Alphaviruses synthesize large amounts of both genomic and subgenomic RNA in infected cells, but usually only the genomic RNA is packaged. This 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 the viral capsid protein. This 132-nucleotide (nt) fragment lies within the coding region of the nsP1 gene (nt 945 to 1076). We proposed that the

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... er is important for capsid recognition and initiates the formation of the viral nucleocapsid. To study the encapsidation of Sindbis virus RNAs in infected cells, we designed a new assay that uses the self-replicating Sindbis virus genomes (replicons) which lack the viral structural protein genes and contain heterologous sequences under the control of the subgenomic RNA promoter. These replicons can be packaged into viral particles by using defective helper RNAs that contain the structural protein genes (P. Bredenbeek, I. Frolov, C. M. Rice, and S. Schlesinger, J. Virol. 67:6439-6446, 1993). Insertion of the 132-mer into the subgenomic RNA significantly increased the packaging of this RNA into viral particles. We have used this assay and defective helpers that contain the structural protein genes of Ross River virus (RRV) to investigate the location of the encapsidation signal in the RRV genome. Our results show that there are several fragments that could act 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 packaging signal for Semliki Forest virus genomic RNA. on May 9, 2020 by guest http://jvi.asm.org/ Downloaded from FIG. 4. Effect of the insertion of sequences from the 5Ј-terminal region of the Sindbis virus genome on packaging of the lacZ subgenomic RNA. (A) Cloning diagram. The fragments were obtained from either DH-BB (5ЈSIN) or DH-EB (5ЈSIN) plasmids (1) with the restriction enzymes shown. After blunt ending (with Klenow fragment), each was inserted into the blunt-ended XbaI site of the SINrep/LacZ replicon plasmid. Fragment S1 contains the 5Ј terminus of the Sindbis virus genome and 5 nt upstream. (B) Autoradiogram of the RNAs present in released particles. The replicon-and helper-containing particles were pelleted and RNAs were isolated as described in Materials and Methods. The fragment inserted is indicated above each lane. (C) Relative efficiency of packaging of the replicon subgenomic RNA. The efficiency of packaging was calculated as described in Materials and Methods. The data were calculated from three experiments, one transfection and two infections (MOI, 5 PFU/cell).