Characterization and template properties of RNA dimers generated during flock house virus RNA replication

Flock house virus (FHV) is the best studied member of the Nodaviridae, a fa mily of small, nonenveloped, isometric RNA viruses of insects and fish. Nod avirus genomes comprise two sing] e-stranded positive-sense RNA segments (R NAs I and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp replicates both genomic RN As and also generates a subgenomic RNA (RNA3) that is not encapsidated. Alt hough genomic RNAs replicate through negative-sense intermediates, little i s known about these RNAs or the details of the replication mechanism. Negat ive-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, hav e been detected in previous studies. In this study we detected dimers of RN As 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected D rosophila cells, as well as from mammalian and yeast cells supporting FHV R NA replication. Characterization of these RNA species by RT-PCR and sequenc e determination showed that they contained head-to-tail junctions of FHV RN As. RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication. To examine the template properties of these di meric RNAs, we made corresponding cDNAs and transcribed them from a T7 prom oter in mammalian cells constitutively expressing T7 RNA polymerase, togeth er with RNA1 to provide the RdRp. Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated fr om complete or partial homodimer templates and from an RNA2-RNA3 heterodime r. (C) 2001 Academic Press.