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Characterization of HCV particles produced in BE-KO cells expressing either ApoE, Erns or NS1.

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posted on 2017-06-23, 17:49 authored by Takasuke Fukuhara, Tomokazu Tamura, Chikako Ono, Mai Shiokawa, Hiroyuki Mori, Kentaro Uemura, Satomi Yamamoto, Takeshi Kurihara, Toru Okamoto, Ryosuke Suzuki, Kentaro Yoshii, Takeshi Kurosu, Manabu Igarashi, Hiroshi Aoki, Yoshihiro Sakoda, Yoshiharu Matsuura

(A) Experimental procedure. (B) HA-tagged complexes were immunoprecipitated from the supernatants of BE-KO cells expressing either HA-ApoE, HA-Erns, Hel, or HA-NS1 by using a control anti-IgG or anti-HA antibody. Expression of HA-ApoE, HA-Erns, Hel, or HA-NS1 in the immunoprecipitated samples was determined by immunoblotting. (C) The HCV RNA associated with ApoE, Erns, and NS1 was determined by qRT-PCR. (D) The supernatants of BE-KO cells expressing either HA-ApoE, HA-Erns, Hel, or HA-NS1 upon infection with HCV at an MOI of 1 were subjected to density gradient fractionation, and density (upper), HCV RNA levels (middle) and infectious titers (bottom) for each fraction were determined by qRT-PCR and focus forming assay, respectively. (E) Transcomplemented HCV particles (HCVtcp) were produced in BE-KO cells expressing either HA-ApoE, HA-Erns, HA-Hel or HA-NS1 at 72-h post-transfection with pCAGC-NS2/JFH1am and PHH SGR-JFH1/Gluc/NS3m. HCVtcp were inoculated into Huh7.5.1 cells, and gaussia luciferase (gLuc) activities were determined at 72-h post-infection. In all cases, asterisks indicate significant differences (* p < 0.01) versus the results of the control cells.

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