IDENTIFICATION OF A REGION IN THE SINDBIS VIRUS NUCLEOCAPSID PROTEIN THAT IS INVOLVED IN SPECIFICITY OF RNA ENCAPSIDATION

The specific encapsidation of genomic RNA by an alphavirus requires re cognition of the viral RNA by the nucleocapsid protein. In an effort t o identify individual residues of the Sindbis virus nucleocapsid prote in which are essential for this recognition event, a molecular genetic analysis of a domain of the protein previously suggested to be involv ed in RNA binding in vitro was undertaken. The experiments presented d escribe the generation of a panel of viruses which contain mutations i n residues 97 through 111 of the nucleocapsid protein. All of the viru ses generated were viable, and the results suggest that, individually, the residues mutated do not play a critical role in encapsidation. Ho wever, one mutant which had lost the ability to specifically encapsida te the genomic RNA was identified. This mutant virus, which contained a deletion of residues 97 to 106, encapsidated both-the genomic RNA an d the subgenomic mRNA of the virus. It is proposed that the encapsidat ion of this second species of RNA, which is not present in wild-type v irions, is the result of the loss of a domain of the nucleocapsid prot ein required for specific recognition of the genomic RNA packaging sig nal. The results suggest that this region of the protein is important in dictating specificity in the encapsidation reaction in vivo. The is olation and preliminary characterization of two independent second-sit e revertants to this deletion mutant are also described.