UL30 was incubated with primer–template (1 μM) in the presence of either 100 μM dGTP or 100 μM dGTP and varying concentrations of dCTP, the … Assays (10 μl) contained 1 μM ssDNA template, [α-32P]NTPs, 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 10 mM MgCl2. Then the samples were transferred to nitrocellulose and incubated with primary and HRP-conjugated secondary antibodies. HisΔOBP and ICP 8 were included as described in the figure legends. MRC-5 cells were treated with Hsp90 inhibitor (0.8 µM) and infected with HSV-1 (MOI = 10) for 4 h. Electrophoresis was performed as described above. Fixation was performed with 3.7% formaldehyde-phosphate buffered saline (PBS) for 30 min, followed by permeabilization in 1% Triton X-100-PBS for 10 min after a brief wash in PBS.
The supernatant was transferred to a new tube, and the mitochondrion-containing heavy membrane fraction was isolated by centrifugation at 12,000 × g for 10 min at 4°C. The efficiency of the energy transfer depends on the distance R between the donor and acceptor chromophores as (R0/R)6 (Förster, 1948), where R0 is the critical distance between the donor and the acceptor wherein the probability of the nonradiative energy transfer from the donor to the acceptor is equal to the probability of spontaneous light emission by the excited donor in the absence of the acceptor. Bromphenol blue gel loading buffer type I (9) was added, and samples were subjected to non-denaturing electrophoresis at 4 °C on 6% polyacrylamide gels (Hoeffer/ Amersham Pharmacia Biotech) in 50 mm Tris (pH 8.3), 45 mm boric acid, and 50 mm EDTA. Quantitative plaque autoradiography of viruses. In order to highlight variations between virions and purified capsids, a difference map (Fig. The structures of these viruses were analyzed by restriction enzyme digestion to confirm that the Uc-DR1-Ub fragment had been inserted by homologous recombination into the BamHI P fragment in opposite orientations. Among African American women, the proportion positive for either HSV–1 or HSV–2 was 97.0% in those with the IFNL4-TT/TT genotype and 97.4% in the IFNL4-ΔG allele carriers (P = 0.75).
To isolate well DNA from HCMV-infected cells, confluent monolayers of 106 HFF cells were infected with HCMV at a multiplicity of infection of >5 PFU/cell and harvested 72 h postinfection. Animals were infected intravaginally with 5 × 105 PFU of HSV-2 strain MS (>1,000 LD50) and scored for acute disease on a scale of 0 to 4, where 0 reflects no disease, 1 redness, 2 a single lesion, 3 coalesced lesions, and 4 ulcerated lesions (23). Presumably, the shorter products were Okazaki fragments, and the long products were leading strand DNA. Sequencing analysis revealed that each of these mutants contained different mutations. 2, only the 93-kDa complex was formed, similar to that observed with equimolar amounts of UL9 protein and Box I. The results are shown as percent input values and normalized percent input values. The position of the AT-rich spacer sequence is also shown.
Reactions were quenched as described above and resolved by electrophoresis through 1% agarose-Tris acetate EDTA, pH 8.3, gels at 1.5 V/cm for 12 h. Southern blot analysis was performed on DNA extracted from both KOS- and K26GFP-infected cells to confirm the genotypes of the recombinant viruses (Fig. DNA helicase gel assays.The synthetic oligonucleotide GO135 (5′-GCAGCAAGCGGTCCACGCTGGTTTG-3′), which is complementary to nucleotides 5902 to 5926 of M13mp18(+) DNA, was annealed to M13 DNA and 3′ end labeled with [α-32P]dCTP and Klenow enzyme. θ and Lf were calculated from the titration curves in Fig. After addition of the oligonucleotides, the samples were heated to 90°C for 5 min and allowed to cool slowly to room temperature to anneal the oligonucleotide. Arrows identify HSV-infected cells. Consensus regions were generated with the PRETTY program, and the HSV-1 UL52 zinc finger region was aligned with those of 10 other alphaherpesviruses.
R. Figure Figure11 shows that the highest efficiency (kcat/KmDNA) for the exonuclease occurred on the substrate containing two mismatches. Most disseminated herpes zoster in HIV-seropositive patients have occurred in those who have had prior opportunistic infections associated with AIDS6. These observations can now be integrated into the model for initiation of Herpes simplex virus, type I DNA replication alluded to above. Such sequences have not been observed in known miRNAs. 1B), which is thought to unwind during the initiation of DNA replication. For example, 13.2% of Sardinian (Italian) children between the ages of 6 and 14 were seropositive for KSHV (Serraino et al., 2000), yet Classic KS is associated with advanced age, with the highest risk for those at least 50 years old (Santarelli et al., 2001).
HSV-1 virion is composed of a capsid containing the genome surrounded by an envelope, both nucleocapsid and envelope being separated by the tegument. Genetic studies have implicated a role for ICP8 in gene regulation in HSV-infected cells (18, 20-22, 57), and evidence that ICP8 promotes homologous pairing and strand transfer, suggesting a role in recombination, has been presented (2, 4, 13,14).