RSV, a member of the paramyxovirus family, is an enveloped virus that has as genome a single-stranded negative-sense RNA of <15 kb. RSV is the most important viral agent of pediatric respiratory tract disease worldwide. Accompanying reports describe the application of molecular techniques for the development of attenuated RSV vaccine viruses. The goal of the present report is to characterize the functional sequence elements in the viral genome and to understand their role in encapsidation, RNA replication, transcription and virion assembly. A better knowledge of the organization and expression of the viral genome will help guide efforts to make attenuated recombinant viruses as vaccines. We previously described a simplified approach to (i) identifying and characterizing functional regions in genomic RNA (this report) and (ii) characterizing the functions of RSV proteins (accompanying report) This is based on helper dependent cDNA- encoded minireplicons, which are versions of genomic or antigenomic (i.e., replicative intermediate) RNA in which the viral genes have been deleted and replaced by one or more marker genes. Plasmid encoding the minireplicon is transfected into cells together with plasmids which individually encode the various RSV proteins. We previously showed that complementation with the N, P, L and M2-1 proteins was necessary and sufficient to reconstitute transcription and RNA replication. The genetic map of the 15,222-nucleotide RSV genome is 3'- [leader region]-NS1-NS2-N-P-M-SH-G-F-M2(ORF1, ORF2)-L-[trailer region]-5', and thus encodes ten mRNAs which encode eleven proteins due to the presence of two translational open reading frames (ORFs) in the M2 mRNA. The RSV proteins are described in an accompanying report. Each gene begins with a 10-nucleotide conserved gene-start (GS) motif and ends with a 12-13 nucleotide gene-end (GE) motif. Mutagenic analyses of these motifs in monocistronic and dicistronic minigenomes showed that they are self-contained, transportable signals. In a dicistronic minigenome, transcription of the downstream gene was dependent on stop-start transcription from the upstream one, providing the first direct confirmation that the RSV polymerase does not enter the genome independently at internal genes. Rather, the polymerase enters the genome at the 3' end and transcribed by an """"""""on-off"""""""" sequential mode: the GS signal switches the polymerase into the """"""""on"""""""" or transcribing mode in which it recognizes only a GE signal, and the GE signal switches it into the """"""""off"""""""" mode in which it moves along the genome without synthesis until encountering a GS signal. The importance of each nucleotide position and each nucleotide assignment to the activity of the GS signal was characterized by detailed mutational analysis, and a similar project is in progress for the GE signal. We also showed that the naturally-occurring intergenic regions of RSV did not influence sequential transcription and thus do not represent control elements. In another study, we are comparing the promoters of genomic and antigenomic RNA, a comparison which is of interest because, although the two promoters are 81% identical, the genomic promoter supports both transcription and RNA replication whereas in nature the antigenomic promoter supports only replication. These promoters had been mapped as described below by insertional mutagenesis, and detailed mutagenic analysis is in progress, also as described below. We are investigating sequences which might be involved in encapsidation, transcription and RNA replication. We found that N and P are necessary and sufficient for genome encapsidation, but found no evidence for a specific encapsidation signal. Within the promoter, we have found that certain point mutations affect transcription and RNA replication disproportionately, implying that the two processes utilize somewhat different elements in the promoter. We also found that the antigenome promoter can support abundant transcription if GS and GE signals are placed in the antigenome, showing that there is not a fundamental difference between the promoters. Because of the sequence identity between the two 3' ends, the genome exhibits terminal complementarity. It has been suggested by others that these ends might base pair to form a structure important for replication or transcription. We have investigated this using a variety of different domain-swap mutations and concluded that the terminal base-pairing does not play a detectable role in RSV RNA synthesis. The last two genes in the RSV map, M2 and L, overlap by 68 nucleotides and thus it was not clear how their transcription is accommodated by the sequential mechanism. Mutational analysis provided evidence that the polymerase first transcribes the M2 gene in its entirety and then, from the M2 GE signal, has the capability of scanning in either direction to access a GS signal. In another approach, we modified the minigenome so that it contained single point mutations in the trailer. We selected specific mutations which did not affect encapsidation and which, when copied into antigenomic RNA, inactivated the antigenome promoter. Thus, this limits RNA replication to a single step (i.e., synthesis of antigenome from genome), and blocks amplification by the RSV polymerase. This is an important modification for some applications, such as examining encapsidation or the partitioning of the polymerase between the synthesis of mRNA and antigenome. This made it possible to directly compare the efficiencies of the genomic and antigenomic promoters, and showed that the amount of positive-sense RNA produced by the two was very similar. We previously initiated """"""""scanning"""""""" mutagenesis of the RSV-CAT minigenome. A nonviral dimer or hexamer was inserted individually at more than 50 sites throughout the RSV-specific sequence of the minigenome. This approach was complemented by deletional analysis. Work thus far indicates that the 3'- terminal 26 nucleotides are critical for both RNA replication and transcription, whereas the GS and GE signals are required in addition for transcription.
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