A 38-Amino-Acid Sequence Encompassing the Arm Domain of the Cucumber Necrosis Virus Coat Protein Functions as a Chloroplast Transit Peptide in Infected Plants

Abstract

Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed.

Induction of Particle Polymorphism by Cucumber Necrosis Virus Coat Protein Mutants In Vivo

“…Such studies should therefore assist in future work aimed at elucidating the in vivo assembly process. In addition, as suggested in a previous study, specific CNV CP sequences along with specific host factors have been implicated in the CNV assembly/disassembly pathway (41). It is anticipated that the studies described here should facilitate further analyses of these aspects of the assembly process.…”

“…Virus particles were denatured and electrophoresed through NuPage BisTris gels (Invitrogen) essentially according to the manufacturer's recommended conditions. Proteins were blotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and probed with a mixture of three polyclonal antibodies known to be specific to the CNV S-and P domains (41) c The approximate Tϭ3:IS:Tϭ1 particle ratio was assessed by TEM of uranyl acetate-stained particles. At least 250 particles were examined using several different areas of one or more TEM grids.…”

Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection

“…Coat protein-GFP fusion protein constructs have also been shown to target mitochondria, wherein 38 aa of the N-terminal region of the CP are sufficient for mitochondrial targeting (39). We have previously shown that 3 specific cleavage events occur during chloroplast targeting of the CNV CP (38,39) (Fig. 1B).…”

“…Approximately 1 to 5% of the CNV CP in a CNV-infected cell is present in chloroplasts (38). Although the biological significance of chloroplast targeting is not known, it was suggested that CNV particles may uncoat on the cytoplasmic side of the chloroplast outer membrane as CP subunits are reeled into the chloroplast during CP uptake (38).…”

“…Although the biological significance of chloroplast targeting is not known, it was suggested that CNV particles may uncoat on the cytoplasmic side of the chloroplast outer membrane as CP subunits are reeled into the chloroplast during CP uptake (38). Using green fluorescent protein (GFP) fusion protein constructs, it has been determined that most of the arm domain plus 4 aa of the S domain are minimally required for chloroplast targeting (38).…”