Figure 7. MARCH8 Mediates HCV Envelopment.

(A) Sucrose density-gradient analysis of intracellular viral particles derived from control (WT) and MARCH8^KO cells 3 days after transfection with HCV RNA by isopycnic separation (see Figure 5A for protein expression). Thirteen fractions were collected and analyzed. The experiment was conducted twice. Representative membranes blotted with anti-core antibody are shown. Plotted are the core content along the gradient normalized to the total core protein (black) and levels of infectivity (focus-forming units [FFU] per milliliter; red).

(B) Proteolytic digestion protection assays in lysates of HCV RNA transfected cells depleted of MARCH8 or NT controls (see Figure 4F for protein expression). Shown are representative membranes and quantitative data pooled from two independent experiments demonstrating the level of protease-resistant core after no treatment or treatment with Triton (T) and/or PK.

(C) Fraction 6 of a rate zonal-like gradient was subjected to RNase digestion protection assays. Samples were left untreated or treated with the S7 RNase either alone, after PK, or after Triton and PK. Plotted is residual HCV RNA measured by qRT-PCR assays pooled from two experiments.

(D) NS2-HRS binding in MARCH8^KO and WT cells measured via PCAs pooled from two experiments. Shown are means ± SD; ***p < 0.001 relative to the corresponding untreated or WT control by one-way ANOVA with Tukey’s post hoc test (C) and Student’s t test (D).