Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry

English version České info

The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

Vyšlo v časopise: Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry. PLoS Pathog 9(12): e32767. doi:10.1371/journal.ppat.1003835
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003835

Souhrn

Zdroje

1. Kuhn RJ (2007) Togaviridae: The Viruses and Their Replication. In: Knipe DM, Howley PM, editors. Fields Virology. Fifth ed. Philadelphia, PA: Lippincott, Williams and Wilkins. pp. 1001–1022.

2. EnserinkM (2007) Infectious diseases. Chikungunya: no longer a third world disease. Science 318: 1860–1861.

3. SchwartzO, AlbertML (2010) Biology and pathogenesis of chikungunya virus. Nat Rev Micro 8: 491–500.

4. JoseJ, SnyderJE, KuhnRJ (2009) A structural and functional perspective of alphavirus replication and assembly. Future Microbiol 4: 837–856.

5. VaneyMC, DuquerroyS, ReyFA (2013) Alphavirus structure: activation for entry at the target cell surface. Curr Opin Virol 3: 151–158.

6. KielianM, Chanel-VosC, LiaoM (2010) Alphavirus entry and membrane fusion. Viruses 2: 796–825.

7. RosePP, HannaSL, SpiridigliozziA, WannissornN, BeitingDP, et al. (2011) Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammlian hosts. Cell Host Microbe 10: 97–104.

8. CollinetC, StoterM, BradshawCR, SamusikN, RinkJC, et al. (2010) Systems survey of endocytosis by multiparametric image analysis. Nature 464: 243–249.

9. BornerGH, AntrobusR, HirstJ, BhumbraGS, KozikP, et al. (2012) Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles. J Cell Biol 197: 141–160.

10. KlimstraWB, HeidnerHW, JohnstonRE (1999) The furin protease cleavage recognition sequence of sindbis virus PE2 can mediate virion attachment to cell surface heparan sulfate. J Virol 73: 6299–6306.

11. OrvedahlA, SumpterRJr, XiaoG, NgA, ZouZ, et al. (2011) Image-based genome-wide siRNA screen identifies selective autophagy factors. Nature 480: 113–117.

12. ThompsonJW, BruickRK (2012) Protein degradation and iron homeostasis. Biochim Biophys Acta 1823: 1484–1490.

13. BethuneJ, WielandF, MoellekenJ (2006) COPI-mediated transport. J Membr Biol 211: 65–79.

14. CherryS, KunteA, WangH, CoyneC, RawsonRB, et al. (2006) COPI activity coupled with fatty acid biosynthesis is required for viral replication. PLoS Pathog 2: e102.

15. CuretonDK, Burdeinick-KerrR, WhelanSP (2012) Genetic inactivation of COPI coatomer separately inhibits vesicular stomatitis virus entry and gene expression. J Virol 86: 655–666.

16. ZhangL, LeeSY, BeznoussenkoGV, PetersPJ, YangJS, et al. (2009) A role for the host coatomer and KDEL receptor in early vaccinia biogenesis. Proc Natl Acad Sci USA 106: 163–168.

17. DaroE, SheffD, GomezM, KreisT, MellmanI (1997) Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component epsilon-COP. J Cell Biol 139: 1747–1759.

18. GrayRS, AbituaPB, WlodarczykBJ, Szabo-RogersHL, BlanchardO, et al. (2009) The planar cell polarity effector Fuz is essential for targeted membrane trafficking, ciliogenesis and mouse embryonic development. Nature Cell Biol 11: 1225–1232.

19. HeydeckW, ZengH, LiuA (2009) Planar cell polarity effector gene Fuzzy regulates cilia formation and Hedgehog signal transduction in mouse. Dev Dyn 238: 3035–3042.

20. BrooksER, WallingfordJB (2012) Control of vertebrate intraflagellar transport by the planar cell polarity effector Fuz. J Cell Biol 198: 37–45.

21. ZilberY, BabayevaS, SeoJH, LiuJJ, MootinS, et al. (2013) The PCP effector Fuzzy controls cilial assembly and signaling by recruiting Rab8 and Dishevelled to the primary cilium. Mol Biol Cell 24: 555–565.

22. SerruV, DessenP, BoucheixC, RubinsteinE (2000) Sequence and expression of seven new tetraspans. Biochim Biophys Acta 1478: 159–163.

23. LevyS, ShohamT (2005) The tetraspanin web modulates immune-signalling complexes. Nat Rev Immunol 5: 136–148.

24. EnnsCA, RutledgeEA, WilliamsAM (1996) The transferrin receptor. Biomembranes 4: 255–287.

25. KielianM, JungerwirthS, SayadKU, DeCandidoS (1990) Biosynthesis, maturation, and acid-activation of the Semliki Forest virus fusion protein. J Virol 64: 4614–4624.

26. CollierS, GubbD (1997) Drosophila tissue polarity requires the cell-autonomous activity of the fuzzy gene, which encodes a novel transmembrane protein. Development 124: 4029–4037.

27. SeoJH, ZilberY, BabayevaS, LiuJ, KyriakopoulosP, et al. (2011) Mutations in the planar cell polarity gene, Fuzzy, are associated with neural tube defects in humans. Hum Mol Genet 20: 4324–4333.

28. GerdesJM, DavisEE, KatsanisN (2009) The vertebrate primary cilium in development, homeostasis, and disease. Cell 137: 32–45.

29. WallingfordJB, MitchellB (2011) Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia. Genes Dev 25: 201–213.

30. van der SchaarHM, RustMJ, WaartsBL, van der Ende-MetselaarH, KuhnRJ, et al. (2007) Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking. J Virol 81: 12019–12028.

31. ZaitsevaE, YangST, MelikovK, PourmalS, ChernomordikLV (2010) Dengue virus ensures its fusion in late endosomes using compartment-specific lipids. PLoS Pathog 6: e1001131.

32. WuC, MacleodI, SuAI (2013) BioGPS and MyGene.info: organizing online, gene-centric information. Nucleic Acids Res 41: D561–565.

33. MercerJ, SchelhaasM, HeleniusA (2010) Virus entry by endocytosis. Annu Rev Biochem 79: 803–833.

34. SunE, HeJ, ZhuangX (2013) Live cell imaging of viral entry. Curr Opin Virol 3: 34–43.

35. ThomasJM, KlimstraWB, RymanKD, HeidnerHW (2003) Sindbis virus vectors designed to express a foreign protein as a cleavable component of the viral structural polyprotein. J Virol 77: 5598–5606.

36. LiljeströmP, LusaS, HuylebroeckD, GaroffH (1991) In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol 65: 4107–4113.

37. HinrichsenL, HarborthJ, AndreesL, WeberK, UngewickellEJ (2003) Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J Biol Chem 278: 45160–45170.

38. WildT, HorvathP, WylerE, WidmannB, BadertscherL, et al. (2010) A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export. PLoS Biol 8: e1000522.

39. BirminghamA, SelforsLM, ForsterT, WrobelD, KennedyCJ, et al. (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6: 569–575.

40. LiaoM, KielianM (2005) Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus-membrane fusion. J Cell Biol 171: 111–120.

41. KamentskyL, JonesTR, FraserA, BrayMA, LoganDJ, et al. (2011) Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27: 1179–1180.

42. Ayala-NunezNV, WilschutJ, SmitJM (2011) Monitoring virus entry into living cells using DiD-labeled dengue virus particles. Methods 55: 137–143.