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Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

ABSTRACT: Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence.

Importance

Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

SUBMITTER: Frericks N

PROVIDER: S-EPMC10949430 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Frericks Nicola N Brown Richard J P RJP Reinecke Birthe M BM Herrmann Maike M Brüggemann Yannick Y Todt Daniel D Miskey Csaba C Vondran Florian W R FWR Steinmann Eike E Pietschmann Thomas T Sheldon Julie J

Journal of virology 20240206 3

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a p ...[more]

PMID: 38319104

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Project description:Despite the long-standing success of the yellow fever virus (YFV) 17D vaccine, the precise virus and host determinants which confer its virulence attenuation remain incompletely defined. To provide insights, we performed YFV genome-level evolutionary analyses combined with investigations of vaccine hepatotropism. Evolutionary analyses confirmed vaccine-specific mutations are selected against in virulent YFV isolates, with genomic signatures of purifying selection most pronounced in the envelope (E) protein, where vaccine-specific mutations are concentrated. Structural prediction and mapping using Alphafold3 confirmed vaccine-specific mutations form a surface-exposed patch on domain III of the E protein, likely overlapping the flavivirus receptor binding domain. In tissue culture, 17D infections exhibited enhanced cell-to-cell spread and cytolysis compared to virulent YFV strains. In humans, YFV infections cause severe liver damage while vaccination has an excellent safety profile. Consequently, we next investigated whether 17D displays impaired tropism for primary human hepatocytes (PHH) or induces distinct cell-intrinsic responses to virulent strains. Surprisingly, ex vivo infections confirmed robust propagation of both 17D and virulent YFV strains in PHH. RNA-sequencing revealed consistent and shared induction of IFNB and IFNL1-4, modulating gene programs associated with antiviral responses, cellular immunity, chemotaxis and inflammation, cell-death, metabolism and protein translation. Consistent with differences in spreading efficiency, subtle differences in virion production kinetics and the magnitude and tempo of PHH transcriptional responses were observed. Antiviral responses to 17D were activated earlier while responses to virulent YFV were delayed but enhanced. In summary, we confirm the YFV vaccine exhibits comparable hepatotropism to virulent strains and induces virtually identical host responses. Mechanistically, these data suggest that impaired hepatotropism and associated cytopathology are not correlates of 17D’s virulence attenuation, implying unknown barriers to systemic dissemination mitigate viscerotropic spread and associated liver pathology upon vaccination.

Project description:Chronic infection of hepatitis B virus (HBV) remains a major health burden worldwide. While the immune response has been recognized to play crucial roles in HBV pathogenesis, the direct cytopathic effects of HBV infection and replication on host hepatocytes and the HBV-host interactions are only partially defined due to limited culture systems. Here, based on our recently developed 5 chemical-cultured primary human hepatocytes (5C-PHHs) model that supports long-term HBV infection, we performed multiplexed quantitative analysis of temporal changes of host proteome and transcriptome on PHHs infected by HBV for up to 4 weeks. We showed that metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels during long-term HBV infection, which led to cytopathic effects and could be partially rescued by early, rather than late, nucleot(s)ide analog (NA) administration and could be significantly relieved by blocking viral antigens with RNA interference (RNAi). Overexpression screening of the dysregulated proteins identified a series of host factors that may contribute to pro- or anti-HBV responses of the infected hepatocytes. In conclusion, our results suggest that long-term HBV infection in primary human hepatocytes leads to cytopathic effects through remodeling the proteome and transcriptome and early antiviral treatment may reduce the extent of such effects, indicating a role of virological factors in HBV pathogenesis and a potential benefit of early administration of antiviral treatment. IMPORTANCE Global temporal quantitative proteomic and transcriptomic analysis using long-term hepatitis B virus (HBV)-infected primary human hepatocytes uncovered extensive remodeling of the host proteome and transcriptome and revealed cytopathic effects of long-term viral replication. Metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels, which could be partially rescued by early, rather than late, NA therapy and could be relieved by blocking viral antigens with RNAi. Overexpression screening identified a series of pro- or anti-HBV host factors. These data have deepened the understanding of the mechanisms of viral pathogenesis and HBV-host interactions in hepatocytes, with implications for therapeutic intervention.

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Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Importance

Publications

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

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