Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus

In order to study whether flavivirus RNA packaging is dependent on RNA repl ication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN 1dGDD, allowing continuous production of replicating (wild-type) and nonrep licating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-le ngth Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Tran sfection of pKUN1dGDD DNA into BHK cells, however, did not result in the re covery of any secreted virus particles containing encapsidated dGDD RNA, de spite an apparent accumulation of this RNA in cells demonstrated by Norther n blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both i n cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much sma ller amounts in PKUN1dGDD DNA-transfected repBHK cells (where it was replic ated via complementation), it was packaged into secreted virus particles, T hus, packaging of defective Kunjin virus RNA could occur only when it was r eplicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C, I. Nugent et al,, J, Virol, 73:427-435, 19 99), both demonstrating the necessity for the RNA to be replicated before i t can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quas ispecies in positive-strand RNA viruses, This mechanism may thus help allev iate the high-copy error rate of RNA-dependent RNA polymerases.