Inhibition of Endosomal Transport Results in Decreased Synthesis of Nucleocapsid Protein of PHV Virus

M. Muyangwa*, E.E. Garanina**

Kazan Federal University, Kazan, 420008 Russia

E-mail: *musalwam@yahoo.co.uk, **kathryn.cherenkova@gmail.com

Received February 1, 2017

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Abstract

This paper is devoted to investigation of molecular mechanisms of transport of nucleocapsid protein of non-pathogenic virus Prospect Hill. Prospect Hill virus (PHV) belongs to the family Bunyaviridae, genus Hantavirus. Hantaviruses utilize the intracellular trafficking pathways for delivery of viral RNA and proteins to the site of viral assembly. While significant progress has been achieved in understanding PHV replication, the role of intracellular transport in virus replication remains largely unknown. Co-localization of PHV nucleocapsid protein and Rab5, Rab7, and Rab11 (early, late, and recycling endosomes, respectively) has been demonstrated using immunofluorescence assay. Western blot analysis has revealed decreased accumulation of nucleocapsid protein when dominant negative (DN) mutant protein forms Rab5 and Rab11were expressed in PHV infected cells. Our data suggest that the functional activity of early, late, and recycling endosomes is essential for replication of HPV proteins. The obtained results are of great importance for comprehension of molecular and cellular mechanisms of pathogenesis of hantaviral infection.

Keywords: recombinant lentivirus, nucleocapsid protein, hantavirus, Prospect Hill Virus, PHV, Rab proteins, endosomes

Acknowledgments. This study was funded by the subsidy allocated to Kazan Federal University as part of the state program for increasing its competitiveness among the world's leading centers of science and education. The work was also supported by the Russian Science Foundation (project no. 15-14-00016).

Figure Captions

Fig. 1. The analysis of the efficiency of transfection of the culture of A549 cells with DNA plasmid encoding for Rab wild-type proteins, 48 h after transfection: A – untransfected control, B – A549 cells culture transfected with DsRed-Rab5-WT plasmid, C – A549 cells culture transfected with DsRed-Rab7-WT plasmid, D – А549 cells culture transfected with DsRed-Rab11-WT plasmid. Scale: 20 ?m.

Fig. 2. Intercellular co-localization of nucleocapsid proteins of PHV hantavirus and proteins of various endosomes. Confocal fluorescence microscopy. Blue fluorescence – DAPI. Green fluorescence – staining to nucleocapsid protein of PHV virus. Red fluorescence – DsRed-Rab5, DsRed-Rab7, or DsRed-Rab11, allows to detect localization of early, late, and recycling endosomes. A – co-localization of nucleocapsid protein of LV-PHV-S recombinant lentivirus with Rab5 protein of early endosomes. B – co-localization of nucleocapsid protein of LV-PHV-S recombinant lentivirus with Rab7 protein of late endosomes. C – co-localization of nucleocapsid protein of LV-PHV-S recombinant lentivirus with Rab11 protein of recycling endosomes. Scale: 20 ?m.

Fig. 3. The analysis of intracellular content of nucleocapsid protein in A549 cell lysates transfected with the plasmid constructions of dominant negative mutant or wild-type Rab5 and Rab11 and transduced with LV-PHV-S recombinant lentivirus. Western blot analysis. A – analysis of the synthesis of nucleocapsid proteins in A549 cell lysates transfected with the plasmid constructions of dominant negative mutant and wild-type Rab5 and Rab11 and transduced after 48 h with LV-PHV-S recombinant lentivirus. NTC – control, non-transduced cells. 1, 3, 5 – cells transfected with DsRed-Rab5-DN. 2, 4, 6 – DsRed-Rab5-WT. 8, 10, 12 – DsRed-Rab11-DN. 9, 11, 13 – DsRed-Rab11-WT. 7, 14 – non-transfected cells. Cells were collected 48 h (1, 2, 7, 8, 9), 72 h (3, 4, 10, 11), and 96 h (5, 6, 12, 13, 14) after transduction. Main band – nucleocapsid protein of hantavirus with the molecular weight of 50 kDa. B – quantitative analysis of intracellular content of nucleocapsid protein of hantavirus of A549 cells transduced with LV-PHV-S recombinant lentivirus 48 h after transfection with the plasmid constructions of dominant negative or wild-type Rab5 and Rab11. Data are presented as mean value ? standard error, p < 0.05. The asterisk marks statistically significant data.

References

  1. Elliott R.M. Molecular biology of the Bunyaviridae. J. Gen. Virol., 1990, vol. 71, no. 3, pp. 501–522. doi: 10.1099/0022-1317-71-3-501.
  2. Jonsson C.B., Figueiredo L.T., Vapalahti O. A global perspective on hantavirus ecology, epidemiology, and disease. Clin. Microbiol. Rev., 2010, vol. 23, no. 2, pp. 412–441. doi: 10.1128/CMR.00062-09.
  3. Vaheri A., Strandin T., Hepojoki J., Sironen T., Henttonen H., Makela S., Mustonen J. Uncovering the mysteries of hantavirus infections. Nat. Rev. Microbiol., 2013, vol. 11, no. 8, pp. 539–550. doi: 10.1038/nrmicro3066.
  4. Vapalahti O., Kallio-Kokko H., Narvanen A., Julkunen I., Lundkvist A., Plyusnin A., Lehvaslaiho H., Brummer-Korvenkontio M., Vaheri A., Lankinen H. Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response. J. Med. Virol., 1995, vol. 46, no. 4, pp. 293–303. doi: 10.1002/jmv.1890460402.
  5. Ramanathan H.N., Jonsson C.B. New and Old World hantaviruses differentially utilize host cytoskeletal components during their life cycles. Virology, 2008, vol. 374, no. 1, pp. 138–150. doi: 10.1016/j.virol.2007.12.030.
  6. Mellman I. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol., 1996, vol. 12, pp. 575–625. doi: 10.1146/annurev.cellbio.12.1.575.
  7. Stenmark H. Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol., 2009, vol. 10, no. 8, pp. 513–525. doi: 10.1038/nrm2728.
  8. Bucci C., Parton R.G., Mather I.H., Stunnenberg H., Simons K., Hoflack B., Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell, 1992, vol. 70, no. 5, pp. 715–728. doi: 10.1016/0092-8674(92)90306-W.
  9. Gorvel J.-P., Chavrier P., Zerial M., Gruenberg J. rab5 controls early endosome fusion in vitro. Cell, 1991, vol. 64, no. 5, pp. 915–925. doi: 10.1016/0092-8674(91)90316-Q.
  10. Rink J., Ghigo E., Kalaidzidis Y., Zerial M. Rab conversion as a mechanism of progression from early to late endosomes. Cell, 2005, vol. 122, no. 5, pp. 735–749. doi: 10.1016/j.cell.2005.06.043.
  11. Ganley I.G., Carroll K., Bittova L., Pfeffer S. Rab9 GTPase regulates late endosome size and requires effector interaction for its stability. Mol. Biol. Cell, 2004, vol. 15, no. 12, pp. 5420–5430. doi: 10.1091/mbc.E04-08-0747.
  12. Grant B.D., Donaldson J.G. Pathways and mechanisms of endocytic recycling. Nat. Rev. Mol. Cell Biol., 2009, vol. 10, no. 9, pp. 597–608. doi: 10.1038/nrm2755.
  13. van Ijzendoorn S.C. Recycling endosomes. J. Cell Sci., 2006, vol. 119, no. 9, pp. 1679–1681. doi: 10.1242/jcs.02948.
  14. Nielsen E., Severin F., Backer J.M., Hyman A.A., Zerial M. Rab5 regulates motility of early endosomes on microtubules. Nat. Cell Biol., 1999, vol. 1, no. 6, pp. 376–382. doi: 10.1038/14075.
  15. Pal A., Severin F., Lommer B., Shevchenko A., Zerial M. Huntingtin-HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease. J. Cell Biol., 2006, vol. 172, no. 4, pp. 605–618. doi: 10.1083/jcb.200509091.
  16. Vanlandingham P.A., Ceresa B.P. Rab7 regulates late endocytic trafficking downstream of multivesicular body biogenesis and cargo sequestration. J. Biol. Chem., 2009, vol. 284, no. 18, pp. 12110–12124. doi: 10.1074/jbc.M809277200.
  17. Lombardi D., Soldati T., Riederer M.A., Goda Y., Zerial M., Pfeffer S.R. Rab9 functions in transport between late endosomes and the trans Golgi network. Eur. Mol. Biol. Organ. J., 1993, vol. 12, no. 2, pp. 677–682.
  18. Schmaljohn C.S., Nichol S.T. Fields Virology. Bunyaviridae. Knipe D.M., Howley P.M., Griffin D.E., Lamb R.A., Martin M.A., Roizman B., Straus S.E. (Eds.). Philadelphia, Lippincott, Williams & Wilkins, 2007, pp. 1741–1789.
  19. Chen W., Feng Y., Chen D., Wandinger-Ness A. Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. Mol. Biol. Cell, 1998, vol. 9, no. 11, pp. 3241–3257. doi: 10.1091/mbc.9.11.3241.
  20. Ravkov E.V., Nichol S.T., Compans R.W. Polarized entry and release in epithelial cells of Black Creek Canal virus, a New World hantavirus. J. Virol., 1997, vol. 71, no. 2, pp. 1147–1154.
  21. Muyangwa M., Garanina E.E., Martynova E.V., Rizvanov A.A. Lentivirus expression of hantavirus nucleocapsid proteins. BioNanoScience, 2016, vol. 6, no. 4, pp. 403–406. doi: 10.1007/s12668-016-0250-9.
  22. Rizvanov A.A. Viral and non-viral methods of administration of recombinant nucleic acids into the organism. Doct. Biol. Sci. Diss. Kazan, 2010. 283 p. (In Russian)
  23. Townsley A.C., Weisberg A.S., Wagenaar T.R., Moss B. Vaccinia virus entry into cells via a low-pH-dependent endosomal pathway. J. Virol., 2006, vol. 80, no. 18, pp. 8899–8908. doi: 10.1128/JVI.01053-06.
  24. Manna D., Aligo J., Xu C., Park W.S., Koc H., Heo W.D., Konan K.V. Endocytic Rab proteins are required for hepatitis C virus replication complex formation. Virology, 2010, vol. 398, no. 1, pp. 21–37. doi: 10.1016/j.virol.2009.11.034.
  25. Engel S., Heger Т., Mancini R., Herzog F., Kartenbeck J., Hayer A., Helenius A. Role of endosomes in simian virus 40 entry and infection. J. Virol., 2011, vol. 85, no. 9, pp. 4198–4211. doi: 10.1128/JVI.02179-10.
  26. Lozach P.Y., Mancini R., Bitto D., Meier R., Oestereich L., Overby A.K., Pettersson R.F., Helenius A. Entry of bunyaviruses into mammalian cells. Cell Host Microbe, 2010, vol. 7, no. 6, pp. 488–499. doi: 10.1016/j.chom.2010.05.007.
  27. Rowe R.K., Suszko J.W., Pekosz A. Roles for the recycling endosome, Rab8, and Rab11 in hantavirus release from epithelial cells. Virology, 2008, vol. 382, no. 2, pp. 239–249. doi: 10.1016/j.virol.2008.09.021.
  28. Sharma D.K., Choudhury A. Singh R.D. Wheatley C.L. Marks D.L., Pagano R.E. Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling. J. Biol. Chem., 2003, vol. 278, no. 9, pp. 7564–7572. doi: 10.1074/jbc.M210457200.
  29. Choudhury A., Dominguez M., Puri V., Sharma D.K., Narita K., Wheatley C.L., Marks D.L.,   Pagano R.E. Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J. Clin. Invest., 2002, vol. 109, no. 12, pp. 1541–1550. doi: 10.1172/JCI15420.
  30. Ren M., Xu G., Zeng J., De Lemos-Chiarandini C., Adesnik M., Sabatini D.D. Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc. Natl. Acad. Sci. U. S. A., 1998, vol. 95, no. 11, pp. 6187–6192.
  31. Schlierf B., Fey G.H., Hauber J., Hocke G.M., Rosorius O. Rab11b is essential for recycling of transferrin to the plasma membrane. Exp. Cell Res., 2000, vol. 259, no. 1, pp. 257–265. doi: 10.1006/excr.2000.4947.
  32. Apodaca G., Katz L.A., Mostov K.E. Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. J. Cell Biol., 1994, vol. 125, no. 1, pp. 67–86. doi: 10.1083/jcb.125.1.67.
  33. Jones J.C., Turpin E.A., Bultmann H., Brandt C.R., Schultz-Cherry S. Inhibition of influenza virus infection by a novel antiviral peptide that targets viral attachment to cells. J. Virol., 2006, vol. 80, no. 24, pp. 11960–11967. doi: 10.1128/JVI.01678-06.

For citation: Muyangwa M., Garanina E.E. Inhibition of endosomal transport results in decreased synthesis of nucleocapsid protein of PHV virus. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 1, pp. 58–71. (In Russian)


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