomerization is mediated by a homologous recombination mechanism which is intimately associated with the act of viral DNA synthesis. Moreover, they demonstrate that the sequences which comprise the L-S junction appear to be inherently recombinogenic and, therefore, do not contain specific signals required for HSV-1 genome isomerization.     

Manipulation of herpes simplex virus type 1 by dielectrophoresis

The frequency-dependent dielectrophoretic behaviour of an enveloped mammalian virus, herpes simplex virus type 1 is described. It is demonstrated that over the range 10 kHz–20 MHz, these viral particles, when suspended in an aqueous medium of conductivity 5 mS m^?1, can be manipulated by both positive and negative dielectrophoresis using microfabricated electrode arrays. The observed transition from positive to negative dielectrophoresis at frequencies around 4.5 MHz is in qualitative agreement with a simple model of the virus as a conducting particle surrounded by an insulating membrane.     

The immune response to ocular herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) is a prevalent microbial pathogen infecting 60% to 90% of the adult world population. The co-evolution of the virus with humans is due, in part, to adaptations that the virus has evolved to aid it in escaping immune surveillance, including the establishment of a latent infection in its human host. A latent infection allows the virus to remain in the host without inducing tissue pathology or eliciting an immune response. During the acute infection or reactivation of latent virus, the immune response is significant, which can ultimately result in corneal blindness or fatal sporadic encephalitis. In fact, HSV-1 is one of the leading causes of infectious corneal blindness in the world as a result of chronic episodes of viral reactivation leading to stromal keratitis and scarring. Significant inroads have been made in identifying key immune mediators that control ocular HSV-1 infection and potentially viral reactivation. Likewise, viral mechanisms associated with immune evasion have also been identified and will be discussed. Lastly, novel therapeutic strategies that are currently under development show promise and will be included in this review. Most investigators have taken full advantage of the murine host as a viable working in vivo model of HSV-1 due to the sensitivity and susceptibility to viral infection, ease of manipulation, and a multitude of developed probes to study changes at the cellular and molecular levels. Therefore, comments in this review will primarily be restricted to those observations pertaining to the mouse model and the assumption (however great) that similar events occur in the human condition.     

A DNA helicase induced by herpes simplex virus type 1.

We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.     

Characterization of multiple nuclear localization signals in herpes simplex virus type 1 thymidine kinase.

We have reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degrève et al. (1998) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or selective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-terminal region of HSV-1 TK, resulted in the loss of the specific nuclear localization of HSV-1 TK. Utilizing information on the crystallographic structure of HSV-1 TK, we have now identified three additional putative nuclear localization signals and evaluated their potential role in the nuclear trafficking of HSV-1 TK by site-directed mutagenesis. We found that the sites containing the amino acids R236-R237 and K317-R318 are absolutely required for specific nuclear targeting of HSV-1 TK. The K317-R318 region, located at the interface between the two monomers in the dimeric HSV-1 TK structure, could act as a nuclear localization signal for monomeric HSV-1 TK. Alternatively, crystallographic data indicate that R318 might be essential for the formation of the TK dimer, and therefore it is required if HSV-1 TK is transported as a dimer.     

Cellular localization of nectin-1 and glycoprotein D during herpes simplex virus infection

During viral entry, herpes simplex virus (HSV) glycoprotein D (gD) interacts with a specific cellular receptor such as nectin-1 (PRR1/HveC/CD111) or the herpesvirus entry mediator A (HVEM/HveA). Nectin-1 is involved in cell-to-cell adhesion. It is located at adherens junctions, where it bridges cells through homophilic or heterophilic interactions with other nectins. Binding of HSV gD prevents nectin-1-mediated cell aggregation. Since HSV gD affects the natural function of nectin-1, we further investigated the effects of gD expression on nectin-1 during HSV infection or in transfected cells. We also studied the importance of the interaction between nectin-1 and the cytoplasmic protein afadin for HSV entry and spread as well as the effects of infection on this interaction. In these investigations, we used a panel of cells expressing nectin-1 or nectin-1-green fluorescent protein fusions as the only mediators of HSV entry. During HSV infection, nectin-1 localization at adherens junction was dramatically altered in a manner dependent on gD expression. Nectin-1 and gD colocalized at cell contact areas between infected and noninfected cells and at the edges of plaques. This specific accumulation of gD at junctions was driven by expression of nectin-1 in trans on the surface of adjacent cells. Reciprocally, nectin-1 was maintained at junctions by the trans expression of gD in the absence of a cellular natural ligand. Our observations indicate that newly synthesized gD substitutes for nectin-1 of infected cells at junctions with noninfected cells. We propose that gD attracts and maintains the receptor at junctions where it can be used for virus spread.     

Study of early events during herpes simplex virus type 1 infection by confocal microscopy

Laser scanning confocal microscopy is a powerful technique that can be applied to study the localisation and behaviour of proteins and nucleic acids in many experimental situations. It is a particularly useful technique for the study of virus infections because of the changes that occur in the distribution and amounts of both viral and cellular proteins as infection develops. These changes reflect key stages and important regulatory events that govern the efficiency of infection. Using herpes simplex virus type 1 infected cells as an experimental model, this article provides guidance for users new to confocal microscopy on basic principles and techniques. The emphasis is on recognising, diagnosing and avoiding potential artifacts, and the workflow of the production of high quality, technically correct images.     

Soluble glycoprotein D blocks herpes simplex virus type 1 infection of rat eyes.

Herpes simplex virus type 1 (HSV-1) ocular infection in rats was blocked by treating the eyes with UV-inactivated virions containing glycoprotein D (gD) prior to ocular challenge. In contrast, rats treated with UV-inactivated virions lacking gD were not protected. A soluble, truncated form of HSV-2 gD (gD-2t) also protected against ocular infection. Treatment with gD-2t not only reduced mortality but also restricted progression of pathology and reduced the amount of viral antigen in the cornea. Host antibody or alpha/beta interferon responses to the gD-2t treatment were not detected. These results are similar to those observed in cell culture (D. C. Johnson, R. L. Burke, and T. Gregory, J. Virol. 64:2569-2576, 1990). The in vivo effect of exogenous gD is consistent with blocking of a cell surface gD receptor or with an inhibitory interaction of gD with virions.     

Molecular cloning of herpes simplex virus type 2 DNA

Restriction enzyme HindIII digestion of the whole genome of herpes simplex virus type 2 strain 186 yielded 10 DNA fragments with molecular weights ranging from approximately 22 X 10(6) to 1.2 X 10(6), which were cloned into the HindIII site of bacterial plasmid pACYC 184. The cloned fragments were identified by hybridization to HSV-2 virus DNA and by double digestion with restriction endonucleases. The recombinant plasmids, even if they carried DNA sequences with molecular weights of more than 10(7), were efficiently replicated in E. coli HB101.     

Biochemical transformation by temperature-sensitive mutants of herpes simplex virus type 1.

Biochemical transformation assays of herpes simplex virus type 1 temperature-sensitive (ts) mutants distinguished three groups of mutants with regard to their thymidine kinase (TK) transforming ability: those incapable of transferring the TK gene at either the permissive or restrictive temperatures (group I); those resembling the wild-type virus, and therefore able to transform at both the permissive and nonpermissive temperatures (group II); and those that failed to transform or exhibited very low transformation frequencies at the permissive temperature but were able to transform at the nonpermissive temperature (group III). Two mutants in group II exhibited greatly enhanced transformation efficiency at the permissive temperature. The ts lesions in the majority of the mutants tested map between 0.30 and 0.60 units on the viral genome. Mutants with TK-positive (TK+), but DNA-negative, phenotypes at the nonpermissive temperature produced no TK+ transformants at the permissive temperature and only unstable transformants at the nonpermissive temperature. This suggests that a function which is required for viral DNA synthesis is also required to obtain stable expression or to transfer the TK+ gene or both when transfer is mediated by the entire viral genome.