Distinct Segregation between High and Low Viremic HBV Individuals Is Correlated to Changes at Nucleotide Position 1752

<p>Comparison of DNA sequences from nucleotide 1720–1769 with the HBV DNA concentration levels of the participants are illustrated. Sera was collected from 60 participants; DNA was isolated from sera and amplified with two rounds of PCR. Results of the sequences were aligned and compared.</p

Identification of Host Cellular Protein as hnRNP K

<div><p>(A) Specific protein spots were cored out and de-stained, following which the gel plug was digested with trypsin. Sequence query of peptide fragments was carried out by Proteomic Research Services, using LC/MS/MS analysis. Results of the 21 sequenced peptides are illustrated.</p> <p>(B) Results of peptide sequencing of the 56-kDa protein by LC/MS/MS showed high homology scores to hnRNP K in sequence alignments.</p></div

Evidence for the Involvement of a Host Cellular Protein in Enh II Activity and HBV Replication

<div><p>(A) Electrophoretic mobility shift assays were performed using HepG2 nuclear extracts with four different probes. Probe 1, lanes 1–4; probe 2 (1752A), lanes 5–8; probe 3, lanes 9–12; probe 4 (1752G), lanes 13–16. Each set of probes contains increasing concentrations (0.0 μg, 0.05 μg, 0.10 μg, and 0.15 μg) of non-specific competitor DNA [poly-(dI)-poly-(dC)], respectively.</p> <p>(B) 40 μg of nuclear protein extracts obtained from HepG2 cells was allowed to bind onto 5 mg Dynabeads M-280 streptavidin-biotin-oligonucleotides in the presence of 2:1 (w/w) ratio of non-specific competitor DNA poly (dI–dC). 1-D isoelectric focusing was followed by 2-D vertical separation on SDS-PAGE (10%). The estimated molecular weight of the specific protein spots detected by silver staining (arrow) is indicated.</p></div

hnRNP K Is Involved in Modulating Viral Replication

<p>HepG2 cells were co-transfected with full-length replicative HBV clones (indicated by “+”) 1752A, 1752ΔG, 1752ΔT, and 1752ΔC (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020163#s2" target="_blank">Methods</a> and <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020163#sg001" target="_blank">Figure S1</a>) with increasing dosages (50, 250, and 1,250 ng/μl) of hnRNP K variant 2 (v2) or variant 3 (v3) as indicated. pcDNA 3.1 serves as a control. Transfections were performed in duplicate; standard deviations are shown.</p

hnRNP K siRNAs Down-Regulate HBV Viral Replication

<div><p>(A) HepG2 cells were co-transfected with 1752A full-length replicative HBV clone either with or without hnRNP K siRNA (2 μg). Non-silencing (Non-T) and lamin A/C (Lamin) siRNAs were used as controls. hnRNP K expression was measured by quantitative real-time RT-PCR.</p> <p>(B) HBV viral load was quantitated by real-time PCR in cells transfected as described in (A).</p> <p>(C) Lamin A/C expression was measured from real-time RT-PCR. Ratios were normalized to 100% for the non-transfected cells. The results represent two independent samples; standard deviations are shown.</p> <p>Black columns represent either non-transfected cells or cells transfected with non-silencing siRNA. White columns represent cells co-transfected with HBV and lamin A/C siRNA. Grey columns represent cells co-transfected with HBV and hnRNP K siRNAs (A, Dharmacon; B, Qiagen; C, Proligo).</p></div

MS Spectrums of the Three Distinct Genotypes at SNV Position 1,727

<p>The T example is from the cultured viral isolate from patient Sin849, the T/C example is from the uncultured lung tissue sample from patient Sin849, and the C example is from the cultured viral isolate from patient Sin852.</p

The Molecular Relationship among 13 Singapore SARS-CoV Isolates Based on the Genotype Sharing Pattern of the Viral Isolates

<p>The Molecular Relationship among 13 Singapore SARS-CoV Isolates Based on the Genotype Sharing Pattern of the Viral Isolates</p