Replication of alphaviruses requires a pseudoknot that involves the poly(A) tail.

Alphaviruses, such as the Sindbis virus and the 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 nt in the 3' noncoding 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 poly(A) tail and is similar to the human telomerase pseudoknot with which it shares 17 nt. Mutants that alter the stability of the pseudoknot were investigated in the context of a replicon of the 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.