RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus

Abstract

The 63 kDa ' 63K ' movement protein encoded by the triple gene block of poa semilatent virus (PSLV) comprises the C-terminal NTPase/helicase domain and the N-terminal extension domain, which contains two positively charged sequence motifs, A and B. In this study, the in vitro RNAbinding properties of PSLV 63K and its mutants were analysed. Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound RNA at high NaCl concentrations. In contrast, C-63K (isolated NTPase/helicase domain) was able to bind RNA only at NaCl concentrations of up to 50 mM. In gel-shift assays, C-63K bound RNA to form complexes that were unable to enter an agarose gel, whereas complexes formed by N-63K could enter the gel. Full-length 63K formed both types of complexes. Visualization of the RNA-protein complexes formed by 63K, N-63K and C-63K by atomic force microscopy demonstrated that each complex had a different shape. Collectively, these data indicate that 63K has two distinct RNA-binding activities associated with the NTPase/helicase domain and the N-terminal extension domain. Mutations in either of the positively charged sequence motifs A and B had little effect on the RNA binding of the N-terminal extension domain, whereas mutations in both motifs together inhibited RNA binding. Hybrid viruses with mutations in motifs A and B were able to infect inoculated leaves of Nicotiana benthamiana plants, but were unable to move systemically to uninoculated leaves, suggesting that the RNA-binding activity of the N-terminal extension domain of PSLV 63K is associated with virus long-distance movement.

Domain organization of the N-terminal portion of hordeivirus movement protein TGBp1

“…The full-length 63K protein and its separated N-terminal extension region were shown previously by a NorthWestern assay to bind ssRNA efficiently and nonspecifically at NaCl concentrations ranging from 50 to 500 mM (Kalinina et al, 2001), which is in agreement with previous results on the RNA-binding properties of BSMV TGBp1 (Donald et al, 1997). To determine whether the proposed domains of the 63K N-terminal region exhibited a ssRNA-binding activity, the NTD and ID expressed in E. coli as separate polypeptides were immobilized on a nitrocellulose membrane and incubated with a non-specific 32 P-labelled RNA transcript.…”

“…As shown in vitro, two RNA-binding activities of hordeivirus TGBp1 can be involved in the formation of such RNPs, namely those specified by the C-terminal NTPase/helicase domain and the two clusters of positively charged amino acid residues in the protein N-terminal extension region (Donald et al, 1997;Kalinina et al, 2001). These clusters are crucial for both the in vitro RNA-binding ability of the extension region and the long-distance transport of virus during infection, but are not necessary for viral cell-to-cell movement (Kalinina et al, 2001). However, the role of the TGBp1 N-terminal region in viral vascular transport remains unknown.…”

“…Interestingly, BSMV and BNYVV TGBp1s are able to bind both ssRNA and dsRNA (Bleykasten et al, 1996;Donald et al, 1997). A double mutation in PSLV TGBp1 that disrupts two clusters of positively charged amino acids responsible for RNA binding blocks long-distance but not cell-to-cell transport of viral infection (Kalinina et al, 2001), whereas deletion of the 24 aa N-terminal region containing an RNA-binding site prevents BNYVV cell-to-cell transport (Bleykasten et al, 1996). Thus, the RNA-binding ability of the TGBp1 Nterminal extension region is necessary for viral transport in plants.…”

“…Deletion analysis of BSMV TGBp1 has revealed that the protein has multiple RNA-binding sites, some of which map to the N-terminal extension region (Donald et al, 1997). As shown for TGBp1 of poa semilatent virus (PSLV; genus Hordeivirus), the Nterminal extension region expressed as a separate polypeptide binds RNA in a non-cooperative manner (Kalinina et al, 2001). The N-terminal 24 aa of TGBp1 of beet necrotic yellow vein virus (BNYVV; genus Benyvirus), another virus with a hordei-like TGB, also binds RNA (Bleykasten et al, 1996).…”

“…The TGB is a conserved module of overlapping genes found in a number of virus groups (Morozov & Solovyev, 2003). Hordeivirus TGBp1 binds viral genomic RNA forming a cell-to-cell transportcompetent complex (Brakke et al, 1988;Kalinina et al, 2001;Lim et al, 2008;Jackson et al, 2009). TGBp2 and TGBp3 are small membrane proteins necessary for intracellular transport of complexes containing TGBp1 and viral RNA to plasmodesmata (Morozov & Solovyev, 2003;Zamyatnin et al, 2004; Haupt et al, 2005;Jackson et al, 2009 Lim et al, 2008;Jackson et al, 2009).…”