HCC risk mutations in genotype C sequences across different disease phases.

<p>We checked the distribution of HBV genotype C HCC risk mutations among sequences from different disease phases (ASC, 18; CHB, 38; LC, 27; HCC, 144). Those mutations showed characteristics among different phases: 1) except three mutations (1479T, 1631T, and 1800C) all the other mutations pre-existed in ASC, among which 1383C and 1719T were most pronounced. More than 50% ASC possessed either one of these two mutations and 42% ASC possessed both; 2) the frequency of four mutations (1485T, 1631T, 1762T, and 1764A) showed an increasing trend accompanied with the disease progression though the changes among groups were not significant (Jonckheere-Terpstra trend test, <i>P</i> > 0.05); and 3) ratios of 4 mutations (1383C, 1479C, 1479T, and 1719T) fluctuated among different disease phases. No mutants were observed in the cases denoted by asterisks.</p

Flowchart for screening HBx sequences downloaded from an online global database.

<p>We downloaded HBx sequences from Hepatitis Virus Database (HVDB, <a href="http://s2as02.genes.nig.ac.jp/" target="_blank">http://s2as02.genes.nig.ac.jp/</a>). The version we exploited was DDBJ Rel. 95, containing 5956 HBx sequences in total. Sequences were then screened successively by attached information such as publication, origin, and diagnosis. Sequences enrolled should be 1) Full length HBV X sequence; 2) human sera origin; and 3) with diagnosis information and thus could be classified to non-HCC or HCC group. Finally 1115 full length HBx sequences (HCC, 161; and Non-HCC, 954) from 57 publications were extracted for further analyses.</p

Seven nucleotide mutations of HBx sequences were independent risk factors for genotype C HBV-related <b>HCC</b>.

<p>^aMutated nucleotides are shown in bold.</p><p>B cell epitope: region (aa positions 29–48); BH3-like motif: region (aa positions 116–132); Box α, region (nt1646-1668); C/EBP, CCAAT/enhancing binding protein, region (nt1643-1658); CP, core promoter, region (nt1613-1849); Enh2: enhancer 2, region (nt1636-1744); HNF3, hepatocyte nuclear factor 3, region (nt1713-1723); NRE, negative regulatory element, region (nt1611-1634); T-cell epitope: region (aa positions 116–127).</p><p>Seven nucleotide mutations of HBx sequences were independent risk factors for genotype C HBV-related <b>HCC</b>.</p

GBF1 is required for HCV replication.

<p>(A) Huh7.5.1 cells were treated with different dosages of BFA or GCA as indicated for 1 hour and then infected with JFH1 for 2 days. Total RNA was isolated and reverse transcribed and then quantitive PCR was performed. (B) Huh7.5.1 cells were treated with 20 µM GCA for 1 hour and then infected with JFH1 for 3 days. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) Huh7.5.1 cells were treated with siRNA against GBF1 or control siRNA for 3 days and then infected with JFH1 for 3 days. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D) Huh7.5.1 cells were treated with siRNA against GBF1 or control siRNA for 3 days and then infected with JFH1 for 3 days. Total RNA was isolated and reverse transcribed and then quantitive PCR was performed.</p

ARF1 is required for HCV replication.

<p>(A) Huh7.5.1 cells were treated with siRNA against PI4KIIIβ or control siRNA for 3 days and then infected with JFH1 for 3 days. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Huh7.5.1 cells were treated with siRNA against ARF1 or control siRNA for 3 days and then infected with JFH1 for 3 days. Total RNA was isolated and reverse transcribed and then quantitive PCR was performed. (C) Huh7.5.1 cells were treated with siRNA against ARF1(#6 sequence or #8 sequence) or control siRNA and then infected with Jc1FLAG2(p7-nsGluc2A) for 3 days. Gaussia luciferase activity and cellular ATP levels were measured. The normalized luciferase activities were then divided by the normalized luciferase activity from mock treatment.</p

PI4KIIIβ colocalizes with ARF1 or GBF1.

<p>(A) Huh 7.5.1 cells transfected with a HA-tagged ARF1 construct and infected with JFH1 or mock were stained with antibodies against HA (red) or PI4KIIIβ (green). (B) Huh 7.5.1 cells infected with JFH1 or mock were stained with antibodies against PI4KIIIβ (red) or GBF1 (green).</p

Sac1 overexpression inhibits HCV replication.

<p>(A) Huh 7.5.1 cells transfected with pEGFP-Sac1 or mock and then cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Huh 7.5.1 cells transfected with pEGFP-Sac1 and infected with JFH1 were stained with antibodies against PI4P (red). (C) Huh 7.5.1 cells transfected with pEGFP-Sac1 and infected with JFH1 were stained with antibodies against HCV core (red). (D) Huh 7.5.1 cells transfected with pEGFP-Sac1 and infected with JFH1 were stained with antibodies against HCV NS5A (red). (E) Huh 7.5.1 cells were transfected with pEGFP-Sac1 and infected with JFH1. Total RNA was isolated and reversely transcribed and then quantitive PCR was performed. (F) Huh7.5.1 cells were transfected with pEGFP-Sac1 and infected with Jc1FLAG2(p7-nsGluc2A) for 3 days. Gaussia luciferase activity and cellular ATP levels were measured. The normalized luciferase activities were then divided by the normalized luciferase activity from mock treatment.</p

PI4P is rearranged during HCV infection.

<p>(A) Huh 7.5.1 cells infected or uninfected with JFH1 were stained with antibodies against PI4P (red) or β-COP (green). (B) Huh 7.5.1 cells infected with JFH1 were treated with 90 IU/ml IFN or mock infection and then stained with antibodies against PI4P (red) or β-COP (green). (C) Huh 7.5.1 cells infected or uninfected with JFH1 were stained with antibodies against PI4P (red) or NS-5A (green).</p

Primers used for quantitative RT-PCR.

a<p>F, forward; R, reverse.</p

PI4KIIIβ is required for HCV replication.

<p>(A) Huh7.5.1 cells were treated with siRNA against PI4KIIIβ or control siRNA for 3 days and then infected with JFH1 for 3 days. Total RNA was isolated and reversely transcribed and then quantitive PCR was performed. (B) Huh7.5.1 cells were treated with siRNA against PI4KIIIβ or control siRNA for 3 days and then infected with Jc1 for 3 days. Gaussia luciferase activity and cellular ATP levels were measured. The normalized luciferase activities were then divided by the normalized luciferase activity from mock treatment. (C) Huh7.5.1 cells were treated with different dosages of PIK93 as indicated for 1 hour and then infected with JFH1 for 3 days. Total RNA was isolated and reversely transcribed and then quantitive PCR was performed. (D) Huh7.5.1 cells were treated with different dosages of PIK93 as indicated for 1 hour and then infected with Jc1FLAG2(p7-nsGluc2A) for 3 days. Gaussia luciferase activity and cellular ATP levels were measured. The normalized luciferase activities were then divided by the normalized luciferase activity from mock treatment.</p