DELETION ANALYSIS OF A DEFECTIVE INTERFERING SEMLIKI-FOREST-VIRUS RNAGENOME DEFINES A REGION IN THE NSP2 SEQUENCE THAT IS REQUIRED FOR EFFICIENT PACKAGING OF THE GENOME INTO VIRUS-PARTICLES

The 1,244-nucleotide genome of Semliki Forest virus (SFV) defective in terfering (DI) RNA 19 (DI-19) is coterminal with the infectious genome and contains two major deletions. One deletion removes the end of the nsP1 gene and the beginning of the nsP2 gene, and the other removes t he end of the nsP2 gene, the nsP3 and nsP4 genes, and all of the struc tural protein genes (M. Thomson and N. J. Dimmock, Virology 199:354-36 5, 1994). Like all DI SFV RNAs, DI-19 contains three regions that are conserved. Region a comprises the 5' terminus continuous with part of the nsP1 gene, region b comprises a central part of the nsP2 gene, and region c comprises the 3' terminus and the associated untranslated re gion. A deletion analysis of the 265-nucleotide b region (nucleotides 679 to 943, inclusive) was undertaken to determine its role in genome replication and packaging into DI virus particles. Deleted plasmids we re constructed and transcribed, and the resulting DI RNAs were transfe cted into SFV-infected BHK cells. Putative progeny DI virus particles that had been released into the tissue culture fluid were then seriall y passaged in new monolayers together with added high-multiplicity SFV , and cells and tissue culture fluids were tested for the presence of DI RNA by reverse transcription-PCR. DI RNA that had all of the b regi on deleted was replicated well in BHK-21 cells, as shown by the presen ce of large amounts of negative-sense DI RNA and an increase in the am ount of positive-sense RNA in the cytoplasm, but was packaged very ine fficiently, as indicated by very low amounts of DI RNA in the tissue c ulture fluid. The genome of a deletion mutant that retained the 3' 224 nucleotides of region b was packaged successfully, but one that retai ned only the 5' 41 nucleotides was not detected in the tissue culture fluid. These and other data suggest that nucleotides 720 to 777 of reg ion b are of particular importance in the packaging process. This find ing agrees with data obtained with Ross River virus and contrasts with the well-studied Sindbis alphavirus major packaging signal that is lo cated within the nsP1 gene.