Functional Analyses and Identification of Two Arginine Residues Essential to the ATP-Utilizing Activity of the Triple Gene Block Protein 1 of Bamboo Mosaic Potexvirus

Abstract

The TGBp1 of bamboo mosaic potexvirus (BaMV) is encoded by the first overlapping gene of the triple-gene-block (TGB), whose products are thought to play roles in virus movement between plant cells. This protein forms cytoplasmic inclusions associated with virus particles in the BaMV-infected tissues. It has been proposed that the inclusion is one of the active forms of TGBp1. To prove this idea, we purified the TGBp1 inclusions from both the BaMV-infected Chenopodium quinoa and Escherichia coli cells overexpressing this protein to test some of their biochemical activities. We found that the TGBp1 inclusions isolated from the infected plant leaves, but not from E. coli, possess the NTP-binding and NTPase activities. However, they lack the RNA-binding activity possessed by the soluble TGBp1. These results indicate that the TGBp1 proteins in the BaMV-infected tissues assume two different functional forms. Mutational analyses and competition experiments show that the two arginine residues, Arg-16 and Arg-21, essential to RNA binding, are also required for the ATP-utilizing activity of the soluble TGBp1. This indicates that a same-structure motif is required for the two functions of the soluble TGBp1. The location of the two arginine residues outside the seven conserved motifs of the NTP-utilizing superfamily I RNA helicases, to which TGBp1 belongs, suggests that an extra-structure motif, besides the seven conserved ones, is required for the NTP-utilizing activity of the TGBp1 protein of BaMV.

Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement

“…With the aid of infectious plasmid clones of BaMV, we have shown that a single or double ArgRAla substitution at positions 16 and 21, which diminishes the RNA-binding and ATP-utilizing activities of TGBp1 (Wung et al, 1999;Liou et al, 2000), also eliminates the ability of BaMV to move from cell to cell. Furthermore, our ability to transcomplement the mutant TGBp1 with its wild-type counterpart supports the idea that the RNA-binding activity and the NTPase activity of TGBp1 are necessary for the movement of potexviruses from cell to cell.…”

“…TGBp1 can move from cell to cell (Lough et al, 1998;Yang et al, 2000) and mediates cell-to-cell movement of TGBp2 and TGBp3 (Krishnamurthy et al, 2002). Further analysis of the soluble TGBp1 of Bamboo mosaic virus (BaMV) has revealed that Arg-11, Arg-16 and Arg-21 in the N-terminal region of this protein are essential for its RNA-binding activity (Liou et al, 2000). Although BaMV TGBp1 belongs to the RNA helicase superfamily I (Kadare & Haenni, 1997;Morozov et al, 1999;Liou et al, 2000), Arg-16 and Arg-21, which are located outside the seven conserved motifs of the superfamily I RNA helicase, are also critical for the NTPase activity of TGBp1 (Liou et al, 2000).…”

“…Further analysis of the soluble TGBp1 of Bamboo mosaic virus (BaMV) has revealed that Arg-11, Arg-16 and Arg-21 in the N-terminal region of this protein are essential for its RNA-binding activity (Liou et al, 2000). Although BaMV TGBp1 belongs to the RNA helicase superfamily I (Kadare & Haenni, 1997;Morozov et al, 1999;Liou et al, 2000), Arg-16 and Arg-21, which are located outside the seven conserved motifs of the superfamily I RNA helicase, are also critical for the NTPase activity of TGBp1 (Liou et al, 2000). TGBp1s of potexviruses are in fact mainly associated with cytoplasmic inclusions in infected tissues (Chang et al, 1997;Davies et al, 1993;Rouleau et al, 1994).…”

“…TGBp1s of potexviruses are in fact mainly associated with cytoplasmic inclusions in infected tissues (Chang et al, 1997;Davies et al, 1993;Rouleau et al, 1994). The TGBp1 obtained from the cytoplasmic inclusions also possesses NTP-binding and NTPase activities but lacks the RNA-binding activity (Liou et al, 2000).…”

“…With the aid of the infectious cDNA clones of BaMV-S and a satellite replicon based on satBaMV (Lin et al, 1996), we have demonstrated that Arg-16 and Arg-21 in the N-terminal region of the BaMV TGBp1, residues that are essential for the RNAbinding and NTPase activities of TGBp1 (Wung et al, 1999;Liou et al, 2000), are also crucial for cell-to-cell movement of BaMV in host plants.…”