Selective assay for herpes simplex viruses expressing thymidine kinase.

A technique for selecting herpes simplex viruses expressing the viral thymidine kinase (TK+) from a population of predominantly TK- viruses was developed. This was accomplished by infecting TK- cells and incubating the cultures under a liquid overlay medium containing methotrexate. Since the TK- cells survive in this medium for only a limited period of time, it was necessary to add fresh uninfected TK- cells 48 h after infection. The technique allowed the detection and quantitation of the TK+ virus fraction in mixtures of TK+ and TK- viruses where the TK+ fraction was present in frequencies as low as 10(-5). It was also used to estimate reversion frequencies and to obtain and analyze TK+ revertants from TK- mutant strains of herpes simplex virus type 1.     

The study of DNA-repair defects using [125I]iododeoxycytidine incorporation as an assay for the growth of herpes simplex virus

[125I]Iododeoxycytidine incorporation was used to measure herpes virus (HSV-1) DNA synthesis following specific DNA damage. Xeroderma pigmentosum fibroblasts were less able to replicate UV-irradiated viral DNA than were normal fibroblasts, indicating the necessity for excision repair for the survival of UV-irradiated virus. Because of its rapidity and ease of quantitation, this assay had advantages over standard viral mediated assays of DNA excision repair. It was possible to monitor viral replication as a function of the cellular cell cycle. Other genetic defects which have been proposed to reflect deficiencies in DNA-repair capacity were not detected by this assay. DNA-repair inhibitors, caffeine and 3-aminobenzamide, also did not show synergistic lethal effects on the replication of damaged viral DNA.     

Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv(r) clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv(r) mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.     

Growth, plaque assay and immunofluorescent studies on Tataguine virus in cell culture.

The growth characteristics of Tataguine virus were studied in Cercopithecus monkey kidney (Vero); rhesus monkey kidney (LLC-MK2), baby hamster kidney (BHK-21); porcine kidney (PK-15), mouse fibroblasts (L-929) and Aedes albopictus cell monolayers. The virus replicated without producing any cytopathology in Vero, BHK-21 and Aedes albopictus: but not in the other three cell culture systems. Two or three subsequent serial blind passages in those cultures supporting the growth of the virus did not produce any appreciable increase in virus titre. Immunofluorescent staining of inoculated Vero cells demonstrated the presence of Tataguine virus antigen in the cytoplasm of infected cells. Plaques 1--1.5 mm in diameter were produced only in Vero cell culture. In neutralization tests performed on Tataguine virus, immune mouse and hamster sera, higher antibody titres were obtained by plaque reduction than mouse protection tests.     

Expression of an early, nonstructural antigen of herpes simplex virus in cell transformed in vitro by herpes simplex virus.

Hyperimmune rabbit antiserum to an early, nonstructural herpes simplex virus type 2 (HSV-2)-induced polypeptide (VP143) reacted in immunofluorescence tests with a variety of cell lines transformed by HSV-2. Cytoplasmic fluorescence was observed in 10 to 50% of HSV-2-transformed cells, whereas no fluorescence was observed in cells transformed by other oncogenic DNA viruses or by a chemical carcinogen. VP143-specific reactivity could be absorbed from anti-VP143 serum with HSV-2-transformed cells but not with cells transformed by other agents. When HSV-2-transformed cells were synchronized in mitosis and examined at various times postmitosis for VP143-specific fluorescence, the expression of VP143 was shown to be cell cycle dependent.     

Advance in herpes simplex viruses for cancer therapy

Oncolytic virotherapy is an attractive approach that uses live viruses to selectively kill cancer cells. Oncolytic viruses can be genetically engineered to induce cell lyses through virus replication and cytotoxic protein expression. Herpes simplex virus (HSV) has become one of the most widely clinically used oncolytic agent. Various types of HSV have been studied in basic or clinical research. Combining oncolytic virotherapy with chemotherapy or radiotherapy generally produces synergic action with unclear molecular mechanisms. Arming HSV with therapeutic transgenes is a promising strategy and can be used to complement conventional therapies. As an efficient gene delivery system, HSV has been successfully used to deliver various immunomodulatory molecules. Arming HSV with therapeutic genes merits further investigation for potential clinical application.     

Interactions of target-sensitive immunoliposomes with herpes simplex virus. The foundation of a sensitive immunoliposome assay for the virus

Interactions between target-sensitive (TS) immunoliposomes and herpes simplex virus (HSV) were investigated. Target sensitivity of phosphatidylethanolamine (PE) immunoliposomes is a result of the ability of acylated monoclonal anti-HSV glycoprotein D (gD) to stabilize the bilayer phase of PE, whereas by itself, PE does not form stable liposomes (Ho, R. J. Y., Rouse, B. T., and Huang, L. (1986) Biochemistry 25, 5500-5506). Upon binding of these immunoliposomes to HSV antigen-containing gD, destabilization of PE immunoliposomes was observed. By encapsulating either a self-quenching fluorescent dye, calcein, or alkaline phosphatase inside the liposomal compartment, the HSV-induced destabilization of TS immunoliposomes was shown to be target-specific. Neither Sendai, Semliki Forest, nor Sindbis virus could significantly destabilize the TS immunoliposomes. Moreover, HSV-induced liposome destabilization could be inhibited by free anti-gD (the same antibody used in TS immunoliposomes) but not by monoclonal anti-HSV glycoprotein B, indicating that the interaction was antigen-specific. Destabilization could also be induced by binding to truncated gD (tgD), but only when in a multivalent form immobilized on latex beads. Truncated gD is a cloned, 312-amino acid fragment of HSV-gD that lacks the transmembrane segment. Preincubation of soluble tgD with the TS immunoliposomes failed to induce destabilization and, in addition, abolished the tgD-bead-induced destabilization. This finding strongly indicated that multivalent binding is essential for TS immunoliposome destabilization. Using alkaline phosphatase encapsulated in the liposomes, TS immunoliposomes could be used to detect HSV in fluid phase with 50% signal recorded at 5 microliters of 3.2 x 10(3) pfu/ml; at least 10-fold more sensitive than the standard double-antibody sandwich enzyme-linked immunosorbent assay. The interactions described here may be useful in designing a homogeneous and sensitive immunoliposome assay.     

Glycoproteins E and I facilitate neuron-to-neuron spread of herpes simplex virus.

Two herpes simplex virus (HSV) glycoproteins E and I (gE and gI) form a heterooligomer which acts as an Fc receptor and also facilitates cell-to-cell spread of virus in epithelial tissues and between certain cultured cells. By contrast, gE-gI is not required for infection of cells by extracellular virus. HSV glycoproteins gD and gJ are encoded by neighboring genes, and gD is required for both virus entry into cells and cell-to-cell spread, whereas gJ has not been shown to influence these processes. Since HSV infects neurons and apparently spreads across synaptic junctions, it was of interest to determine whether gD, gE, gI and gJ are also important for interneuronal transfer of virus. We tested the roles of these glycoproteins in neuron-to-neuron transmission of HSV type 1 (HSV-1) by injecting mutant viruses unable to express these glycoproteins into the vitreous body of the rat eye. The spread of virus infection was measured in neuron-rich layers of the retina and in the major retinorecipient areas of the brain. Wild-type HSV-1 and a gJ- mutant spread rapidly between synaptically linked retinal neurons and efficiently infected major retinorecipient areas of the brain. gD mutants, derived from complementing cells, infected only a few neurons and did not spread in the retina or brain. Mutants unable to express gE or gI were markedly restricted in their ability to spread within the retina, produced 10-fold-less virus in the retina, and spread inefficiently to the brain. Furthermore, when compared with wild-type HSV-1, gE- and gI- mutants spread inefficiently from cell to cell in cultures of neurons derived from rat trigeminal ganglia. Together, our results suggest that the gE-gI heterooligomer is required for efficient neuron-to-neuron transmission through synaptically linked neuronal pathways.