Replication of alphaviruses requires a pseudoknot that involves the polyA tail

Summary Alphaviruses, such as Sindbis virus and Chikungunya virus, are RNA viruses with a positive sense single-stranded RNA genome that infect various vertebrates including humans. A conserved sequence element (CSE) of ∼19 nucleotides (nts) in the 3’ non-coding region is important for replication. Despite extensive mutational analysis of the CSE no comprehensive model of this element exists to date. Here it is shown that the CSE can form an RNA pseudoknot with part of the polyA tail and is similar to the human telomerase peudoknot with which it shares 17 nts. Mutants that alter the stability of the pseudoknot were investigated in the context of a replicon of Sindbis virus and by native gel electrophoresis. These studies reveal that the pseudoknot is required for virus replication and is stabilized by UAU base triples. The new model is discussed in relation to previous data on Sindbis virus mutants and revertants lacking (part of) the CSE.