Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein

The negative-sense segmented RNA genome of influenza virus is transcribed i nto capped and polyadenylated mRNAs, as well as full-length replicative int ermediates (cRNAs). The mechanism that regulates the two forms of transcrip tion remains unclear, although several lines of evidence imply a role for t he viral nucleoprotein (NP). In particular, temperature-shift and biochemic al analyses of the temperature-sensitive viruses A/WSN/33 ts56 and A/FPV/Ro stock/34/Giessen tsG81 containing point mutations within the NP coding regi on have indicated specific defects in replicative transcription at the nonp ermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both m utants were temperature sensitive for influenza virus gene expression in tr ansient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-asso ciate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temp erature but not when expressed at 30 degrees C. This suggests that the RNA- binding activity of NP is required for replicative transcription.