The role of the capsid protein in Semliki Forest virus assembly

Abstract: Enveloped viruses, like the Alphaviruses, encapsidate their genomes into a nucleocapsid (NC) or core structure which is surrounded by a lipid bilayer (envelope) with spike proteins. The alphavirus Semliki Forest virus (SFV), is an RNA virus with a genomic 42 S RNA of positive polarity. SFV aquires its envelope by budding at the plasmamembrane (PM) where interactions between spike protein complexes in the membrane and preformed NCs in the cytoplasm are thought to drive formation of new virus particles. In addition to these spike-C interactions, lateral (horisontal) spikespike interactions on the external surface of the lipid bilayer have been implicated. During virus entry, NCs from the incoming virus are delivered in the cytoplasm and the RNA uncoated by an as yet poorly defined mechanism. The C protein of SFV consists of two domains: an N-terminal domain, which is thought to mediate RNA binding and a chymotrypsin-like C-terminal domain which is suggested to bind to the spike complexes. The N-terminal domain contain three regions (I-III) with high concentrations of residues with positively charged side chains and two other conserved regions with many uncharged residues. To this end I have engineered mutants with various deletions in the N-terminus of C. I have tested the role of these regions in RNA encapsidation, NC assembly, budding and RNA uncoating. I found that the bulk of the positively charged residues were needed to condense the viral RNA into NCs. The positive charges will probably bind to negatively charged phosphate groups in the RNA and thus pack the RNA chain. When the number of positive charges was decreased under a certain treshold the genomic 42 S RNA could not be encapsidated but instead, the subgenomic 26 S RNA was encapsidated. The conserved, uncharged regions were necessary for NC assembly and seemed to be involved in C-C interactions. A deletion, which removed both uncharged regions and the charged regions 11 and III from the C protein resulted in a virus that had lost its ability, to preform NCs in cytoplasm. Surprisingly, this virus could still form enveloped virus particles with correct icosahedral symmetry. This demonstrates that the membrane proteins have the capacity to direct icosahedral virus assembly. I also tested the generality of an RNA encasidation signal-binding activity which is proposed to reside in the second conserved region with many uncharged residues. My results showed that this region is not essential for genome encapsidation in SFV although this seems to be the case in the related Sindbis virus. Finally, I could show that the same conserved region is not essential for genome uncoating as was earlier suggested by others for both SFV and Sindbis virus.