Refinement of the procapsid isolation technique. Vero cells infected with wild-type HSV-1(F) (A to C) and YK536 (MEF-UL47) (A), YK539 (MEF-UL31) (B), or YK538 (MEF-UL34) (C) at an MOI of 5 for 18 h were harvested, immunoprecipitated with anti-Myc antibody, and analyzed by immunoblotting with the indicated antibodies. However, when reconstituted SCID mice were repeatedly infected at days 0, 2, and 4 with either ICP4−mutant d120, which expressed little if any UL6, or an ICP8− mutant (which expressed UL6 at levels about one-third that of the wild-type virus) (Fig.3), neither infection resulted in the expression of HSK (Fig. 4b, lanes 1 and 2) and in the recruitment of a population of Nup358 into MAb414-precipitable complexes (Fig. Red arrowheads indicate viral nuclear replication sites, white arrowheads indicate the nuclear membrane, and the white arrow indicates the plasma membrane. This virus, 5/B, exhibited resistance to 5652, similar to that of 5652r-1 (Fig. Effects of Sam B on HSV-1 replication and Vero cell viability and growth.
2C and D). 2A and B, lane 6) while permitting the low-level expression of gB from its native promoter in WT RE and the rescue virus (Fig. In contrast, in HSV-1-infected cells at 16 h postinfection, clear recruitment of PKCα to the nuclear rim and colocalization with lamin B staining was observed. However, Myb34.5 and other strains with intact γ34.5 (wild-type F and hrR3) prevented the infected-cell response (shutoff of protein synthesis), thus leading to viral protein production. MβCD inhibited HSV entry into A10 and C10 cells in a dose-dependent fashion (Fig. These results were reminiscent of those presented in Fig. The latter explanation is the more likely, and the results also suggest that conformation B is H1819+/SB1low/H420−, as discussed later.
2B, upper panel). In contrast, MAb DL16 reacted selectively with multimeric gB(730t) under native conditions of electrophoresis. Cells were then either left on ice or shifted to 37°C for various times before being washed with cold PBS. Alexa 488-, 568-, and 647-conjugated goat anti-mouse secondary antibodies were used at a concentration of 2 μg/ml. In addition, YK421 (ΔICP22) replicated in HEL cells much less efficiently than wild-type HSV-1F or YK422 (ΔICP22-repair) (), whereas growth of YK421 (ΔICP22) in Vero cells was comparable to that of wild-type HSV-1F and YK422 (ΔICP22-repair) (). Detection was carried out using an HRP substrate kit (Bio-Rad, Milan, Italy). Cells were postfixed in 1% osmium tetroxide, washed in cacodylate buffer, dehydrated, embedded in Spurr’s resin, and cut into 95-nm sections.
Immunoblotting for US3 expression was performed with rabbit polyclonal anti-US3 primary antibody, followed by horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (Jackson Immunoresearch) and developed by ECL (Pierce) chemiluminescence. To isolate nuclear proteins, the cell monolayer was washed twice with ice-cold PBS, scraped off the plate, and spun down (2,000 × g for 1 min). Viral protein expression was assessed by immunohistochemistry using a rabbit polyclonal antibody that recognizes multiple HSV-1-infected cell proteins; LAT transcripts were detected by RNA in situ hybridization. It has been tested in pancreatic adenocarcinoma, mesothelioma, rhabdomyosarcoma, osteosarcoma, bladder, lung, gastric, and colorectal cancer, and in metastases to the liver and peritoneum [20,22,23,25,27,35,53,54] (). Moreover, the results also suggested that specificity for virus is not required for the persistence of T-cell infiltration into the latent phase of infection, although virus recognition may result in elevated T-cell numbers at this time. HEp-2 cells were either mock-infected (A) or infected with HSV-1 at a MOI of 3 PFU/cell, fixed at 3 hours (B), 6 hours (C), 9 hours (D), 12 (E) and 15 hours (F) post infection, and then analysed for NuMA localisation. To prevent synthesis of progeny virus, 0.5 mM cycloheximide (Sigma-Aldrich) was added.
HSV-1 envelope proteins gE, gG, gI, gJ, gM, and Us9 are not viral determinants of the pH-dependent entry pathway. Sections were cut with either a Sorvall MT-2 or MT-5000 ultramicrotome and collected on nickel grids. The positions of insertions are indicated by the inverted triangles. Citrate buffer was then added to inactivate virus that had not bypassed the plasma membrane. 1.5 coverglass that has the optimal thickness for z-axis resolution during confocal microscopy. The glass coverslip with the dry cells on it was laid on an adhesive film on an SEM sample holder. In reactivation studies, recurrent ocular virus shedding was detected by culture of tear film swab material on Vero cells, which were then monitored for virus-induced cytopathic effects 48 and 96 h later.
identified a mutant mouse strain, called the triple deficient or “3d” mouse, with altered function of UNC93B1 (27). Two independent studies have demonstrated that tetherin expression inhibits the release of Kaposi’s sarcoma-associated herpesvirus (KSHV; a gammaherpesvirus) (12, 13). Peggy Marconi, University of Ferrara, Italy). Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB.