PACKAGING SIGNALS IN ALPHAVIRUSES

Alphaviruses synthesize large amounts of both genomic and subgenomic R NA in infected cells, but usually only the genomic RNA is packaged. Th is implies the existence of an encapsidation or packaging signal which would be responsible for selectivity, Previously; we had identified a region of the Sindbis virus genome that interacts specifically with t he viral capsid protein, This 132-nucleotide (nt) fragment lies within the coding region of the nsP1 gene (nt9-15 to 1076), We proposed that the 132-mer is important for capsid recognition and initiates the for mation of the viral nucleocapsid. To study the encapsidation of Sindbi s virus RNAs in infected cells, we designed a new assay that uses the self-replicating Sindbis virus genomes (replicons) which lack the vira l structural protein genes and contain heterologous sequences under th e control of the subgenomic RNA promoter, These replicons can be packa ged into viral particles by using defective helper RNAs that contain t he structural protein genes (P. Bredenbeek, I. Frolov, C. M. Rice, and S. Schlesinger, J. Virol. 67:6439-6446, 1993), Insertion of the 132-m er into the subgenomic RNA significantly increased the packaging of th is RNA into viral particles, We have used this assay and defective hel pers that contain the structural protein genes of Ross River virus (RR V) to investigate the location of the encapsidation signal in the RRV genome. Our results show that there are several fragments that could a ct as packaging signals. They are all located in a different region of the genome than the signal for the Sindbis virus genome, For RRV, the strongest packaging signal lies between nt 2761 and 3062 in the nsP2 gene, This is the same region that was proposed to contain the packagi ng signal for Semliki Forest virus genomic RNA.