A fourth capsid shell protein, VP26, forms six-membered rings that decorate the external surface of capsid hexons but not pentons (7, 13, 73). To verify and extend the interaction data obtained with mass spectrometry-based proteomics screening, we performed two series of experiments. Viable cells were blocked with heat-inactivated fetal bovine serum and washed with flow cytometry buffer (1× PBS with 1% bovine serum albumin and 0.05% sodium azide). Although the precise identity of all of the proteins detected by this antibody are not known, many have been shown to be nucleoporins (6, 14, 28, 44). Samples from fractions 5, 8, and 10 of each gradient were subjected to SDS-PAGE. For 5652, the reduction of plaques was dose dependent, and the dose that reduced plaque formation by 50% (ED50) was 1 to 2 μM (Fig. The 5 × 106 Vero cells were infected with HSV-1 at an MOI of 3 or were not infected in the presence or absence of Sam B (25 μM) for 16 h.
1D). This plasmid was linearized and cotransfected with infectious HSV-1 RE DNA, and recombinant viruses were picked and purified based on the gain of EGFP fluorescence when visualized by blue UV light. Digital images were acquired by confocal microscopy, and representative examples are shown in Fig. MGH1 (26) was generated by recombining a lacZ cDNA into the ICP6 locus of the γ34.5 deletion mutant R3616 (11). A cocktail of antibiotics was added, and the supernatants from the CVL fluid samples were aliquoted and stored at −80°C. Cells were stained on ice for 1 h by the addition of 5 μg of CK41 per ml directly labeled with phycoerythrin to detect nectin-1. Consistent with the reduced nuclear translocation of p65 observed in IKKb−/− cells, IκBα was reduced in these cells only 1.3- and 1.1-fold following infection or TNF-α treatment.
The method used for quantitation of dual antibody binding could not be used for quantitating the labeling of gB in acidic structures. Fractions were collected from the gradient at the top of the tube. They were then incubated for 1 h at 4°C with serial dilutions of soluble glycoproteins diluted in DMEM containing 5% FCS and 30 mM HEPES (DFH). The ectodomain comprising the first 730 amino acids of gB was expressed by recombinant baculovirus-infected insect cells. The mouse monoclonal antibodies (MAbs) against human CD1d, CD1d51, and D5 were from Steven Porcelli (Albert Einstein College of Medicine, Bronx, NY) and Steven Balk (Harvard Medical School, Boston, MA), respectively. 293T cells were transfected with pcDNA-MEF-ICP22 using polyethylenimine as described previously (39), harvested at 48 h posttransfection, and lysed in 0.1% NP-40 buffer (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 50 mM NaF, 0.1% NP-40) containing a protease inhibitor cocktail (Nacalai Tesque). Titers are expressed as PFU/ml.
(A) Schematic diagram of the HSV-1 genome in prototype arrangement, showing the unique sequences (lines) flanked by inverted repeats (boxes). gB-deficient virus and adenovirus expressing gB protein were generous gifts from Konstantin Kousoulas (Louisiana State University, New Orleans, LA). Inflammatory macrophages were induced by injection of 2 ml of 10% thioglycolate (TG) into the peritoneal cavities of the mice. Recombinant BAC8411 DNA was cotransfected with pRB4867 into Vero cells (24). Much like surgical resection, ablative techniques are limited by size, location, and number of tumors in the liver. Identifying CD8+ T cells in the dorsal root ganglia (DRGs).Mice were sacrificed and perfused with phosphate-buffered saline, and the ganglia innervating the site of cutaneous inoculation were removed with the aid of a dissecting microscope. HSV-2 infection reactivations vary substantially in and among individuals according to duration and peak HSV DNA copy number, 2 measures that strongly correlate.
The motility of the NuMA band was slower than that of mitotic cell extracts (not shown). By day 3, we had not yet observed the previously described pattern of increased relative quantities of HSV-2 in sacral and HSV-1 in lumbar spinal cord, implying that this phenotype may develop later in infection. However, in epithelial cells, depolymerization of the MT network inhibits HSV-1 infection significantly but not completely (45, 77). Wild-type HSV-1 strains KOS and F had reduced plaquing efficiency on B78 receptor cells (Fig. In the absence of a fully effective HSV vaccine (1), topical microbicides represent an important potential strategy for preventing the sexual and mother-to-child spread of HSV. This may not be the complete list of references from this article. For some other HSV glycoproteins, such as gC, gD, and gH, functional domains have been mapped by linker-insertion mutagenesis, in which short oligonucleotides are ligated into restriction sites within the gene (7, 12, 15, 35).
Although the entry of many viruses is cholesterol dependent (5–8), the molecular features of cholesterol that are important for viral entry remain understudied. If one of the intracellular conformations is the fusion-active form, another factor required for fusion is presumably absent from wherever that conformation is present in infected cells so that inappropriate fusion is avoided.