Differential RNA replication from the paramyxovirus genomic and antigenomic promoters

Both negative, as well as positive, sense RNA viruses generate more genomic than antigenomic RNA during the course of infection. In this thesis I have utilized a reverse genetics system that reconstitutes RNA synthesis from cDNA-derived components to study the cis-acting signals that govern differential RNA replication.; We have previously shown for the prototypic paramyxovirus simian virus 5 (SV5) that a functional antigenomic promoter for RNA replication requires proper spacing between two discontinuous elements: a 19-base segment at the 3′ terminus (Conserved Region I [CRI]) and an 18-base internal region (CRII) that is contained within the coding region of the L protein gene. The results from our mutational analysis indicate that in addition to CRI and CRII, optimal replication from the SV5 antigenomic promoter requires a third sequence-dependent element located 51–66 bases from the 3 ′ end of the RNA.; Using a minigenome system, I have shown that the genomic promoter (GP) for the paramyxovirus SV5 directs RNA replication ∼14 fold lower than that seen from the antigenomic promoter (AGP). Minigenomes containing chimeric promoters were constructed to test the hypothesis that transfer of discrete cis-acting AGP elements to the GP could confer higher replication properties to the GP. The result indicates that transfer of both the AGP 3′ terminal CRI and Region III elements into the corresponding sites of the GP led to a minigenome which replicated to ∼40% of the levels seen with the AGP. This enhanced RNA replication from the GP was further increased up to AGP levels by also including the intervening AGP segment (bases 20–50) located between CRI and Region III. Importantly, transfer of non-viral sequences in place of GP bases 20–50 also increased RNA replication to levels approaching that of the AGP, but only in the context of the AGP CRI and Region III substitutions. (Abstract shortened by UMI.)...