Pathway for the Formation of Semliki Forest Virus Rna Replication Complex

INTRODUCTION ............................................................................................................ 1 1.Alphaviruses ....................................................................................................................1 1.1.Alphavirus expression vectors ........................................................................ 1 1.2.Life cycle .......................................................................................................... 2 1.3.Replication ........................................................................................................4 1.4.Nonstructural polyprotein ...............................................................................4 1.5.Properties of individual nonstructural proteins ............................................. 6 1.5.1.nsP4 ..................................................................................................... 6 1.5.2.nsP3 ..................................................................................................... 6 1.5.3.nsP2 ..................................................................................................... 6 1.5.4.nsP1 ..................................................................................................... 7 2.Morphological features of infected cells ...................................................................... 9 2.1.CPV structures and spherules in alphavirus infected cells ..........................9 2.2.Spherules are not unique to alphaviruses ................................................... 10 AIMS OF THE STUDY ................................................................................................ 11 MATERIALS AND METHODS ................................................................................. 12 1.Cells, viruses and plasmids ......................................................................................... 12 2.Yeast two-hybrid analysis (unpublished) ................................................................... 12 3.Infection and transfection ........................................................................................... 13 4.Radioactive labelling, cell fractionation and chemical cross-linking ..................... 14 5.Flotation and extraction analysis ................................................................................ 14 6.Antisera and cell staining reagents ............................................................................ 14 7.Immunoprecipitation and immunoblotting ................................................................ 14 8.Enzyme assays ............................................................................................................. 15 9.Immunofluorescence and electron microscopy ........................................................ 15 10.Virus production from SFV infectious cDNA ........................................................ 15 RESULTS ................................................................................................................... 16 1.SFV replication complex in infected cells ................................................................ 16 1.1.Localization (II) ............................................................................................ 16 1.2.Composition (II, unpublished) ..................................................................... 16 1.3.Morphogenesis (II) ....................................................................................... 18 2.Interactions between SFV nsPs .................................................................................. 18 2.1.Yeast two-hybrid analysis (unpublished) .................................................... 18 2.2.Co-expression of nsPs in mammalian cells ................................................ 19 2.2.1.Co-immunoprecipitation experiments (III) .................................. 19 2.2.2.Immunofluorescence studies (III, unpublished) .......................... 19 3.Characterization of the nonstructural polyproteins (III) .......................................... 19 4.Membrane association and targeting of the replication complex ........................... 21 4.1.Early events: Polyprotein stage (III) ........................................................... 21 4.2.Role of the membrane binding peptide of nsP1 in RNA replication (IV) 22 DISCUSSION ................................................................................................................. 23 1.SFV replication structures .......................................................................................... 23 2.Properties of the alphavirus replication complex ..................................................... 25 2.1.Formation of the complex ............................................................................ 25 2.2.Composition and conversion from RC minus to RC plus ................................... 27 3.Functional signif icance of the nsP1 membrane binding peptide ............................ 30 4.Regulation of replication ............................................................................................ 31 CONCLUDING REMARKS ....................................................................................... 33 ACKNOWLEDGEMENTS .......................................................................................... 34 REFERENCES ............................................................................................................. 35 REPRINTS OF ORIGINAL PUBLICATIONS ORIGINAL PUBLICATIONS This thesis is based on the following articles, which are referred to in the text by their Roman numerals, and on unpublished data presented in the text. I Salonen A, Ahola T and Kääriäinen L. 2004. Viral RNA Replication in Association with Cellular Membranes. Review. Curr. Top. Microbiol. Immunol. 285, in press. II Kujala P, Ikäheimonen A, Ehsani N, Vihinen H, Auvinen P and Kääriäinen L. 2001. Biogenesis of the Semliki Forest Virus RNA Replication Complex. J. Virol. 75:3873-84. III Salonen A, Vasiljeva L, Merits A, Magden J, Jokitalo E and Kääriäinen L. 2003. Properly Folded Nonstructural Polyprotein Directs the Semliki Forest Virus Replication Complex to the Endosomal Compartment. J. Virol. 77:1691-702. IV Salonen A, Merits A, Kääriäinen L and Ahola T. The significance of the Membrane Binding Peptide of Semliki Forest Virus Replicase Protein nsP1. Manuscript.