Lasar flow cytophotometric immunoperoxidase detection of herpes simplex virus type 2 antigens in infected cultured human cells.

Human cells in culture (HEp-2) were infected with herpes simplex virus type 2 (HSV-2) at multiplicities of infection varying from 0.2 to 10, and fixed 6, 12, 18 and 24 hr after infection. Infection-related antigens were detected by an indirect double antibody (peroxidase conjugated goat anti-rabbit to rabbit anti-herpes simplex virus type 2) immunoenzymatic staining reaction that rendered infection-related antigens visible by light microscopy. A corresponding series of laser flow cytophotometric experiments yielded reproducible large-angle (1-19 degrees) laser-light scattering distributions that depended upon multiplicities of infection and the location of the infection-related antigens in the infected cells.     

Comprehensive quantification of herpes simplex virus latency at the single-cell level.

To date, characterization of latently infected tissue with respect to the number of cells in the tissue harboring the viral genome and the number of viral genomes contained within individual latently infected cells has not been possible. This level of cellular quantification is a critical step in determining (i) viral or host cell factors which function in the establishment and maintenance of latency, (ii) the relationship between latency burden and reactivation, and (iii) the effectiveness of vaccines or antivirals in reducing or preventing the establishment of latent infections. Presented here is a novel approach for the quantitative analysis of nucleic acids within the individual cells comprising complex solid tissues. One unique feature is that the analysis reflects the nucleic acids within the individual cells as they were in the context of the intact tissue-hence the name CXA, for contextual analysis. Trigeminal ganglia latently infected with herpes simplex virus (HSV) were analyzed by CXA of viral DNA. Both the type and the number of cells harboring the viral genome as well as the number of viral genomes within the individual latently infected cells were determined. Here it is demonstrated that (i) the long-term repository of HSV-1 DNA in the ganglion is the neuron, (ii) the viral-genome copy number within individual latently infected neurons is variable, ranging over 3 orders of magnitude from <10 to >1,000, (iii) there is a direct correlation between increasing viral input titer and the number of neurons in which latency is established in the ganglion, (iv) increasing viral input titer results in more neurons with greater numbers of viral-genome copies, (v) treatment with acyclovir (ACV) during acute infection reduces the number of latently infected ganglionic neurons 20-fold, and (vi) ACV treatment results in uniformly low (<10)-copy-number latency. This report represents the first comprehensive quantification of HSV latency at the level of single cells. Beyond viral latency, CXA has the potential to advance many studies in which rare cellular events occur in the background of a complex solid tissue mass, including microbial pathogenesis, tumorigenesis, and analysis of gene transfer.     

Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.     

The physical state of herpes simplex virus DNA in infected human cells.

Treatment of herpes simplex virus type 1 (HSV-1)-infected human embryo lung (HEL) cells with phosphonoacetic acid (PAA) resulted in complete inhibition of HSV DNA replication. DNA was extracted from PAA-treated HEL cells infected with HSV-1 and centrifuged in a neutral CsCl density gradient. The HSV DNA sequences in the nuclei of PAA treated cells at 24 hr post infection banded at the same density as free HSV DNA (1.725 g/cm3), but a significant amount of viral DNA sequences were detected in the regions of cell DNA (1.700 g/cm3) as well as in the intermediate fractions as determined by hybridization with 3H HSV complementary RNA. The viral DNA sequences of lower deisntiy did not change in density by recentrifugation in a CsCl density gradient, but did change to the density of free viral DNA after treatment with EcoR1 restriction endonuclease. When the DNA from the nuclei of PAA treated cells was analyzed in an alkaline glycerol gradient, more than 95% of the viral DNA sequences were found in the free viral DNA fractions. Since the viral and cellular hybrid DNA represented approximately 33% of the total viral DNA sequences, it is concluded that some of the HSV DNA sequences in PAA treated, infected cells are associated with cell DNA by alkali-labile bonds.     

Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).     

Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro.

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Treatment with acyclovir for the first 7 days after viral inoculation prevented lytic infections in 100% of the cultures and resulted in viral latency in 100% of the cultures; reactivation occurred as the result of nerve growth factor (NGF) deprivation. Treatment of the cultures with several different inhibitors of viral DNA polymerase (acyclovir, aphidicolin, and phosphonoacetic acid) for 7 days after viral inoculation did not prevent the establishment of latency, which suggests that viral DNA replication was not required. During the latent phase of the infection, viral antigens were not detected with HSV-specific immunohistochemistry. However, 24 h after NGF deprivation, viral antigens were detected in essentially all of the neurons, indicating that the majority of neurons harbored latent HSV. The establishment of latency was not strain or type specific since latency was established with HSV type 2 and four strains of HSV type 1 and reactivation occurred in response to NGF deprivation.     

Modulation of apoptosis during herpes simplex virus infection in human cells

Human herpes simplex virus (HSV) is cytolytic and has profound impacts on its host cells. Consequences of HSV infection include the induction of apoptosis and the concomitant synthesis of proteins which act to block this process. We review recent evidence showing how this important human pathogen modulates the fundamental cell death process.     

Expression of herpes simplex virus type 1 glycoproteins in interferon-treated human neuroblastoma cells.

Human alpha interferon (IFN) significantly inhibits the replication of herpes simplex virus type 1 in human neuroblastoma cells. This inhibitory effect can be blocked by pretreatment with antiserum to IFN. We observed no significant differences in the expression of major nucleocapsid proteins, including VP5, between IFN-treated and untreated neuroblastoma cells. Electron micrographs demonstrated that there were distinct viral nucleocapsids within IFN-treated neuroblastoma cells. The expression of glycoproteins B and E was significantly reduced in these IFN-treated cells. On the other hand, glycoprotein D, although reduced in quantity, was expressed after IFN treatment. An immunofluorescence assay of the IFN-treated and virus-infected cells detected glycoprotein D in the Golgi complexes and in the nuclear membranes. Our results indicate that human alpha IFN may be useful in the study of gene expression in IFN-treated cells of neuronal origin.     

Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells.

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circle replication of circular duplex DNA molecules. The products of the reaction are longer than monomer unit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance to DpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on the gradient consistent with those of molecules composed mainly of one parental DNA strand and one newly synthesized DNA strand, and (iii) the appearance in the electron microscope of molecules consisting of duplex circles with multiunit linear appendages, a characteristic of a rolling circle mode of DNA replication. The reaction requires ATP and is dependent on herpes simplex virus type 1-encoded DNA polymerase.     

Inhibition of herpes simplex virus type 1 replication in fibroblast cultures by human blood mononuclear cells.

An assay was developed to test the effect of human blood mononuclear cells (MNCs) on herpes simplex virus (HSV) replication. In this assay, human fibroblast monolayers were inoculated with HSV and then cultured with or without blood MNCs. Fewer HSV-infected cells were recovered from human fibroblasts cultured in the presence than in the absence of blood MNCs. This inhibition of viral replication by MNCs was independent of HLA matching between the MNCs and fibroblasts and persisted even when T cells were depleted by antibody and complement. However, depletion of Leu11+ MNCs either by panning or with antibody and complement reduced the ability of the cells to suppress HSV infection, whereas enrichment of Leu11+ cells by fluorescence-activated cell sorting increased the viral suppression. Depletion of OKM1+ MNCs also reduced the viral suppression. After coculturing of MNCs and HSV-infected fibroblasts for 3 days, alpha interferon (IFN) and gamma IFN were detected in the supernatants. Predepletion of Leu11+ MNCs reduced the amount of gamma IFN produced in these cultures. Incubation of the MNCs and HSV-infected fibroblasts with antibody specific for either alpha or gamma IFN resulted in reduced viral suppression. Preincubation of MNCs for 18 h with either interleukin 2 or alpha IFN or for 7 days with antigen increased the suppression of HSV infection. These results suggest that natural killer cells with the Leu11+ phenotype participate in the recognition of HSV-infected cells at a point sufficiently early to interfere with the spread of infection in vitro and may inhibit viral replication by natural killer cell cytotoxicity, by generation of interferon, and by lymphokine-activated killing.