The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 5′-bulged promoter structure, as caused by the central unpaired residue A10 in its 5′ branch. Upon insertion of two uridine residues in the 3′ branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 3′-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 5′-bulged conformation. In this promoter variant it is exclusively the cRNA that is found packaged in the progeny virions. Upon insertion of only a single uridine nucleotide opposite 5′A10, the two debulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio, but at lower rates. We propose that the binding interactions of viral polymerase with either of the two differently bulged vRNA and cRNA promoter structures result in two different conformations of the enzyme protein. Only the 5′ bulged RNA-associated polymerase conformation appears to be recognized for nuclear export, which depends on nuclear matrix protein M1 and nonstructural protein NS2. And the respective wild-type vRNP- or insertion mutant cRNP complex is observed to enter the cytoplasm and hence is included in the viral encapsidation process, which takes place at the plasma membrane.

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (−)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (−)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (−)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine–adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (−)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3–20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (−)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.