Efficiency of E2-p7 processing modulates production of infectious hepatitis C virus

Abstract

Previous studies indicate that the processing of hepatitis C virus (HCV) E2-p7-NS2 precursor mediated by host signal peptidase is relatively inefficient, resulting in the accumulation of E2-p7-NS2 and E2-p7 precursors in addition to E2 in mammalian cells. In this study, we discovered that a significant inhibition of the processing at an E2-p7 junction site is detrimental for HCV production, whether it was caused by the mutations in p7 or by the strategic introduction of a mutation at a terminal residue of E2 to block the signal peptidase-mediated cleavage of this junction site. However, complete separation of E2 and p7 by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between these two proteins also moderately inhibited virus production. These results indicate that optimal processing of the E2-p7 junction site is critical for efficient HCV production. We further demonstrated that disrupting E2-p7 processing inhibits both NS2 localization to the putative virus assembly sites near lipid droplets (LD) and NS2 interaction with NS3 and E2. However, the impact, if any, of the p7-NS2 processing efficiency on HCV production seems relatively minor. In conclusion, these results imply that effective release of E2 and p7 from the precursor E2-p7 promotes HCV production by enhancing NS2-associated virus assembly complex formation near LD.

Fig 1

The effect of the HA epitope tag at the N terminus of p7 on infectious HCV production and p7-NS2 processing. (A) Organization of the gt1a/gt2a HCV chimera HJ3-5 modified to encode the HA epitope tag at the N terminus of p7 with or without the insertion of a stop codon followed by the EMCV IRES at the junction site of p7 and NS2. The location of p7(KRAA) mutations is also indicated. The gt1a-based sequence is shaded. C, core protein. (B) Intracellular and extracellular virus titers expressed as infectious focus-forming units (FFU) (see Materials and Methods). The dotted line indicates infectious virus detection limit. Mean titers ± standard deviations from four different experiments are shown. (C) The expression of NS2, NS3, ^HAp7, and ^HAp7-NS2 determined by Western blot analysis by using anti-NS2 (α-NS2), anti-NS3 (α-NS3), and anti-HA (α-HA) antibodies, at day 2 postelectroporation of the indicated HCV RNAs.

Fig 2

E2-p7 processing efficiency. (A) E2 and its precursors were detected by using Western blot analyses of cell lysates collected at day 2 postelectroporation of the indicated HCV RNAs to Huh-7 cells by using anti-E2 (α-E2) and anti-HA (α-HA) antibodies. The arrowheads indicate the locations of E2-^HAp7-NS2. The Western blots of endoglycosidase H (Endo-H)-treated samples to better separate the E2 and p7 are shown in the bottom panel. E2 is detected in doublet bands upon Endo-H treatment, similar to results from previous studies (49–51). GND represents replication-defective HCV RNA. (B) The percentage of E2 in unprocessed E2-p7 precursor forms, including E2-p7, E2-^HAp7, and E2-p7_HA, was calculated from Western blot analyses of Endo-H-treated samples following quantification of E2-related protein bands by using an Odyssey Infrared Imaging system (Li-Cor) after background correction. Mean percentages of E2-p7 precursors and the standard deviations from three to five experiments are shown. Statistical analyses were performed by using GraphPad Prism, version 6, software (see Materials and Methods). Asterisks indicate statistically significant differences between two paired values: ***, P < 0.0005; **, P < 0.005; *, P < 0.05. The differences with a P value of >0.05 were considered not significant (ns).

Fig 3

Time course of HCV RNA replication and virus production. (A) The results of the quantitative TaqMan RT-PCR assays for HCV RNAs in lysates of electroporated Huh-7 cells at the indicated time points. The relative HCV RNA represents the copy number for each construct relative to the value present at 4 h after electroporation.) Extracellular (B) and intracellular (C) virus titers were determined at the indicated time points. Mean titers ± standard deviations from four different experiments are shown. The dotted line is the limit of virus titration assays.

Fig 4

The effect of E2-p7 processing on HCV production. (A) The organization of HJ3-5 with and without E2 terminal residue mutation A384R (AR) to block E2-p7 processing by the signal peptidase and HJ3-5/E2/IRES, which is modified to encode a stop codon followed by an EMCV IRES at the junction of E2 and p7 to separate E2 and p7. (B) Intracellular and extracellular virus titers determined at day 2 postelectroporation of indicated HCV RNAs. Mean titers ± standard deviations from four different experiments are shown. The dotted line is the limit of virus titration. (C) Western blot analysis of cell lysates collected at the time point indicated above following Endo-H treatment to detect E2 and E2-p7 by using an anti-E2 antibody.

Fig 5

The impact of E2-p7 processing on NS2 localization and its interaction with NS3 from the following in panel A: frame a, HJ3-5; b, HJ3-5/p7(KRAA); c, HJ3-5/E2(AR); d, HJ3-5/E2/IRES; e, HJ3-5/E2/IRES/p7(KRAA); f, HJ3-5/^HAp7; and g, HJ3-5/^HAp7(KRAA). Confocal image analysis using an Olympus FluoView FV1000 laser scanning confocal microscope of cells at day 2 postelectroporation with HCV RNAs encoding indicated genomes. Anti-NS2 antibody (green) and LipidTOX deep red neutral lipid stain (red) were used to detect NS2 and lipid droplets. The numbered images at the bottom are enlargements of the corresponding areas from the images at the top. (B) The relative percentages of NS2-positive cells displaying different NS2 localization patterns NS2 localization patterns were analyzed from 50 NS2 immunostaining-positive cells from two independent (lanes a, b, c, d, and e) or single (lanes f and g) experiments. Punctate, NS2 localization similar to that detected from frames a, d, f, and g in panel A; intermediate, NS2 localization similar to that observed from frame e' in panel A; nonpunctate, NS2 localization similar to that detected from frames b and c in panel A.

Fig 6

The impact of E2-p7 processing on the interaction of NS2 with NS3 and E2. (A) The interaction of NS2 with NS3 and E2 was determined by Western blot analysis following NS2 pulldown assays. (B) The level of immunoprecipitated NS2 (IP-NS2) was normalized to that of NS2 present in input lysates (NS2-input). IP-NS2 (normalized with by NS2-input) from HJ3-5 was set to 100. The standard deviations from five different experiments are shown. Asterisks indicate statistically significant differences between a and b, c, d, or e paired values: ***, P < 0.0005; **, P < 0.005; *, P < 0.05. The differences with a P value of >0.05 were considered not significant (ns). (C) The same experiment as shown in panel B except that the level of immunoprecipitated NS3 (IP-NS3) was normalized to that of NS3 present in input lysates (NS3-input). Results from three different experiments are shown. (D) The data shown in panel C were normalized to those shown in panel B from three different experiments to determine the efficiency of NS2 and NS3 interaction. (E) The same experiment as shown in panel B except that the level of immunoprecipitated E2 (IP-E2) was normalized to that of E2 present in input lysates (E2-input). (F) The data shown in panel E were normalized to those shown in panel B.