Cooperation between the Hepatitis C Virus p7 and NS5B Proteins Enhances Virion Infectivity

Abstract

The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly.

Importance: Hepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection.

FIG 1

Increase in HCV-JFH-1 titers with cell culture adaptation. (A) Viral titers were determined by focus formation assay (focus-forming units [FFU]/ml) at 72 h after infection with JFH-1 or cell culture-adapted JFH-1 (C. Adap). (B) The percentage of HCV NS5A-positive cells was determined at 72 h postinfection by counting over 100 cells from each of three different fields of view. (C) The intracellular HCV RNA levels in infected cells were measured by qRT-PCR at the indicated time points. Values are presented as means ± SD (n = 3). ***, P ≤ 0.001 by unpaired Student t test (A and B) or by one-way ANOVA (C). The data presented in panels A and C are representative of three different experiments. (D) Schematic diagram of the JFH-1 open reading frame. The positions of the 9 identified cell culture-adapted mutations are indicated with triangles. Table 1 gives the exact positions in the HCV genome.

FIG 2

The JFH-1-M9 infectious clone with culture-adapted mutations has increased viral titer and specific infectivity compared to JFH-1. (A) Intracellular HCV RNA levels in Huh-7.5 cells transfected with in vitro-transcribed JFH-1 RNA constructs (wild-type [Wt], 9 culture-adapted amino acid changes [M9], or with an inactivating mutation in the NS5B active site [GND]) were measured by qRT-PCR at the indicated time points. (B) Time course of HCV RNA release into the supernatant was determined by qRT-PCR following transfection of indicated constructs. (C) Release of HCV virions into the supernatant from transfected cells was determined at 72 h postinfection by focus formation assay. (D) Intracellular titer of HCV at 72 h after transfection of the indicated RNA constructs. (E) Specific infectivity of the indicated viruses was calculated as a ratio of the titer to the HCV RNA in the supernatant. Values are presented as means ± SD (n = 3). *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001, by unpaired Student t test (C to E) or by one-way ANOVA comparing Wt to M9 (A and B). Panels shown are representative of three independent experiments.

FIG 3

Culture-adapted mutations in both structural and nonstructural genes of JFH-1 slightly enhance specific infectivity. (A) Schematic diagram of the JFH-1 open reading frame with mutations indicated by asterisks. Table 1 has the exact positions in the HCV genome. (B to E) At 72 h after transfection of the indicated RNA constructs into Huh-7.5 cells, HCV intracellular RNA levels were determined by qRT-PCR (B), extracellular titer was determined by focus formation assay (C), HCV RNA release into the supernatant was determined by qRT-PCR (D), and specific infectivity was determined (E). Values are presented as means ± SD (n = 3). Panels shown are representative of three independent experiments.

FIG 4

Culture-adapted mutations in p7 and NS5B enhance the titer and specific infectivity of JFH-1. (A) Schematic diagram of the JFH-1 open reading frame with mutations indicated by asterisks. Table 1 gives exact positions in the HCV genome. (B to E) At 72 h after transfection of the indicated RNA constructs into Huh-7.5 cells, HCV intracellular RNA levels were determined by qRT-PCR (B), extracellular titer was determined by focus formation assay (C), HCV RNA release into the supernatant was determined by qRT-PCR (D), and specific infectivity was determined (E). Values are presented as means ± SD (n = 3). Panels shown are representative of three independent experiments.

FIG 5

Specific culture-adapted mutations in HCV affect lipid droplet size and circularity. (A) Confocal micrographs of Huh-7.5 cells transfected with indicated in vitro-transcribed HCV RNAs that were stained for lipid droplets (BODIPY, green), HCV core protein (red), and nuclei (DAPI, blue). Crop images are taken from the region with the white box in the merged image. Bar, 10 μm. (B) Percentage of lipid droplets larger than 1.5 μm² is graphed in box-and-whisker plots, representing the minimum, first quartile, median, third quartile, and maximum. (C) The average circularity of lipid droplets is graphed in box-and-whisker plots. Values are presented as in panel B. For panels B and C, all quantifications were conducted on at least 8 cells per HCV clone. **, P ≤ 0.01, and ***, P ≤ 0.001, by unpaired Student t test.

FIG 6

Role of sphingomyelin in the infectivity of HCV. At 72 h after transfection of the indicated constructs into Huh-7.5 cells, the sphingomyelin content of virions was determined by a fluorometric sphingomyelin assay and the sphingomyelin content relative to the extracellular RNA was calculated (A), the extracellular titer was determined by focus formation assay (B), HCV RNA release into the supernatant was measured by qRT-PCR (C), and the specific infectivity was determined (D). Values are presented as means ± SD (n = 3). ***, P ≤ 0.001, by one-way ANOVA comparing p7+NS5B to the other viruses.