Analysis Of Wuhan Nodavirus Genomic Structure And Characterization Of Its Nonstructural Protein B2

Wuhan nodavirus (WhNV) was recently isolated from dead larvae of Pieris rapae infected by Granulosis virus (PrGV), in a cabbage field near Wuhan city, Hubei province, China. Based on the physiochemical characterization of WhNV and the nucleotide sequence of its genome, it was classified into the family Nodaviridae.Firstly, the WhNV ProA, B2 and Proa were successfully expressed and purified as fusions with His-tag. We prepared three polyclonal antisera using the three purified recombination proteins, respectively. RT-PCR, western blot and transmission electron microscopy analyses showed that purified WhNV could multiply efficiently in the natural host Pieris rapae larvae under laboratory conditions. WhNV replication in the host cells resulted in the expression of viral proteins, ProA, B2 and Proa, with the absence of B1 production. Northern blot hybridization assay revealed the existence of subgenomic RNA3 which is 5'capped and 3'co-terminal with RNA1. The subgenomic RNA3 which is 370 nucleotides in length contains only one ORF (B2) with two putative in-frame initiation codons locating at nt 2795 and nt 2855 (nomenclature corresponding to RNA1), respectively, and may thus encode two isoforms of B2 (B2-85 and B2-65).The results of site-directed mutagenesis and western blot analysis indicated that the homologous Pr-E cells only synthesize B2-85 in the presence of WhNV RNA3. As a consequence, we conclude that the first AUG codon in WhNV RNA3 is the authentic translation initiation codon. Detection of fluorescent and RT-PCR showed that WhNV B2 protein could inhibit dsRNA and siRNA inducing RNAi in cultured Drosophila cell line S2. The amino-terminal region (aa 1-20) of B2 is essential for this RNAi inhibition activity. Furthermore, RNA binding and Dicer protection assays indicated that the proposal of a mechanism by which WhNV B2 inhibited RNAi at two distinct steps. First, by effectively coating long dsRNAs, B2 prevents their cleavage by Dicer and thus inhibits formation of siRNAs. Secondly, by virtue of its ability to bind tightly to siRNAs, B2 prevents incorporation of siRNAs into multiprotein RNA-inducing silencing complex (RISC) thereby inhibiting cleavage of target RNAs.Because the genomic RNA terminus is very important to virus replication, we re-determined the terminal sequence of WhNV RNA1 and RNA2.5'RACE and 3'RACE showed that WhNV RNA1 and RNA2 consisted of 3151 and 1572 nucleotides, respectively. Based on the newly determined sequences, the pWh1[G,0] and pWh2[G,0], with which the full length WhNV RNA1 and RNA2 containing the authentic termini could be transcribed, respectively, in the presence of T7 RNA polymerase, have been constructed. The results of RT-PCR indicated that the replicon and infectious cDNA clone of WhNV were constructed successfully.