Biology subjects

Vondran, F.

Publications and source records attributed to Vondran, F..

3 recordsLinked to original sources

bioRxiv2023

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes

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 which 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 establishing persistence. Author SummaryHCV infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms which 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.

molecular biology↗

bioRxiv2022

Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a read-out for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in a broad range of cell culture models, independently of cytopathogenic effect formation. Compared to other cell culture models, the Caco-2 subline Caco-2-F03 displayed superior performance, as it possesses a stable SARS-CoV-2 susceptible phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of PHGDH, CLK-1, and CSF1R. The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the HK2 inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false positive hits.

pharmacology and toxicology↗

bioRxiv2019

HBV DNA is a substrate for the cGAS/STING pathway but is not sensed in infected hepatocytes

HBV chronic infection is a critical risk factor for hepatocellular carcinoma. Although debated, the absence of innate immune response to HBV infection in hepatocytes is becoming the current view. However the underlying reasons are poorly understood. This study aims to define potential viral pathogen-associated molecular patterns (PAMPs) and the pattern recognition receptors (PRRs), and to elucidate whether HBV counteracts the innate pathways. The innate immune response to HBV infection was monitored by interferon-stimulated gene 54 (ISG54) mRNA, a direct downstream transcriptional target of Interferon Regulatory Factor 3 (IRF3), or IRF3 phosphorylation. The immunostimulatory potential of naked HBV DNAs or RNAs and the respective PRRs were determined upon viral nucleic acid transfection in immunocompetent cells including knockout cells lacking key molecules of innate pathways. The expression and functionality of DNA and RNA sensing pathways in primary human hepatocytes (PHH) were assessed. The inhibition of the DNA-sensing pathway by HBV was tested using IRF3 nuclear translocation assay. Our study revealed that HBV infection does not induce an innate response in infected hepatocytes, even in absence of HBV X protein. HBV relaxed-circular DNA (rcDNA) and DNA replication intermediates, but not HBV RNAs, are immunostimulatory and sensed by Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase (cGAS) and Stimulator of Interferon Genes (STING). Although PHH express DNA sensors to reduced levels compared to myeloid cells, they can respond to naked HBV rcDNA. However, we show that the absence of innate response to HBV infection in hepatocytes is not due to an active inhibition of the DNA sensing pathway by the virus. HBV passively evades the innate immune response in infected hepatocytes by (i) producing non-immunostimulatory RNAs, (ii) avoiding sensing of its DNAs by cGAS/STING without active inhibition of the pathway, possibly through shielding of the viral DNAs by the capsid. Author summaryInnate immune responses are the first line of defense against viral infections. They lead to the production of antiviral factors after recognition of specific viral features by the infected cells. Here we show that HBV, a major cause of liver cirrhosis and cancer, avoids recognition by infected hepatocytes through different means. First, HBV RNAs, contrarily to other viral RNAs, are not immunostimulatory. Second, we show that naked HBV DNAs are recognized by cGAS/STING and induce an innate immune response. Furthermore, we demonstrate that this pathway is active in hepatocytes and is not inhibited by the virus. Instead, we propose that HBV DNAs are not accessible to cGAS/STING in the context of an infection. This might be due to shielding of the viral DNA by the viral capsid.

microbiology↗