1887

Abstract

The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000511
2016-08-01
2021-07-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/97/8/1877.html?itemId=/content/journal/jgv/10.1099/jgv.0.000511&mimeType=html&fmt=ahah

References

  1. Berger K. L., Cooper J. D., Heaton N. S., Yoon R., Oakland T. E., Jordan T. X., Mateu G., Grakoui A., Randall G. 2009; Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication. Proc Natl Acad Sci U S A 106:7577–7582 [View Article][PubMed]
    [Google Scholar]
  2. Blagoev B., Kratchmarova I., Ong S. E., Nielsen M., Foster L. J., Mann M. 2003; A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21:315–318 [View Article][PubMed]
    [Google Scholar]
  3. Blanchard E., Belouzard S., Goueslain L., Wakita T., Dubuisson J., Wychowski C., Rouillé Y. 2006; Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80:6964–6972 [View Article][PubMed]
    [Google Scholar]
  4. Boulant S., Douglas M. W., Moody L., Budkowska A., Targett-Adams P., McLauchlan J. 2008; Hepatitis c virus core protein induces lipid droplet redistribution in a microtubule- and dynein-dependent manner. Traffic 9:1268–1282 [View Article][PubMed]
    [Google Scholar]
  5. Bukong T. N., Momen-Heravi F., Kodys K., Bala S., Szabo G. 2014; Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90. PLoS Pathog 10:e1004424 [View Article][PubMed]
    [Google Scholar]
  6. Carrère-Kremer S., Montpellier C., Lorenzo L., Brulin B., Cocquerel L., Belouzard S., Penin F., Dubuisson J. 2004; Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions. J Biol Chem 279:41384–41392 [View Article][PubMed]
    [Google Scholar]
  7. Coller K. E., Berger K. L., Heaton N. S., Cooper J. D., Yoon R., Randall G. 2009; RNA interference and single particle tracking analysis of hepatitis C virus endocytosis. PLoS Pathog 5:e1000702 [View Article][PubMed]
    [Google Scholar]
  8. Cosset F. L., Dreux M. 2014; HCV transmission by hepatic exosomes establishes a productive infection. J Hepatol 60:674–675 [View Article][PubMed]
    [Google Scholar]
  9. Drummer H. E., Maerz A., Poumbourios P. 2003; Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins. FEBS Lett 546:385–390 [View Article][PubMed]
    [Google Scholar]
  10. Eyre N. S., Fiches G. N., Aloia A. L., Helbig K. J., McCartney E. M., McErlean C. S., Li K., Aggarwal A., Turville S. G. et al. 2014; Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection. J Virol 88:3636–3652 [View Article][PubMed]
    [Google Scholar]
  11. Ferraris P., Beaumont E., Uzbekov R., Brand D., Gaillard J., Blanchard E., Roingeard P. 2013; Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection. Cell Mol Life Sci 70:1297–1306 [View Article][PubMed]
    [Google Scholar]
  12. González-Carmona M. A., Vogt A., Heinicke T., Quasdorff M., Hoffmann P., Yildiz Y., Schneider C., Serwe M., Bartenschlager R. et al. 2011; Inhibition of hepatitis C virus gene expression by adenoviral vectors encoding antisense RNA in vitro and in vivo. J Hepatol 55:19–28 [View Article][PubMed]
    [Google Scholar]
  13. Gosert R., Egger D., Lohmann V., Bartenschlager R., Blum H. E., Bienz K., Moradpour D. 2003; Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77:5487–5492[PubMed] [CrossRef]
    [Google Scholar]
  14. Gower E., Estes C., Blach S., Razavi-Shearer K., Razavi H. 2014; Global epidemiology and genotype distribution of the hepatitis C virus infection. J Hepatol 61:S45–57 [View Article][PubMed]
    [Google Scholar]
  15. Herbst J. J., Opresko L. K., Walsh B. J., Lauffenburger D. A., Wiley H. S. 1994; Regulation of postendocytic trafficking of the epidermal growth factor receptor through endosomal retention. J Biol Chem 269:12865–12873[PubMed]
    [Google Scholar]
  16. Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. 1993; Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67:4665–4675[PubMed]
    [Google Scholar]
  17. Hocking S. L., Wu L. E., Guilhaus M., Chisholm D. J., James D. E. 2010; Intrinsic depot-specific differences in the secretome of adipose tissue, preadipocytes, and adipose tissue-derived microvascular endothelial cells. Diabetes 59:3008–3016 [View Article][PubMed]
    [Google Scholar]
  18. Ito T., Yasui K., Mukaigawa J., Katsume A., Kohara M., Mitamura K. 2001; Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system. Hepatology 34:566–572 [View Article][PubMed]
    [Google Scholar]
  19. Lai C. K., Jeng K. S., Machida K., Lai M. M. 2008; Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A. J Virol 82:8838–8848 [View Article][PubMed]
    [Google Scholar]
  20. Lindenbach B. D., Evans M. J., Syder A. J., Wölk B., Tellinghuisen T. L., Liu C. C., Maruyama T., Hynes R. O., Burton D. R. et al. 2005; Complete replication of hepatitis C virus in cell culture. Science 309:623–626 [View Article][PubMed]
    [Google Scholar]
  21. Liu Z., Zhang X., Yu Q., He J. J. 2014; Exosome-associated hepatitis C virus in cell cultures and patient plasma. Biochem Biophys Res Commun 455:218–222 [View Article][PubMed]
    [Google Scholar]
  22. Ludwig A., Blume J., Diep T. M., Yuan J., Mateos J. M., Leuthäuser K., Steuble M., Streit P., Sonderegger P. 2009; Calsyntenins mediate TGN exit of APP in a kinesin-1-dependent manner. Traffic 10:572–589 [View Article][PubMed]
    [Google Scholar]
  23. Moradpour D., Evans M. J., Gosert R., Yuan Z., Blum H. E., Goff S. P., Lindenbach B. D., Rice C. M. 2004; Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J Virol 78:7400–7409 [View Article][PubMed]
    [Google Scholar]
  24. O'Quinn P. R., Knabe D. A., Wu G. 2002; Arginine catabolism in lactating porcine mammary tissue. J Anim Sci 80:467–474[PubMed] [CrossRef]
    [Google Scholar]
  25. Pawlotsky J. M. 2015; Therapy: Avoiding treatment failures associated with HCV resistance. Nat Rev Gastroenterol Hepatol 12:673–674 [View Article][PubMed]
    [Google Scholar]
  26. Ponomareva O. Y., Holmen I. C., Sperry A. J., Eliceiri K. W., Halloran M. C. 2014; Calsyntenin-1 regulates axon branching and endosomal trafficking during sensory neuron development in vivo. J Neurosci 34:9235–9248 [View Article][PubMed]
    [Google Scholar]
  27. Ramakrishnaiah V., Thumann C., Fofana I., Habersetzer F., Pan Q., de Ruiter P. E., Willemsen R., Demmers J. A., Stalin Raj V. et al. 2013; Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells. Proc Natl Acad Sci U S A 110:13109–13113 [View Article][PubMed]
    [Google Scholar]
  28. Rindler M. J., Xu C. F., Gumper I., Cen C., Sonderegger P., Neubert T. A. 2008; Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells. J Histochem Cytochem 56:381–388 [View Article][PubMed]
    [Google Scholar]
  29. Romero-Brey I., Merz A., Chiramel A., Lee J. Y., Chlanda P., Haselman U., Santarella-Mellwig R., Habermann A., Hoppe S. et al. 2012; Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog 8:e1003056 [View Article][PubMed]
    [Google Scholar]
  30. Sabahi A. 2009; Hepatitis C Virus entry: the early steps in the viral replication cycle. Virol J 6:117 [View Article][PubMed]
    [Google Scholar]
  31. Shahidi M., Tay E. S., Read S. A., Ramezani-Moghadam M., Chayama K., George J., Douglas M. W. 2014; Endocannabinoid CB1 antagonists inhibit hepatitis C virus production, providing a novel class of antiviral host-targeting agents. J Gen Virol 95:2468–2479 [View Article][PubMed]
    [Google Scholar]
  32. Targett-Adams P., McLauchlan J. 2005; Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. J Gen Virol 86:3075–3080 [View Article][PubMed]
    [Google Scholar]
  33. Vogt D. A., Camus G., Herker E., Webster B. R., Tsou C. L., Greene W. C., Yen T. S., Ott M. 2013; Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein. PLoS Pathog 9:e1003302 [View Article][PubMed]
    [Google Scholar]
  34. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Kräusslich H. G. et al. 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000511
Loading
/content/journal/jgv/10.1099/jgv.0.000511
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error