Immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type 1

Herpes simplex viruses (HSV) have developed several immunoevasive strategies. Here we demonstrate a novel mechanism by which HSV type 1 may interfere with the immune response through infection of immature dendritic cells (DC) and selective downmodulation of costimulatory molecules. In our study we show productive infection of immature monocyte-derived DC, which closely resemble sessile Langerhans cells, by sequential expression of immediate-early, early, and late viral proteins and of glycoprotein D mRNA, as well as production of infectious virus of moderate titers. Infection was cytopathic, with the progressive loss of 20 to 45% of cells from 24 to 48 h after infection, with no more than 80% of DC found to be infected. These results are in contrast to those of previous findings of nonpermissive or abortive infection of monocytes and mature monocyte-derived DC. Infection of immature DC also led to selective and asynchronous downregulation of CD1a, CD40, CD54 (ICAM-1) (12 h postinfection), CD80 (24 h postinfection), and CD86 (48 h postinfection) but not of CD11c or major histocompatibility complex class I and II molecules when compared to DC exposed to UV-inactivated virus. Thus, we propose that productive infection of epidermal Langerhans cells in vivo may lead to delayed activation of T cells, allowing more time for replication of HSV type 1 in epidermal cells.     

Polyphosphoinositide metabolism in baby-hamster kidney cells infected with herpes simplex virus type 1.

The incorporation of [32P]Pi and [3H]inositol into the inositol lipids of baby-hamster kidney cells was studied in herpes-simplex-virus-type-1(HSV-1)-infected and mock-infected cells. The infection was conducted during incorporation of, as well as after prelabelling with, the precursors. These methods were used in order to study both synthesis de novo of, and steady-state changes in, the phosphoinositides. Both with infection during labelling, and after prelabelling, we found increased [32P]- and [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) and decreased [32P]- and [3H]-phosphatidylinositol 4-monophosphate in infected as compared with mock-infected cells, whereas no effect was observed on phosphatidylinositol. This altered inositol-lipid metabolism was (at least in the case of PIP2) not present until 3-6 h after infection and remained stable, or increased slightly, throughout the infection period. Polyphosphoinositide metabolism constitutes an important step in signal processing in many forms of cellular stimulation, and the results obtained suggest that HSV-1 infection may induce such events in our cell system.     

Electrorotation studies of baby hamster kidney fibroblasts infected with herpes simplex virus type 1

The dielectric properties of baby hamster kidney fibroblast (BHK(C-13)) cells have been measured using electrorotation before and after infection with herpes simplex virus type 1 (HSV-1). The dielectric properties and morphology of the cells were investigated as a function of time after infection. The mean specific capacitance of the uninfected cells was 2.0 microF/cm2, reducing to a value of 1. 5 microF/cm2 at 12 h after infection. This change was interpreted as arising from changes in the cell membrane morphology coupled with alterations in the composition of the cell membrane as infection progressed. The measured changes in the cell capacitance were correlated with alterations in cellular morphology determined from scanning electron microscope (SEM) images. Between 9 and 12 h after infection the internal permittivity of the cell exhibited a rapid change, reducing in value from 75epsilono to 58epsilono, which can be correlated with the generation of large numbers of Golgi-derived membrane vesicles and enveloped viral capsids. The data are discussed in relation to the known life cycle of HSV-1 and indicate that electrorotation can be used to observe dynamic changes in both the dielectric and morphological properties of virus-infected cells. Calculations of the dielectrophoretic spectrum of uninfected and infected cells have been performed, and the results show that cells in the two states could be separated using appropriate frequencies and electrode arrays.     

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

How viral infections affect host cell mitochondrial functions is largely unknown. In this study, uptake of radiolabeled precursors was used to assess how a herpes simplex virus type 1 (HSV 1) infection influences synthesis of macromolecules comprising Vero cell mitochondria. Total macromolecular synthesis in infected cells was determined for comparative purposes. Mitochondrial and total cellular DNA syntheses were approximately halved at 1-2.5 h postinfection (PI). Mitochondrial DNA synthesis in infected cells then rose to 3.5-fold that in control cells at 3-4.5 h PI. Total DNA synthesis in infected cells also rose, but more slowly, reaching threefold that for control cells at 5-6.5 h PI. Mitochondrial and total RNA synthesis in infected cells were both decreased by approximately 40% at 1-3 h PI. Over the next 4 h, total RNA synthesis in infected cells slowly continued to decrease, while that in mitochondria recovered to control levels. Synthesis of mitochondrial proteins in infected cells decreased progressively, dropping to about 60% of control levels by 5-6.5 h PI. With the metabolic inhibitors ethidium bromide and cycloheximide, it was determined that nuclear DNA and mitochondrial DNA and mitochondrial DNA directed synthesis of mitochondrial proteins were each partially inhibited in infected cells. Total cellular protein synthesis was decreased by 30% at 1-2.5 h PI and then recovered to control levels by 5-6.5 h PI. Finally, phospholipid synthesis in mitochondria from infected cells was elevated 2.3-fold at 1-5 h PI, but dropped to 14% below control levels during 4-8 h PI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of a virus coded glycoprotein from herpes simplex virus type 1 infected cells

HEp-2 cells, which were infected with HSV-1, excrete besides other proteins a soluble glycoprotein (Mr 125000–130000) related to the virus protein gC. The excretion of the glycoprotein and the production of extracellular virus particles is reduced to a similar extent when the cells were treated with monensin. Possible consequences of the excretion of soluble viral proteins to a modulation of the immune response are discussed.Abbreviations HSV-1 Herpes simplex virus type 1 - PAGE Polyacrylamide gel electrophoresis - SDS Sodium dodecylsulfate     

Fate of microfilaments in vero cells infected with measles virus and herpes simplex virus type 1.

In herpes simplex virus type 1-infected Vero cells, reorganization of microfilaments was observed approximately 4 h postinfection. Conversion of F (filamentous) actin to G (globular) actin, as assessed by a DNase I inhibition assay, was continuous over the next 12 to 16 h, at which time a level of G actin of about twice that observed in uninfected cells was measured. Fluorescent localization of F actin, using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, demonstrated that microfilament fibers began to diminish at about 16 to 18 h postinfection, roughly corresponding to the time that G actin levels peaked and virus-induced cytopathology was first observable. In measles virus-infected cells, no such disassembly of microfilaments occurred. Rather, there was a modest decrease in G actin levels. Fluorescent localization of F actin showed that measles virus-infected Vero cells maintained a complex microfilament network characterized by fibers which spanned the entire length of the newly formed giant cells. Disruption of microfilaments with cytochalasin B, which inhibits measles virus-specific cytopathology, was not inhibitory to measles virus production at high multiplicities of infection (MOI) but was progressively inhibitory as the MOI was lowered. The carbobenzoxy tripeptide SV-4814, which inhibits the ability of Vero cells to fuse after measles virus infection, like cytochalasin B, inhibited measles virus production at low MOI but not at high MOI. Thus, it appears that agents which affect the ability of Vero cells to fuse after measles virus infection may be inhibitory to virus production and that the actin network is essential to this process.     

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.     

Viral DNA synthesis in cells infected with temperature-sensitive mutants of herpes simplex virus type 1.

Temperature-sensitive mutants of herpes simplex virus type 1 representing eight DNA-negative complementation groups were grouped into the following three categories based on the viral DNA synthesis patterns after shift-up from the permissive to the nonpermissive temperature and after shift-down from the nonpermissive to the permissive temperature in the presence and absence of inhibitors of RNA and protein synthesis. (i) Viral DNA synthesis was inhibited after shift-up in cells infected with tsB, tsH, and tsJ. After shift-down, tsB- and tsH-infected cells synthesized viral DNA in the absence of de novo RNA and protein synthesis whereas tsJ-infected cells synthesized no viral DNA in the absence of protein synthesis. The B, H, and J proteins appear to be continuously required for the synthesis of viral DNA. (ii) Viral DNA synthesis continued after shift-up in cells infected with tsD and tsK whereas no viral DNA was synthesized after shift-down in the absence of RNA and protein synthesis. Mutants tsD and tsK appear to be defective in early regulatory functions. (iii) Cells infected with tsL, tsS, and tsU synthesized viral DNA after shift-up and after shift-down in the absence of RNA and protein synthesis. The functions of the L, S, and U proteins cannot yet be determined.     

Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.     

Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity

Mature dendritic cells (DC) are the most potent antigen-presenting cells within the entire immune system. Interference with the function of these cells therefore constitutes a very powerful mechanism for viruses to escape immune responses. Several members of the Herpesviridae family have provided examples of such escape strategies, including interference with antigen presentation and production of homologous cytokines. In this study we investigated the infection of mature DC with herpes simplex virus type 1 (HSV-1) and the way in which infection alters the phenotype and function of mature DC. Interestingly, the T-cell-stimulatory capacity of these DC was strongly impaired. Furthermore, we demonstrated that HSV-1 leads to the specific degradation of CD83, a cell surface molecule which is specifically upregulated during DC maturation. These data indicate that HSV-1 has developed yet another novel mechanism to escape immune responses.     

Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface.

To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.