Mutations in the 5 ' trailer region of a respiratory syncytial virus minigenome which limit RNA replication to one step

The 3' termini of the genomic and antigenomic RNAs of human respiratory syn cytial virus (RSV) are identical at 10 of the first 11 nucleotide positions and 21 of the first 26 positions. These consented S'-terminal sequences ar e thought to contain the genomic and antigenomic promoters. Furthermore, th e complement of each conserved sequence (i.e., the 5' end of the RNA it enc odes) might contain an encapsidation signal. Using an RSV minigenome system , we individually mutated each of the last seven nucleotides in the 5' trai ler region of the genome. We analyzed effects of these mutations on encapsi dation of the T7 polymerase-transcribed negative-sense genome, its ability to function as a template for RSV-driven synthesis of positive-sense anti-g enome and mRNA, and the ability of this antigenome to be encapsidated and t o function as template for the synthesis of more genome. As a technical com plication, mutations in the last five nucleotides of the trailer region wer e found to affect the efficiency of the adjoining T7 promoter over more tha n a 10-fold range, even though three nonviral G residues had been included between the core promoter and the trailer to maximize the efficiency of pro moter activity, This was controlled in all experiments by monitoring the le vels of total and encapsidated genome. The efficiency of encapsidation of t he T7 polymerase-transcribed genome was not affected by any of the trailer mutations. Furthermore, neither the efficiency of positive-sense RNA synthe sis from the genome nor the efficiency of encapsidation of the encoded anti genome was affected by the mutations. However, nucleotide substitution at p ositions 2, 3, 6, or 7 relative to the 5' end of the trailer blocked the pr oduction of progeny genome, whereas substitution at positions 1 and 5 allow ed a low level of genome production and substitutions at position 4 were to lerated. Position 4 is the only one of the seven positions examined that is not conserved between the 3' ends of genomic and antigenomic RNA. The muta tions that blocked the synthesis of progeny genome thus limited RNA replica tion to one step, namely, the synthesis and encapsidation of antigenome. Re storation of terminal complementarity for one of the trailer mutants by mak ing a compensatory mutation in the leader region did not restore synthesis of genomic RNA, confirming that its loss was not due to reduced terminal co mplementarity. Interestingly, this leader mutation appeared to prevent anti genome synthesis with only a slight effect on mRNA synthesis, apparently pr oviding a dissociation between these two synthetic activities, Genomes in w hich the terminal 24 or 325 nucleotides of the trailer have been deleted we re competent fur encapsidation and the synthesis of mRNA and antigenomic RN A, further confirming that terminal complementarity was not required fur th ese functions.