Defective segment 1 RNAs that interfere with production of infectious influenza A virus require at least 150 nucleotides of 5' sequence: evidence from a plasmid-driven system.

The presence of at least 80-90 and more typically around 200 nucleotides (nt) at the 5' end of the virion-sense RNA in all naturally occurring defective influenza A virus RNAs suggests that this is essential sequence, whereas the 3'-end sequence may be as short as 25 nt. The stability of defective RNA on serial passage with infectious helper virus also depends on the length of 5'-end sequence. Here, we have studied the influence of 5'-end sequences of a panel of six defective segment 1 RNAs from H3N8 and H7N7 viruses on their ability to interfere with the multiplication of plasmid-produced infectious A/WSN virus (H1N1). Four of the H3N8 defective RNAs are identical in overall length but vary in the length of 5' sequence. Transfected defective RNAs interfered with infectious virus production in a concentration-dependent manner. The extent of interference also depended on the length of 5'-end sequence in the defective genome. This required at least 150 nt and was maximal with 220 nt of 5' end sequence. The reduction in virus multiplication was highly significant and correlated with the presence of detectable intracellular defective RNA. Packaging of full-length segment 1 RNA by progeny virus was inversely proportional to the packaging of defective segment 1 RNA and may explain the reduction in infectivity. In summary, a critical length of 5'-end sequence is essential for the interfering properties of defective influenza virus RNAs, which indicates that this plays some vital role in the virus life cycle.