Biology subjects

Meuleman, P.

Publications and source records attributed to Meuleman, P..

5 recordsLinked to original sources

bioRxiv2023

Phosphatidylinositol 4-kinase III alpha governs cytoskeletal organization for invasiveness of liver cancer cells

Background and AimsHigh expression of phosphatidylinositol 4-kinase III alpha (PI4KIII) correlates with poor survival rates in patients with hepatocellular carcinoma (HCC). In addition, Hepatitis C virus (HCV) infections activate PI4KIII and contribute to HCC progression. We aimed at mechanistically understanding the impact of PI4KIII on the progression of liver cancer and the potential contribution of HCV in this process. MethodsSeveral hepatic cell culture and mouse models were used to study functional importance of PI4KIII on liver pathogenesis. Antibody arrays, gene silencing and PI4KIII specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. ResultsHigh PI4KIII expression and/or activity induced cytoskeletal rearrangements via increased-phosphorylation of paxillin and cofilin. This led to morphological alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2{gamma}) working downstream of PI4KIII in regulation of the cytoskeleton. PIK3C2{gamma} generates plasma membrane (PM) phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]- enriched, invadopodia-like structures which regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. ConclusionsPI4KIII regulates cytoskeleton organization via PIK3C2{gamma}/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIII and HCV to progression of liver cancer and identify promising targets for therapeutic intervention. IMPACT AND IMPLICATIONSUnderstanding mechanistically how high PI4KIII expression are associated with poor clinical outcomes of liver cancer is important to develop pharmaceutical interventions. Our study sheds light on the importance of the two lipid kinases PI4KIII and PIK3C2{gamma} as well as the contribution of HCV on liver cancer progression, unraveling the signaling pathway governing this process. This preclinical study contributes to better understanding the complex connection of phospholipids, cytoskeleton and liver cancer and suggests strategies to improve therapeutic outcomes by targeting important signaling molecules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/541742v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@16ba717org.highwire.dtl.DTLVardef@a6f681org.highwire.dtl.DTLVardef@181c3cdorg.highwire.dtl.DTLVardef@5df6aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation

ObjectiveHepatitis A virus (HAV) infections are considered not to trigger an innate immune response in vivo, in contrast to hepatitis C virus (HCV). This lack of immune induction has been imputed to strong immune counteraction by HAV proteases 3CD and 3ABC. We aimed at elucidating the mechanisms of innate immune induction and counteraction by HAV and HCV in vivo and in vitro. DesignuPA-SCID mice with humanized liver were infected with HAV and HCV. Hepatic cell culture models were used to assess HAV and HCV sensing by TLR3 and RIG-I/MDA5, respectively. Cleavage of the adaptor proteins TRIF and MAVS was analyzed by transient and stable expression of HAV and HCV proteases and virus infection. ResultsWe detected similar levels of Interferon stimulated genes (ISGs) induction in hepatocytes of HAV and HCV infected human liver chimeric mice. In cell culture, HAV induced ISGs exclusively upon sensing by MDA5 and dependent on LGP2. TRIF and MAVS were only partially cleaved by HAV 3ABC and 3CD, not sufficiently to abrogate signalling. In contrast, HCV NS3-4A efficiently degraded MAVS, as previously reported, whereas TRIF was not cleaved. ConclusionsHAV induces an innate immune response in hepatocytes via MDA5/LGP2, with limited control of both pathways by proteolytic cleavage. HCV activates TLR3 and lacks TRIF cleavage, suggesting that this pathway mainly contributes to HCV induced antiviral response in hepatocytes. Our results shed new light on induction and counteraction of innate immunity by HAV and HCV and their potential contribution to clearance and persistence. SIGNIFICANCE OF THIS STUDYO_ST_ABSWhat is already known on this topic?C_ST_ABS-- Despite sharing biological and molecular similarities, HAV infections are always cleared while HCV infections persist in most cases. -- In infected chimpanzees HAV does not trigger a strong innate immune response, as opposed to HCV. This has been imputed to the action of HAV proteases abrogating the signalling pathways. -- Physiological in vitro and in vivo models, based on human hepatocytes, to assess HAV and HCV mechanisms of induction and interference of innate immunity are still missing. What this study adds-- HAV induces an innate immune response in vitro and in vivo, in systems with intact signalling pathways and devoid of adaptive immunity. -- HAV 3ABC and 3CD proteases do not abolish the host innate immune response. -- HCV NS3-4A protease disrupts the RLRs pathways, but cannot cleave TRIF and has no impact on TLR3 response. How this study might affect research, practice or policy-- This study offers a comprehensive, side-by-side investigation on HAV and HCV infections in physiological models which recapitulate a cytokine response in the human liver, and allows a precise assessment of the viral interference related to the function of the respective signalling pathways. -- Our results elucidate mechanisms, so far controversial or poorly investigated, thus contributing to our understanding of HAV clearance and HCV persistence.

immunology↗

bioRxiv2022

A Hepatitis C virus genotype 1b post transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes, that do not include the highly prevalent gt1b, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, but cell-to-cell spreading efficiency was not higher as in other isolates like JFH-1. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is the first efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines. Author summaryChronic HCV infections remain an important global health issue, despite the availability of highly efficient therapies. So far no protective vaccine is available, which is in part due to the high divergence of HCV variants and the limited possibly to mirror this genetic diversity in cell culture. It has been proven particularly difficult to grow infectious virus in cell culture, requiring extensive adaptation with multiple mutations, which in turn affect infectivity of the adapted variants in vivo. Here we have isolated a genotype 1b variant from a very high titer serum of a patient after liver transplantation (German Liver Transplant 1, GLT1), showing an outstanding genome replication efficiency in cultured hepatoma cells. We were able to adapt this isolate to production of infectious virus, therefore generating the first efficient full-replication cycle cell culture model for highly prevalent HCV genotype 1b. Despite multiple mutations required, adapted GLT1 was still infectious in vivo. GLT1 therefore is not only an important novel development facilitating future efforts in vaccine development. It also provides novel perspectives towards our understanding how liver transplantation drives the evolution of viral isolates with high replication capacity, which might contribute to direct pathogenesis of HCV infection.

microbiology↗

bioRxiv2020

Human hepatocyte PNPLA3 148M exacerbates rapid non-alcoholic steatohepatitis development in chimeric mice

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of chimeric mice respond to a hypercaloric Western-style diet. As early as 4 weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatohepatitis selectively in the human graft, followed by pericellular fibrosis after 8 weeks of hypercaloric feeding. The PNPLA3 148M variant, either from a homozygous 148M human donor or overexpressed in a homozygous 148I donor background, caused widespread microvesicular steatosis and even more severe steatohepatitis. We conclude that PNPLA3 148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variants associated with advanced fatty liver diseases.

pathology↗

bioRxiv2018

New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Recently, we developed an efficient HEV cell culture system and demonstrated that this virus produces three different forms of its capsid protein: (i) the ORF2i form (infectious/intracellular) which is the form associated with the infectious particles, (ii) the ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are massively secreted glycoproteins not associated with infectious particles, but are the major antigens present in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). Although ORF2 protein is the most characterized viral protein, its glycosylation status and the biological relevance of this post-translational modification is still unclear. In the present study, we constructed and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. During our study, we also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. In conclusion, our study led to new insights into the molecular mechanisms of ORF2 expression.

microbiology↗