The Stable Association of Virion with the Triple-gene-block Protein 3-based Complex of Bamboo mosaic virus

Abstract

The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

The Nucleolar Fibrillarin Protein Is Required for Helper Virus-Independent Long-Distance Trafficking of a Subviral Satellite RNA in Plants

“…Like potexviruses, all three TGBps and CP are required for BaMV cell-to-cell and systemic movement (Lin et al, 2004Lan et al, 2010;Lee et al, 2011;Wu et al, 2011;Chou et al, 2013). To examine the interaction of TGBp3 with P20 and/or fibrillarin, we inoculated N. benthamiana leaves with a 35S promoter-driven HV derivative (pCB-P3HA) carrying a TGBp3:: HA fusion ( Figure 6C; Chou et al, 2013) with or without the satBaMV pCBSF4 plasmid (Lin et al, 2004).…”

“…To examine the interaction of TGBp3 with P20 and/or fibrillarin, we inoculated N. benthamiana leaves with a 35S promoter-driven HV derivative (pCB-P3HA) carrying a TGBp3:: HA fusion ( Figure 6C; Chou et al, 2013) with or without the satBaMV pCBSF4 plasmid (Lin et al, 2004). Co-IP assays with an HA antibody precipitated TGBp3HA, TGBp1, TGBp2, and CP but not P20 or fibrillarin in fractions, from pCB-P3HA-infected plants ( Figure 6C, lane 4).…”

“…(C) Genomic map of BaMV infectious clone pCB-P3HA (Chou et al, 2013). N. benthamiana plants were inoculated with wild-type pCB (B), pCB + pCBSF4 (B +S), or pCB-P3HA (B-P3HA), or pCB-P3HA + pCBSF4 (B-P3HA+S).…”

“…For transient expression of mCherry-NbFIB2 in N. benthamiana, we constructed pBIN-mCherryNbFIB2. The mCherry DNA fragment was first amplified from pBA-mCh-p1 (Chou et al, 2013) using primers mCherry-SmaI-F and mCherry-KpnI-R (Supplemental Table 3) and then cloned into the pCass plasmid (Ding et al, 1995) at the SmaI and KpnI sites to generate pCass-mCherry. Then, the FIB2 coding region was amplified from N. benthamiana cDNA using primers NbFIB2-KpnI-F and NbFIB2-EcoRI-R (Supplemental Table 3).…”

“…BaMV HV is a member of the Potexvirus genus that contains a single-stranded, positive-sense RNA genome with five open reading frames. These open reading frames encode a replicase, three MPs (TGBp1, TGBp2, and TGBp3, encoded by a triple gene block), and a capsid protein (CP) , each required for cell-to-cell movement (Lin et al, 2004Lan et al, 2010;Lee et al, 2011;Wu et al, 2011;Chou et al, 2013). However, the factors involved in long-distance trafficking of BaMV remain to be determined.…”