Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes.
Replication of all known positive-strand RNA viruses occurs in replication complexes associated with intracellular membranes. The putative nucleoside triphosphate binding (NTB) protein of Tomato ringspot virus (ToRSV) contains a stretch of hydrophobic residues at its C terminus, suggesting that it may act as a membrane anchor for the replication complex. Anti-NTB antibodies detected two predominant proteins in membrane-enriched fractions (the 66-kDa NTB and 69-kDa NTB-VPg proteins) along with other, larger proteins. The proteins containing the NTB domain cofractionated with markers of the endoplasmic reticulum (ER) and with ToRSV-specific RNA-dependent RNA polymerase activity in sucrose gradients. ToRSV infection induced severe changes in the morphology of the ER in plants expressing an ER-targeted green fluorescent protein (ER-GFP), and proteins containing the NTB domain colocalized with ER-GFP in indirect immunofluorescence assays. The proteins containing the NTB domain have properties of integral membrane proteins. Proteinase K protection assays using purified membranes from infected plants revealed that although the central portion of the NTB domain is exposed to the cytoplasmic face of the membranes, an 8-kDa fragment, recognized by anti-VPg antibodies, is protected by the membranes. This fragment probably consists of the 3-kDa VPg and the 5-kDa stretch of hydrophobic residues at the C terminus of the NTB protein, suggesting a luminal location for the VPg in at least a portion of the molecules. These results provide evidence that proteins containing the NTB domain are transmembrane proteins associated with ER-derived membranes and support the hypothesis that one or several of the proteins containing the NTB domain anchor the replication complex to the ER.
Han
Sumin
S
Department of Botany, University of British Columbia, Vancouver, Canada.
Sanfaçon
Hélène
H
D001665:Binding Sites; D004721:Endoplasmic Reticulum / Q000821:virology*; D018551:Solanum lycopersicum / Q000821:virology*; D017860:Nepovirus / Q000737:chemistry* / Q000502:physiology*; D009711:Nucleotides / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D014758:Viral Core Proteins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D014763:Viral Matrix Proteins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D014779:Virus Replication
D009711:Nucleotides; D012367:RNA, Viral; D014758:Viral Core Proteins; D014763:Viral Matrix Proteins