Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

Abstract

Background: Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined.

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

“…Bright Yellow-2 (BY-2)) suspension-cultured cells in standard Murashige-Skoog (MS) medium and transient co-transformation of cells with 4 g of plasmid DNA using a PDS1000 biolistic particle delivery system (Bio-Rad Laboratories) were performed as described previously (36). For localization experiments, cells were resuspended in transformation buffer (MS medium plus 0.25 M sorbitol and 0.25 M mannitol), spread onto Whatman filter paper prewetted with transformation buffer, biolistically bombarded, and then incubated for ϳ3-4 h to allow for expression and sorting of expressed proteins.…”

A Unique N-Terminal Sequence in the Carnation Italian ringspot virus p36 Replicase-Associated Protein Interacts with the Host Cell ESCRT-I Component Vps23

“…Plant expression plasmids used in transient transformations of Nicotiana tabacum Bright Yellow-2 (BY-2) cells include the following plasmids that have been described previously (11,35): pRTL2/p36, encoding CIRV p36; pRTL2/Myc-p36, encoding p36 with an N-terminal Myc epitope tag; pRTL2/p95, encoding CIRV p95, in which the p36 amber stop codon was mutated to a tyrosine codon; pRTL2/Rep, encoding the overlapping CIRV open reading frame 1 (ORF1) and ORF2 that encode both p36 and p95; p36 1-90 -CAT and p36 -CAT, encoding the N-terminal 90 or 90 to 190 amino acid residues of p36 fused to the N terminus of chloramphenicol acetyltransferase (CAT), respectively; pRTL2/Myc-Vps23, pRTL2/GFPVps23, pUC18/Vps23-GFP, and pRTL2/HA-Vps23, encoding either the Myc or hemagglutinin (HA) epitope tag or the green fluorescent protein fused to the N or C terminus of isoform A of Arabidopsis Vps23 (referred to as Vps23 in this study); pRLT2/Myc-Vps28 and pRTL2/Myc-Vps25, encoding N-terminal Myc-tagged Arabidopsis Vps28 isoform A and Arabidopsis Vps25, respectively. Other previously described plant expression vectors include the following plasmids: pRTL2/RFP, encoding the red fluorescent protein (RFP) (36); pUC18/GFP-Syp21 and pUC18/GFPSyp52, which encode GFP fused to the N termini of the Arabidopsis membrane-bound Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) Syp21 (Syntaxin of plants 21) and Syp52 (37) proteins (kindly provided by M. Sato [Kyoto University]); pSAT2/Cherry-PTS1, encoding the monomeric cherry fluorescent protein fused to the C-terminal 10 amino acid residues of pumpkin hydroxypyruvate reductase, including its type 1 peroxisomal targeting signal (38); pRTL2/TIC40-RFP, encoding the Arabidopsis 40-kDa component of the translocon at the inner membrane of chloroplasts fused to the N terminus of RFP (39); and pSAT4A/AtPAP26-mCherry, encoding the Arabidopsis purple acid phosphatase isoform 26 fused to the N terminus of the monomeric cherry fluorescent protein (40).…”

“…pRTL2/p33 (4). Rub inoculations were performed 2 days after infiltration with 10 g of plasmid DNA encoding full-length infectious TBSV or CIRV cDNA diluted in 30 l of RNA inoculation buffer (11,44). Approximately 4 to 6 days after inoculation or, for experiments involving TRV, 2 or 4 days after infiltration, the leaves were flash frozen in liquid nitrogen followed by total RNA extraction (45).…”

“…Given the ultrastructural similarities of the modified peroxisomal and mitochondrial membranes in TBSV-and CIRV-infected cells, respectively, as well as the ability of chimeric versions of these two viruses to produce the corresponding organelle-specific membrane rearrangements (5,11,26), it seems likely that CIRV relies on ESCRT in a manner similar to TBSV. However, and NP_062898.1) and aligned using ClustalW.…”

“…Consequently, the N-terminal portion of p92/p95 is identical to p33/p36. Both sets of replicase proteins are also integral membrane proteins, each possessing two transmembrane domains (TMDs), as well as unique targeting signals that mediate their specific sorting to either peroxisomes or mitochondria (4,10,11) and thus dictate the intracellular site for viral replication.…”