Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis

Abstract

In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.

Fig 1. Impact of inhibition of SP-catalyzed cleavage at the core-E1 junction on HCV infectious cycle.

Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT), Con1/C3/Sp1mt (Sp1mt), or Con1/C3/ΔE1E2 (ΔE1E2). Culture supernatants were harvested and cells were lysed at the indicated days after transfection. (A) Schematic representation of the WT and mutated versions of HCV polyprotein. The scissors and the arrowhead represent the SP and the SPP, respectively. The asterisk indicates the site of Sp1mt mutation. The SPP-catalyzed cleavage cannot occur when SP-catalyzed cleavage at the core-E1 junction is inhibited by Sp1mt mutation. (B) Cell lysates were subjected to western blot analysis with mAb against HCV core protein (C7-50). Positions on blots of protein molecular mass standards are indicated (in kDa). Bands corresponding to the mature core protein (p21) or to different glycoforms of the core-E1 precursor (Core-E1 precursor) are indicated by arrows. A representative western blot is shown. (C) Cell lysates were probed for core antigen using HCV core-specific ELISA. The threshold of detection of this assay is evaluated at 0.3 log fmol per μg of total protein. (D and E) Culture supernatants were probed for infectivity titer (D) and core antigen (E). The thresholds of detection of the infectivity assay and of HCV core-specific ELISA are evaluated at 0.3 log ffu/mL and 2 log fmol/L, respectively. The mean values and standard errors of at least 3 independent experiments are shown.

Fig 2. In situ EM visualization of HCV-like particle budding.

Representative electron micrographs of ultra-thin sections of BHK-21 cells electroporated with the recombinant RNAs SFV-HCV1b (WT), SFV-HCV1b/Sp1mt (Sp1mt), or SFV-HCV1b/Sp2mt (Sp2mt). Some budding events are indicated on electron micrographs at low magnification (arrows). High-magnification images of such budding events are shown in insets.

Fig 3. In situ EM detection of HCV-like particle budding after immunogold labeling.

Shown are representative electron micrographs of ultra-thin sections of BHK-21 cells electroporated with the recombinant RNAs SFV-HCV1b (WT), SFV-HCV1b/Sp1mt (Sp1mt), or SFV-HCV1b/Sp2mt (Sp2mt), after immunogold labeling with mAbs against HCV core protein (+ anti-core) or HCV E2 envelope glycoprotein (+ anti-E2). High-magnification images of gold-labeled budding events are shown in insets.

Fig 4. Titration of extracellular and intracellular infectivity.

Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT), Con1/C3/ΔE1E2 (ΔE1E2), or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, (A) culture supernatants were harvested and probed for extracellular infectivity titers, and (B) cells were lysed by freeze-thaw cycles and probed for intracellular infectivity titers. Mean values of four independent experiments are shown and the standard errors of the means are presented. The threshold of detection of the intracellular infectivity assay is evaluated at 2 ffu per 10⁶ transfected cells.

Fig 5. Core protein envelopment analysis by membrane protection assay.

Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT), Con1/C3/ΔE1E2 (ΔE1E2) or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, cells were lysed by freeze-thaw cycles and left untreated or treated with 5 μg/mL proteinase K (PK) in the presence or absence of 1% Triton X-100 (Triton). The samples were subjected to (A) western blot analysis with mAb against HCV core protein (a representative western blot is shown) or (B) dot blot for quantification of signal intensities with ImageJ (values were normalized to untreated samples). The mean values and standard errors of four independent experiments are shown. *, P comprised between 0.05 and 0.01; **, P comprised between 0.01 and 0.001; ***, P below 0.001.

Fig 6. EM visualization of intracellular HCV particles.

Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT) or Con1/C3/ΔE1E2 (ΔE1E2) or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, cells were lysed by freeze-thaw cycles and cell lysates were loaded onto iodixanol gradients. After isopycnic ultracentrifugation, fractions with densities ranging from 1.12 to 1.13 g/mL were pooled, and directly subjected to EM visualization after negative staining (left panels) or delipidated with 0.1% Triton X-100 and subjected to immunogold labeling with mAb against HCV core protein before negative staining and EM visualization (right panels).