The gels were transferred by electrophoresis to nitrocellulose blots and blocked as previously described (58). Immunofluorescence staining.HEC-1-A cells cultured on 10-mm glass coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at RT before being permeabilized using 0.2% Triton X-100 for 15 min. After another centrifugation, the supernatants were immunoprecipitated with an anti-Flag monoclonal antibody and the immunoprecipitates were washed three times with wash buffer (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 50 mM NaF). Virus recovery and titrations.At various time points postinfection, swabs of the corneal surface were obtained. After centrifugation, the gradients were fractionated from top to bottom, and aliquots of single fractions were analyzed by Western blotting with the appropriate anti-nucleoporin antibodies. Confocal microscopy.Vero or HFT cells (6 × 105) in four-well chamber slides (Labtek no. K/B was obtained by cotransfecting D6 cells with KΔ4B infectious DNA, prepared from D6 cells as previously described (18), and the wild-type cosmid scKOS20, which contains gB, using Effectene transfection reagent (QIAGEN).
Total, viable, and nonviable cells were counted three times under the microscope with the help of a hemocytometer following staining by trypan blue. The cells were then incubated with phycoerythrin (PE)-conjugated annexin V and/or 7AAD for 30 min in the dark at room temperature. The PCR product was then cut with BamHI and NheI inserted into the corresponding sites engineered upstream of gB in pK1968. Cells were then probed with primary antibodies specific for lamin B, PKC, and HSV-1 ICP4 for 1 h. Virus progeny was harvested 5 to 7 days following transfection when cytopathic effects were evident. Alternatively, the drugs (or phosphate-buffered saline [PBS] as a control) were preincubated with the cells for 1 h at 37°C and then either washed extensively or not washed prior to inoculation with HSV-2(G). Based on the nucleotide sequence of the HSV-1 gB open reading frame, we synthesized two PCR primers in order to amplify and modify the gB ectodomain coding region for cloning and expression in a recombinant baculovirus.
Rabbit polyclonal antibody to PARP (H-250, sc-7150) was used at a 1:1,000 dilution. MAbs SB1 and SB3 were produced in my laboratory, from mice immunized with UV-inactivated HSV-2 (58). Endoglycosidase H (Endo H) and N-glycosidase F (PNGaseF) were purchased from New England Biolabs (Ipswich, MA). The results provide evidence for the existence of an entry receptor specific for gB. Hence, it is of interest to know whether HVEM is constitutively distributed in raft-like structures on cell surfaces or redistributes there as a consequence of virus binding. Mediated by its cytoplasmic tail, cell surface CD1d can be endocytosed to endosomes and then recycle back to the cell surface. In this study, we screened cellular proteins interacting with ICP22 by tandem affinity purification of transiently expressed ICP22 and mass spectrometry-based proteomics technology.
Among novel strategies, delivery of the immunogens by lentiviral vectors proved particularly effective to vaccinate against infectious diseases and cancer (26, 31, 37, 48). It seems likely, therefore, that the UL34 function is widely conserved among herpesviruses. Structurally, CD1 is similar to MHC class I molecules in that it is a heterodimer formed by a 43- to 49-kDa heavy chain and β2 microglobulin (β2m). The synthesis of viral gene products, both RNA and proteins, takes place in three sequential waves. Antiviral mechanisms include direct effector functions as well as interactions between macrophages and other cell types. Notably, the many hundreds of HSV reactivations from latency that occur in sensory ganglia over the lifetime of the infected individual seldom result in ganglionitis, peripheral neuropathy, or spread to the central nervous system (2–4). Untreated patients have a median survival of 6 – 13 months.
The presence of cytokines such as gamma interferon and the finding that persisting lymphocytes localize to neurons within the ganglia (29) has resulted in the proposal that continual or intermittent HSV-1 antigen expression is occurring during latency (10). HSV-2 shedding episodes are notable for rapid expansion and decay and extreme heterogeneity of duration and viral production. This may not be the complete list of references from this article. Herpes simplex virus (HSV) is a large DNA virus. While both viruses establish latency in sensory ganglia and reactivate to cause recurrent disease, HSV-1 reactivates more efficiently from trigeminal ganglia to cause cold sores or keratitis and HSV-2 reactivates more efficiently from lumbosacral dorsal root ganglia (DRG) to cause genital herpes (8). To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Analysis of clonal lines indicated that resistance to superinfecting virus correlates with the expression of glycoprotein D.
The γ genes are further sub-classified into γ1 genes which are expressed prior to DNA replication and γ2 genes which are absolutely dependent on DNA replication for their expression. Thus, the required HSV glycoproteins, gB, gD, and gH-gL, may be sufficient for entry regardless of entry route taken.