Site-specific binding of polymerase-containing particles of the Giardia lamblia double-stranded RNA virus to the viral plus-strand RNA

Abstract

The non-segmented, double-stranded RNA genome of the Giardia lamblia virus (GLV) contains two genes encoding the major capsid protein (gag) and a fusion of gag with the viral RNA-dependent RNA polymerase (pol). Computer analysis of the viral RNA genome revealed three putative stem-loop structures that were predicted to mediate replication, transcription and packaging of the GLV genomic RNA by binding to the pol domain of the virus-encoded fusion protein. To provide evidence of these postulated RNA/protein interactions, gel retardation assays were employed to examine the potential binding capacity of various viral RNA genome-related sequences to native GLV protein(s). Viral proteins were obtained by disrupting purified GLV particles under low-ionic-strength conditions. The resulting viral protein particles maintained their RNA polymerase activity in the presence of GLV genomic RNA and thus appeared to be suitable tools for the analyses of GLV-protein-mediated binding reactions. A 72-nt short single-stranded in vitro transcript containing a putative stem-loop structure predicted to participate in the packaging of GLV (+)-strand RNA bound specifically to the disrupted virus particles. RNAs containing modified motifs of this stem-loop structure failed to bind to the GLV capsid.