Naturally occurring hepatitis C virus subgenomic deletion mutants replicate efficiently in Huh-7 cells and are trans-packaged in vitro to generate infectious defective particles

Abstract

Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.

FIG. 1.

Schematic representation of subgenomic HCV RNAs used in this study. The 5′ NTR, 3′ NTR, and EMCV IRES (solid line) structures are shown, and ORFs are depicted as open boxes with the polyprotein cleavage products indicated. Parental (wt) J6/JFH genome (encoding amino acids 1 to 1030 [J6] and 1031 to 3034 [JFH1]) is shown on top, and the JFH-1 replicon (JFH-neo-rep) is at the bottom.

FIG. 2.

Replication efficiency of subgenomic deletion mutants in Huh-7.5 cells. RNA transcripts were transfected into Huh-7.5 cells, and total cellular RNA was extracted at different times from 24 to 120 h p.t. RNA was quantified by qPCR utilizing rRNA for normalization. Values are medians from at least three independent experiments. Error bars indicate standard deviations.

FIG. 3.

trans-packaging efficiency of subgenomic deletion mutants in Huh-7.5 infected with wt J6/JFH. Subgenomic deletion mutants and JFH-1 replicon (JFH-neo-rep) RNA were transfected into J6/JFH-infected Huh-7.5 cells. Supernatants of each transfection experiment were collected at 24, 48, and 72 h p.t. and used to infect naïve Huh-7.5 cells. Total cellular RNA was extracted at 48 h postinfection, and RNA quantification was performed by quantitative qPCR with J6/JFH (A) or individual subgenome-specific (B) oligonucleotide-probe sets. Quantification of rRNA was used for normalization, and values are numbers of HCV genome copies per 50 ng of total RNA. Values are medians from at least three independent experiments. Error bars indicate standard deviations.

FIG. 4.

Efficiency of packaging of subgenomic deletion mutants and JFH-1 replicon when cotransfected with wt J6/JFH RNA. Subgenomic deletion mutants or the JFH-1 replicon (JFH-neo-rep) was cotransfected (3 μg RNA each per 2 × 10⁶ cells) into Huh-7.5 cells with an equal amount of wt J6/JFH RNA. Supernatants of each transfection were collected at 24, 48, and 72 h p.t. and used to infect naïve Huh-7.5 cells. Total cellular RNA was extracted at 48 h, and RNA of different genomes was quantified by quantitative qPCR with construct-specific oligonucleotide-probe sets. Quantification of rRNA was used for normalization, and HCV RNA amounts are expressed as number of genome copies per 50 ng of total RNA. (A) Results of the infection with supernatants collected at the indicated times p.t. Values are medians from at least three independent experiments. Error bars indicate standard deviations. (B) Ratio of the copy number of subgenomic deletion mutants to that of J6/JFH RNA.

FIG. 5.

Schematic representation of subgenomic HCV mutants expressing a luciferase reporter gene. The 5′ NTR, 3′ NTR, and EMCV IRES (solid line) structures are shown, and ORFs are depicted as open boxes with the polyprotein cleavage products indicated. The luciferase-2A reporter gene was inserted after the codons for amino acid 212 of E1 and amino acid 19 of core in Luc-coreΔ212-NS2 and Luc-nocore-NS2, respectively, and before the codon for amino acid 814 of NS2 in both. The luciferase-UBI reporter gene was inserted after the codons for amino acid 212 of E1 and amino acid 19 of core in Luc-coreΔ212-NS3 and Luc-nocore-NS3, respectively, and before the codon for amino acid 1031 of NS3 in both.

FIG. 6.

Replication of subgenomic deletion mutants expressing a luciferase reporter gene. RNA transcripts were transfected into Huh-7.5 cells, and luciferase activity was measured at different times from 5 to 72 h p.t. Values are medians from at least three independent experiments. Error bars indicate standard deviations. (A) Efficiency of replication of each RNA, measured in luciferase units. (B) Results from panel A, reported as percentages of the value at 5 h for each RNA.

FIG. 7.

trans-packaging of subgenomic deletion mutants expressing a luciferase reporter gene. Subgenomic deletion mutants containing a luciferase reporter were cotransfected (3 μg RNA each per 2 × 10⁶ cells) in Huh-7.5 cells alone or with equal amounts of wt J6/JFH RNA. Transfection supernatants were collected at 48 h p.t. and used to infect naive Huh-7.5 cells. Luciferase activities of each infection were measured at 48 h postinfection. Values are medians from at least three independent experiments. Error bars indicate standard deviations.

FIG. 8.

Summary of naïve Huh-7.5 infections with supernatants generated by DNA-RNA cotransfections of subgenomic deletion mutants with structural protein expression vectors at 48 h p.t. Subgenomic deletion mutants used in the cotransfection experiments are at the tops of the columns, and expression vectors are on the left. The qualitative evaluation of packaging efficiency is proportionally expressed with plus signs. Numbers in parentheses are the numbers of foci (two to four cells each) detected by immunostaining. The results are the averages from four independent experiments. n.t., not tested.

FIG. 9.

Efficiency of replication of J6/JFH-Δ284-736, J6/JFH-ΔE1E2, and J6/JFH in naïve Huh-7.5 and clone 9 cells. RNA transcripts were transfected (10 μg RNA per 2 × 10⁶ cells) in naïve Huh-7.5 and clone 9 cells, and total cellular RNA was extracted at 24, 48, and 72 h p.t. RNA was quantified by qPCR, and quantification of rRNA was used for normalization. Values are numbers of HCV genome copies per 50 ng of total RNA and are averages from at least three independent experiments. Error bars indicate standard deviations.

FIG. 10.

Efficiency of infection and replication in naïve Huh-7.5 or clone 9 cells of subgenomic deletion mutants packaged in naïve Huh-7.5 or clone 9 cells. Supernatants of transfections at 48 h p.t. from the experiment described in the legend to Fig. 9 were used to infect either naïve Huh-7.5 (A) or clone 9 (B) cells. Total cellular RNA was extracted at 24, 48, and 72 h postinfection. RNA quantification was performed by qPCR. Quantification of rRNA was used for normalization. Values are numbers of HCV genome copies per 50 ng of total RNA and are averages from at least three independent experiments. Error bars indicate standard deviations.