Autocleavage of the UL26 protease (16) generates the capsid proteins VP24 (containing the N-terminal protease domain) and VP21 (containing the C-terminal oligomerization domain), and cleavage of the UL26.5 protein generates the capsid protein VP22a (also containing the oligomerization domain) (13, 46, 67). After 20 min of incubation, the cells were rinsed 3 times, and the plates were read using the Hitachi F7000 spectrofluorometer (Tokyo, Japan) (excitation, 488 nm, and emission, 525 nm). Rabbit polyclonal antibody to UL34, UL31, ICP22, Us3 and UL47, mouse polyclonal antibody to UL31, and chicken polyclonal antibody to UL34 (a generous gift from R. Kroeplin GhBH, Schluechtern, Germany) as described elsewhere (14). Cells infected by HSV-1 were fixed at different times after infection and probed with MAb414 (Fig. Cells infected with KOS or with a virus encoding an inactive protease (KUL26H61E) were cultured with or without NFV and analyzed by SDS-PAGE and immunoblotting. To confirm that incubation at 4°C allowed only viral attachment and not entry, cells to which virus had been preattached at 4°C were treated with acidic glycine (0.1 M glycine, 0.14 M NaCl, pH 3) to inactivate virus.
For DNA extraction, the cells were lysed with 0.2 M Tris-HCl (pH 8.5) containing 100 mM EDTA, 100 mM NaCl, 0.5% NP-40, 1% sodium dodecyl sulfate (SDS), and 100 μg of proteinase K/ml. The membranes were stripped between antibodies. Viral DNAs from both viruses were cut with NcoI, AvrII, and NcoI-AvrII double digests and probed with 5′ 32P-radiolabeled oligonucleotides derived from the gB coding sequence or the UL28 coding sequence (data not shown). During this incubation, the lysate was aspirated several times through a 25-gauge needle. Host protein synthesis shutoff studies were performed by infecting cells with viral strains for 16 h. The chip surfaces were regenerated with 100-s injections of 0.05% sodium dodecyl sulfate. Cells were then incubated for 30 min at room temperature with serial dilutions of methyl-β-cyclodextrin (MβCD) or nystatin (both reagents from Sigma) in cell culture medium containing 30 mM HEPES.
Briefly, cells were harvest by trypsin treatment and were resuspended in DMEM supplemented with 10% fetal bovine calf serum, 100 U of penicillin/ml, 1% streptomycin, and 1% l-glutamine. All fluorescent conjugates were obtained from Molecular Probes. Western blots were developed with HRP-conjugated anti-mouse IgG or IgM Abs (Dianova) and ECL (GE Healthcare, Munich, Germany). MAbs DL16 and DL21 were selected among a panel of antibodies generated against cell extracts infected by HSV type 1 (HSV-1) and HSV-2. B78-H1 cells (3E5) expressing a green fluorescent protein (GFP)-tagged form of HVEM (enhanced green fluorescent protein [EGFP] at the C terminus of HVEM) were cultured similarly. In addition, HIV-1 also uses Vpu protein, to relocalize CD1d from the late endosome/lysosome to the early endosomal compartment (48). Vero, rabbit skin, HEL, 293T, and Plat-GP cells and the HSV-1F wild-type strain were described previously (31–33).
MATERIALS AND METHODSCells. Here we report that this deletion virus has defects in plaque formation and single-step growth in cell culture. On the other hand, the activation of NKT cells, mostly by the prototypical NKT cell ligand, α-galactosylceramide (α-GalCer), greatly reduces the replication of mouse cytomegalovirus (69), hepatitis B virus (35), influenza virus (29), respiratory syncytial virus (34), lymphocytic choriomeningitis virus (LCMV), and EMCV (71). The sheer volume of viral RNA transcripts accumulating in infected cells enables viral gene expression. In particular, Sin et al. Our aims in this study were to define the course of events that follows HSV-1 inoculation of DRG xenografts, identify ganglionic cell types permissive for HSV-1 replication, and determine whether HSV-1 achieves latency, based on LAT expression. Prognostic factors include: node-positive primary tumor, disease-free interval < 12 months between colon resection and appearance of metastases, tumor > 5 cm, < 1 tumor, and carcinoembryonic antigen (CEA) level > 200 ng/dl.
Stocks were stored at −70°C until required. The University of Washington Institutional Review Board approved the studies. The nuclear structure temporally and spatially coordinates these processes, as highly condensed chromatin that is organised into discrete loops is attached to a nonchromatin scaffold that has been referred to as the nuclear matrix or nucleoskeleton (Gueth-Hallonet et al., 1998). HSV-2 infection of autonomic neurons can give rise to transient bladder paralysis in humans, and latently infected murine parasympathetic neurons express the latency-associated transcript (12). Early in infection many viruses use microtubules (MT) for efficient nuclear targeting, either for cytosolic transport of naked viral particles or for transport inside vesicles (16), e.g., herpes simplex virus type 1 (HSV-1) (77), human cytomegalovirus (58), human immunodeficiency virus (48), adenovirus (42, 80), parvoviruses (71, 79), simian virus 40 (61), influenza virus (41), or hepatitis B virus (29). Moreover, unlike the viral DNA in BHKtk- cells which was amplified, that in BJ cells decreased in copy number. Using gB as our model antigen, in this work we show that during reactivation, gB expression occurs much sooner than gC expression, indicating that it expressed fairly early even during reactivation.