Nucleic Acid Binding Activity of Pns6 Encoded by Genome Segment 6 of Rice Ragged Stunt Oryzavirus

Abstract

The ORF of genome segment 6 (S6) of rice ragged stunt oryzavirus (RRSV) Philippines isolate was cloned and sequenced based on the S6 sequence of the Thailand isolate. Pns6, the 71 kD product of S6 expressed in E. coli, was demonstrated to be a viral non-structural protein of RRSV by Western blotting. The gel mobility shift assays showed that Pns6 had nucleic acid binding activity. Pns6 could interact with single-and double-stranded forms of DNA and RNA, showing a preference for single-stranded nucleic acid and a slight preference for RRSV ssRNA over the rice ssRNA, as demonstrated by both competition and displacement assays. The binding of Pns6 to nucleic acids is strong and sequence non-specific. By using five truncated derivatives of Pns6, it was found that the basic region from amino acid 201 to 273 of Pns6 was the unique nucleic acid binding domain. Subcellular fractionation of leaf tissues of RRSV-infected rice plants and subsequent Western blotting had shown that Pns6 accumulated predominately in the cytoplasmic membrane fraction. The possible role of RRSV Pns6 in virus replication and assembly is discussed.

Development of Continuous Cell Culture of Brown Planthopper To Trace the Early Infection Process of Oryzaviruses in Insect Vector Cells

“…Previous data showed that Pns6 is a viral movement protein and a viral RNA-silencing suppressor in plant hosts (16,17). Furthermore, Pns6 of RRSV binds to both dsRNA and ssRNA in a sequence-independent manner, showing a preference for RRSV ssRNA over the rice ssRNA (27). Here, we showed that Pns6 rather than Pns10 specifically interacted with core capsid P3 (Fig.…”

p2 of Rice grassy stunt virus (RGSV) and p6 and p9 of Rice ragged stunt virus (RRSV) isolates from Vietnam exert suppressor activity on the RNA silencing pathway

“…The GFP and U6 probes were end-labeled with [c-32 P] ATP using T4 polynucleotide kinase (Promega, http://www. promega.com), and northern blots were performed as described by Lacombe et al [32]. U6 hybridization provides a control for RNA loading of the gels Fig.…”

“…The RNA 7 (s7gp1) and 10 (s10gp1) segments encode non-structural proteins p7 and p10 with molecular weights (MW) of 68 and 33 kDa, respectively [28], the RNA 5 segment (s5gp) encodes the 91-kDa structural protein p5 [29], and the RNA 8 segment (s8gp1) encodes the 67-kDa structural protein p8, which is endowed with self-aggregation and self-cleavage abilities [30,31]. The p9 protein encoded by s9gp1 is a 39-kDa protein that contributes largely as a viral spike protein and plays an important role in virus transmission by the insect vector [32,33].…”

Recent progress in research on cell-to-cell movement of rice viruses

“…The functioning of the non-structural Pns6 protein of RRSV in viral cell-to-cell movement has been confirmed by transient-expression experiments with the GFP-fused Pns6 protein of RRSV in epidermal cells of Nicotiana benthamiana and by trans -complementation experiments using a movement-defective TMV mutant in Nicotiana tabacum (Shao et al, 2004; Wu et al, 2010). Pns6 has a sequence-non-specific binding of single- and double-stranded forms of DNAs and RNAs, but binds sequence-specifically to single-stranded forms of the viral genome, and its binding domain was also determined to be located between amino acids 201 and 273 of the Pns6 of RRSV (Shao et al, 2004).…”

“…Pns6 has a sequence-non-specific binding of single- and double-stranded forms of DNAs and RNAs, but binds sequence-specifically to single-stranded forms of the viral genome, and its binding domain was also determined to be located between amino acids 201 and 273 of the Pns6 of RRSV (Shao et al, 2004). …”