Rapid Intracellular Competition between Hepatitis C Viral Genomes as a Result of Mitosis

Self Cite

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...

“…7.5-H2B-EBFP2 cells (containing a histone H2B-enhanced blue fluorescent protein proviral insertion) were used as "naive" cells (17) (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Magnetic Bead Transfection and Cell Isolationmentioning

confidence: 99%

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

Webster

Ott

Greene

2013

Self Cite

“…In vitro mechanisms of intracellular competition have been identified that facilitate the preferential expansion of one genome over another (59). Competitive lineage suppression of L2 by L1 is evident in the data corresponding to the time from RL1 to RL7 (Table 2).…”

Section: Discussionmentioning

confidence: 87%

Analysis of the Evolution and Structure of a Complex Intrahost Viral Population in Chronic Hepatitis C Virus Mapped by Ultradeep Pyrosequencing

Palmer

Dimitrova

Skums

et al. 2014

Hepatitis C virus (HCV) causes chronic infection in up to 50% to 80% of infected individuals. Hypervariable region 1 (HVR1) variability is frequently studied to gain an insight into the mechanisms of HCV adaptation during chronic infection, but the changes to and persistence of HCV subpopulations during intrahost evolution are poorly understood. In this study, we used ultradeep pyrosequencing (UDPS) to map the viral heterogeneity of a single patient over 9.6 years of chronic HCV genotype 4a infection. Informed error correction of the raw UDPS data was performed using a temporally matched clonal data set. The resultant data set reported the detection of low-frequency recombinants throughout the study period, implying that recombination is an active mechanism through which HCV can explore novel sequence space. The data indicate that polyvirus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low for the daughter virions to compete against the parental genomes. The subpopulations of parental genomes contributing to the recombination events highlighted a dynamic virome where subpopulations of variants are in competition. In addition, we provide direct evidence that demonstrates the growth of subdominant populations to dominance in the absence of a detectable humoral response. IMPORTANCEAnalysis of ultradeep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimized for amplicon analysis, assume random incorporation of sequencing errors, and are focused on achieving higher specificity at the expense of sensitivity. Such analysis is further complicated by the presence of hypervariable regions. In this study, we made use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology, we were able to (i) detect multiple instances of hepatitis C virus intrasubtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (ii) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to coincide with the collapse of specific viral subpopulations.

“…There is some evidence that a similarly closed system operates within the HCV-infected liver. Spatial HCV distribution within the liver is known to be focal (12), and local viral transfer is thought to occur through division of infected cells or cell-to-cell spread (48). Additionally, superinfection exclusion has been shown to prevent viral infection of already-infected cells (49), probably through strain competition at a postentry step (50).…”

Section: Discussionmentioning

confidence: 99%

Determining the Cellular Diversity of Hepatitis C Virus Quasispecies by Single-Cell Viral Sequencing

Leitch

McLauchlan

2013

Single-cell genomics is emerging as an important tool in cellular biology. We describe for the first time a system to investigate RNA virus quasispecies diversity at the cellular level utilizing hepatitis C virus (HCV) replicons. A high-fidelity nested reverse transcription (RT)-PCR assay was developed, and validation using control transcripts of known copy number indicated a detection limit of 3 copies of viral RNA/reaction. This system was used to determine the cellular diversity of subgenomic JFH-1 HCV replicons constitutively expressed in Huh7 cells. Each cell contained a unique quasispecies that was much less diverse than the quasispecies of the bulk cell population from which the single cells were derived, suggesting the occurrence of independent evolution at the cellular level. An assessment of the replicative fitness of the predominant single-cell quasispecies variants indicated a modest reduction in fitness compared to the wild type. Real-time RT-PCR methods capable of determining single-cell viral loads were developed and indicated an average of 113 copies of replicon RNA per cell, correlating with calculated RNA copy numbers in the bulk cell population. This study introduces a single-cell RNA viral-sequencing method with numerous potential applications to explore host-virus interactions during infection. HCV quasispecies diversity varied greatly between cells in vitro, suggesting different within-cell evolutionary pathways. Such divergent trajectories in vivo could have implications for the evolution and establishment of antiviral-resistant variants and host immune escape mutants.