LARP1 binding to hepatitis C virus particles is correlated with intracellular retention of viral infectivity.

Hepatitis C virus (HCV) virions contain a subset of host liver cells proteome often composed of interesting virus-interacting factors. A proteomic analysis performed on double gradient-purified clinical HCV highlighted the translation regulator LARP1 on these virions. This finding was validated using post-virion capture and immunoelectron microscopy, as well as immunoprecipitation applied to in vitro (Huh7.5 liver cells) grown (Gt2a, JFH1 strain) and patient-derived (Gt1a) HCV particles. Upon HCV infection of Huh7.5 cells, we observed a drastic transfer of LARP1 to lipid droplets, inducing colocalization with core proteins. RNAi-mediated depletion of LARP1 using the C911 control approach decreased extracellular infectivity of HCV Gt1a (H77), Gt2a (JFH1), and Gt3a (S52 chimeric strain), yet increased their intracellular infectivity. This latter effect was unrelated to changes in the hepatocyte secretory pathway, as evidenced using a functional RUSH assay. These results indicate that LARP1 binds to HCV, an event associated with retention of intracellular infectivity.

Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

FIG4 is a hepatitis C virus particle-bound protein implicated in virion morphogenesis and infectivity with cholesteryl ester modulation potential.

There is growing evidence that virus particles also contain host cell proteins, which provide viruses with certain properties required for entry and release. A proteomic analysis performed on double-gradient-purified hepatitis C virus (HCV) from two highly viraemic patients identified the phosphatidylinositol 3,5-bisphosphate 5-phosphatase FIG4 (KIAA0274) as part of the viral particles. We validated the association using immunoelectron microscopy, immunoprecipitation and neutralization assays in vitro as well as patient-derived virus particles. RNA interference-mediated reduction of FIG4 expression decreased cholesteryl ester (CE) levels along with intra- and extracellular viral infectivity without affecting HCV RNA levels. Likewise, overexpressing FIG4 increased intracellular CE levels as well as intra- and extracellular viral infectivity without affecting viral RNA levels. Triglyceride levels and lipid droplet (LD) parameters remained unaffected. The 3,5-bisphosphate 5-phosphatase active site of FIG4 was found to strongly condition these results. Whilst FIG4 was found to localize to areas corresponding to viral assembly sites, at the immediate vicinity of LDs in calnexin-positive and HCV core-positive regions, no implication of FIG4 in the secretory pathway of the hepatocytes could be found using either FIG4-null mice, in vitro morphometry or functional assays of the ERGIC/Golgi compartments. This indicates that FIG4-dependent modulation of HCV infectivity is unrelated to alterations in the functionality of the secretory pathway. As a result of the documented implication of CE in the composition and infectivity of HCV particles, these results suggest that FIG4 binds to HCV and modulates particle formation in a CE-related manner.

A noncatalytic function of the ligation complex during nonhomologous end joining.

Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)-DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)-X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4-Cer-XLF complexes on DNA ends.