Evasion of Superinfection Exclusion and Elimination of Primary Viral RNA by an Adapted Strain of Hepatitis C Virus

Abstract

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequencebarcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/ UC) tract of the 3= untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo. In superinfection exclusion, a cell productively infected with a specific virus becomes resistant to infection with a homologous virus. This process has been described for several viruses, including human immunodeficiency virus (HIV) (1), Sindbis virus (2), duck hepatitis B virus (3), and citrus tristeza virus (4), in a broad range of hosts. Cells infected with or actively replicating hepatitis C virus (HCV) also become refractory to further HCV infection (5, 6). The superinfection block occurs at the level of translation or replication of the incoming secondary viral RNA (5-7), which is similar to the case of the related pestivirus bovine diarrhea virus (8). The exact mechanism is unclear. Since HCV RNA levels quickly plateau in infected cells in vitro, a superinfection block at the level of RNA translation or replication suggests that access to the necessary host factor(s) is rate limiting. This has clear implications for HCV treatment. By definition, a rate-limiting host factor is critical for the replication of the virus and may be a unique target in future therapies.Superinfection exclusion itself also has clear implications for treating HCV infection. Since viral recombination requires a host cell to be productively replicating two genomes, superinfection exclusion would be expected to effectively prevent viral recombination. If, howeve...

Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

“…Related to viral entry, some authors have described a superinfection exclusion mechanism by which cells currently infected with HCV are resistant to a secondary infection with the same or a closely related virus (43)(44)(45). These authors suggest that the first virus prevents secondary infections because it sequesters a limiting host factor (44,45). Our results suggest that HCV NS5B might be the HCV protein that mediates Akt/PKB relocalization to fulfill a function in the replication complexes.…”

“…Transient, E2 glycoprotein-mediated Akt/PKB activation has been previously related to HCV entry into the cell (13). Related to viral entry, some authors have described a superinfection exclusion mechanism by which cells currently infected with HCV are resistant to a secondary infection with the same or a closely related virus (43)(44)(45). These authors suggest that the first virus prevents secondary infections because it sequesters a limiting host factor (44,45).…”

A Combined Proteomics/Genomics Approach Links Hepatitis C Virus Infection with Nonsense-Mediated mRNA Decay

“…After selecting cells with puromycin and confirming knockdown by quantitative RT-PCR, we inoculated cells with a monocistronic infectious clone of HCV Jc1 encoding orange fluorescent protein (OFP) (Figures S2A, B) (Webster et al, 2013). After 6 days, we assessed viral spread by flow cytometry to quantify the OFP-expressing cells, giving the observed infection rate.…”

The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion

“…In the latter examples, superinfecting viruses were able to enter the primary virus-infected cells and produce some of the virus-encoded proteins but failed to replicate, suggesting that SIE targets the replication step of the superinfecting virus. Additionally, replicationrelated proteins such as NS4 and 2K of WNV (26) and NS5A of HCV (60) were identified as targets of superinfection exclusion activity. SIE at the level of virus entry into cells may not be conserved between plant and animal viruses, as there is no indication that plant viruses have specific cell surface receptors for virus entry into plant cells.…”