FIG 6.

Characterization of HCV/NPHV p7 chimeras in recombinant HCV cell culture viruses. (A) The p7 sequences from the HCV isolate J6 and the NPHV isolate H14 as well as the designated chimeras were cloned into Jc1 or Jc1 HAHA-L-p7 as depicted. (B) In vitro transcripts of the Jc1 HAHA-L-p7 constructs as well as Jc1 wild type were transfected into Huh-7.5 cells, and the expression of HAHA-tagged p7 in cell lysates was visualized by Western blotting 48 h posttransfection. Additionally, the viral protein NS5A and the cellular protein β-actin were stained. (C) Expression of NS2 and E2 in lysates of cells transfected with in vitro transcripts of the Jc1 HAHA-L-p7 constructs was visualized 48 h posttransfection by Western blotting. (D) Viral titers of the Jc1 p7 constructs were determined in supernatants 24 h, 48 h, and 72 h after transfection into Huh-7.5 cells. Shown are log₁₀ TCID₅₀/ml as mean values from three independent experiments + SD. (E) Intra- and extracellular core amounts were measured 48 h posttransfection of the Jc1 p7 constructs into Huh-7.5 cells. The background level of extracellular core amounts was set to the upper value of Jc1 p7H14, since this construct does not produce any infectious particles. Two independent experiments were performed, and the mean values + SD as log₁₀ core femtomoles/liter are shown. (F) The specific infectivity was calculated based on the viral titers shown in panel C and extracellular core release shown in panel D.