Aminoterminal Amphipathic α-Helix AH1 of Hepatitis C Virus Nonstructural Protein 4B Possesses a Dual Role in RNA Replication and Virus Production
Figure 5
Mutations of the conserved positively charged amino acids in NS4B AH1 affect the morphology of double-membrane vesicles.
(A) Electron microscopy (EM) analyses of H7-T7-IZ cells transfected with empty vector (mock) or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring wild-type (wt) or mutant NS4B. Cells were fixed 24 h post-transfection and processed for EM as described in the Materials and Methods section. The scale bar corresponds to 500 nm. Note that double-membrane vesicles (DMV) induced by mutants K18A, K20A and K18A/K20A are larger as compared to wt and E8A/E15A. (B) Graphical representation of DMV diameter induced by wt and NS4B mutants. The analysis is based on at least 621 DMVs from at least 10 different transfected cells. Mean value ± SEM for wt = 178.4±3.1 nm (n = 666); K18A = 298.3±5.1 (n = 832); K20A = 295.1±5.0 (n = 621); K18A/K20A = 374.0±5.0 (n = 1317). Horizontal lines (red) indicate mean values. * P≤0.0001; ns, non significant (P>0.25).