AAM-B colocalizes with NS4B in the proximity to the lipid droplets.

<p>(A) AAM-B stable cells fixed with paraformaldehyde were incubated with anti-V5 antibody to detect AAM-B and BODIPY. AAM-B colocalized with LD in the cytoplasm. Bars, 10 μm. (B) Either vector stable or AAM-B stable cells were transiently transfected with NS4B plasmid. At 48 h after transfection, cells were fixed in 4% paraformaldehyde and incubated with anti-NS4B antibody and BODIPY for 1 h at 37°C. Samples were analyzed for immunofluorescence staining using the LSM 700 laser confocal microscopy system. Cells were counterstained with DAPI to label nuclei. The insets in the panels show enlarged views of the areas marked in white squares. Bars, 10 μm.</p

AAM-B is not required in the replication step of the HCV life cycle.

<p>Huh7 cells harboring HCV subgenomic replicon derived from genotype 1b were transfected with the indicated siRNAs for 72 h. Both AAM-B mRNA level (A) and intracellular HCV RNA level (B) were quantified by qPCR. (C) Protein expression levels were analyzed by immunoblot analysis using the indicated antibodies. (D) Huh6 cells harboring HCV subgenomic replicon derived from genotype 2a were transfected with the indicated siRNAs for 72 h. Both AAM-B mRNA level (D) and intracellular HCV RNA level (E) were quantified by qPCR. The asterisks indicate significant difference (**, <i>p</i><0.01) from the value for the negative control. (F) Protein expression levels in HCV replicon cells of genotype 2a were analyzed by immunoblotting with the indicated antibodies.</p

AAM-B is required for HCV propagation.

<p>(A) Huh7.5 cells were transfected with 20 nM of scrambled siRNA (Negative) or the indicated AAM-B specific siRNA constructs for 48 h and then infected with Jc1 for 4 h. At 48 h postinfection, AAM-B mRNA levels were analyzed by qPCR. (B) Using total RNAs isolated from (A), intracellular HCV RNA levels were quantified by qPCR. (C) Naïve Huh7.5 cells were infected with Jc1 harvested from the culture supernatants of (A). At 48 h postinfection, intracellular HCV RNA levels were quantified by qPCR. (D) (Left) AAM-B stable cells were transfected with the indicated siRNAs for 48 h and then infected with Jc1 for 4 h. At 48 h postinfection, total cell lysates were immunoblotted with the indicated antibodies. (Right) The band intensities of viral proteins were quantified using ImageJ software and were expressed as relative fold from the negative control. (E) (Left) Naïve Huh7.5 cells were infected with Jc1 harvested from culture supernatants of (D). At 48 h postinfection, total cell lysates were immunoblotted with the indicated antibodies. (Right) The band intensities of viral proteins were determined by using ImageJ software and were shown as relative fold from the negative control.</p

AAM-B is involved in the virion assembly and release stage of the HCV life cycle.

<p>(A, B) Huh7.5 cells were electroporated with in vitro transcribed Jc1 RNA for 24 h and then transfected with the indicated siRNAs. At 48 h after transfection, both AAM-B mRNA level (A) and HCV RNA levels (B) were quantified by qPCR. (C) Naïve Huh7.5 cells were infected with culture supernatant harvested from (A). At 48 h postinfection, intracellular HCV RNA level was quantified by qPCR. (D) AAM-B stable cells were infected with Jc1 for 4 h. At 48 h postinfection, the cells were transfected with the indicated siRNAs. At 48 h after siRNA transfection, both AAM-B mRNA level (D) and HCV RNA levels (E) were quantified by qPCR. (F) Naïve Huh7 cells were infected with culture supernatant harvested from (D). At 48 h postinfection, intracellular HCV RNA level was quantified by qPCR. The asterisks indicate significant differences (*, <i>p</i><0.05;**, <i>p</i><0.01) from the value for the negative control.</p

AAM-B is required for the virion production without affecting lipid droplet formation.

<p>(A) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were further transfected with the indicated siRNAs. At 24 h after siRNA transfection, the culture medium was replaced with fresh medium and cells were further grown for the indicated time points. AAM-B mRNA levels (A), intracellular HCV RNA levels (B), and extracellular HCV RNA levels (C) were quantified by qPCR. (D) (Left) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were further transfected with the indicated siRNAs. At 24 h after siRNA transfection, the cells were transfected with plasmid expressing either wild-type AAM-B or siRNA resistant mutant AAM-B. At 48 h after plasmid transfection, extracellular HCV RNAs were quantified by qPCR. pAAM-B WT, V5-tagged wild-type AAM-B; pAAM-B SR, V5-tagged siRNA resistant mutant AAM-B. (Right) Huh7 cells were treated as described in the left panel and protein expressions were analyzed by immunoblot analysis using the indicated antibodies. (E) Naïve Huh7 cells were infected with culture supernatant harvested from (A) for 4 h. At 48 h postinfection, intracellular HCV RNA levels were analyzed by qPCR. (F) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, the cells were transfected with either negative or AAM-B specific siRNAs. At the indicated time points, the cells were harvested and washed twice with PBS. The cells were subjected to three consecutive freeze-thaw cycles and then centrifuged for 30 min in a microfuge. Supernatant was used as intracellular Jc1. Naïve Huh7 cells were infected with supernatant for 4 h. At 48 h postinfection, relative intracellular HCV infectivity was determined by qPCR. (G) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were further transfected with the indicated siRNAs. The cells were fixed and incubated with BODIPY for 1 h at 37°C. Cells were counterstained with 4',6-diamidino-2-phenylindole(DAPI) to label nuclei. Immunofluorescence images were processed using the Zeiss LSM 700 laser confocal microscopy system. Bars, 10μm.</p

AAM-B interacts with HCV NS4B protein.

<p>(A) HEK293T cells were cotransfected with V5-tagged AAM-B plasmid and Myc-tagged HCV protein as indicated. Protein expressions of input plasmids were confirmed by immunoblot analysis using either anti-Myc antibody (Left, upper) or anti-V5 antibody (Left, lower). (Right) Total cell lysates were immunoprecipitated with anti-Myc antibody and then bound proteins were immunoblotted with anti-V5 antibody. The arrow indicates the interacting band. (B) (Left) HEK293T cells were cotransfected with V5-tagged AAM-B plasmid and Myc-tagged NS4B (genotype 1b) expressing plasmid. At 48 h after transfection, total cell lysates were immunoprecipitated with anti-V5 antibody and then bound proteins were immunoblotted with anti-Myc antibody. (Right) The same cell lysates were immunoprecipitated with anti-Myc antibody and bound proteins were immunoblotted with anti-V5 antibody. (C) Huh7 cells harboring subgenomic replicon derived from genotype 1b were transfected with V5-tagged AAM-B expression plasmid. Total cell lysates were immunoprecipitated with rabbit anti-V5 antibody and then bound proteins were immunoblotted with either mouse anti-V5 antibody (left) or mouse anti-NS4B (right). The arrow indicates the interacting band. (D) (Upper) Schematic representation of both wild-type and mutant constructs of NS4B protein. (Lower) HEK293T cells were cotransfected with V5-tagged AAM-B and Myc-tagged mutant constructs of NS4B. Total cell lysates harvested at 48 h after transfection were immunoprecipitated with anti-Myc antibody and bound proteins were immunoblotted with anti-V5 antibody. Both wild-type and mutant protein expressions of NS4B were verified by immunoprecipitation and immunoblot analysis using anti-Myc antibody (arrows). (E) AAM-B stable cells were transiently transfected with either vector or NS4B expression plasmid. At 48 h after transfection, cells were fixed in 4% paraformaldehyde. Cells were washed twice with PBS and then incubated with either anti-NS4B or anti-AAM-B antibodies for 1 h in room temperature. Immunofluorescence staining was performed by using either FITC-conjugated or TRICT-conjugated secondary antibodies. The inset in each panel shows enlarged image of the area marked in a white square. Bars, 10 μm.</p