Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus.

Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.

The hydrophobic segment of Potato virus X TGBp3 is a major determinant of the protein intracellular trafficking.

Potato virus X (PVX) encodes three movement proteins, TGBp1, TGBp2 and TGBp3. The 8 kDa TGBp3 is a membrane-embedded protein that has an N-terminal hydrophobic sequence segment and a hydrophilic C terminus. TGBp3 mutants with deletions in the C-terminal hydrophilic region retain the ability to be targeted to cell peripheral structures and to support limited PVX cell-to-cell movement, suggesting that the basic TGBp3 functions are associated with its N-terminal transmembrane region. Fusion of green fluorescent protein to the TGBp3 N terminus abrogates protein activities in intracellular trafficking and virus movement. The intracellular transport of TGBp3 from sites of its synthesis in the rough endoplasmic reticulum (ER) to ER-derived peripheral bodies involves a non-conventional COPII-independent pathway. However, integrity of the C-terminal hydrophilic sequence is required for entrance to this non-canonical route.

Localization of Poa semilatent virus cysteine-rich protein in peroxisomes is dispensable for its ability to suppress RNA silencing.

Subcellular localization of the Poa semilatent virus cysteine-rich gammab protein was studied by using different approaches. In infected tissue, gammab was detected mainly in the P30 fraction as monomers, dimers and oligomers. Green fluorescent protein-fused gammab was found to localize in punctate bodies in the cytoplasm. Colocalization with marker proteins demonstrated that these bodies represent peroxisomes. Immunoelectron microscopy revealed that gammab was localized in the peroxisomal matrix and that localization of gammab in peroxisomes required the C-terminal signal tripeptide SKL. An SKL-deletion mutant exhibited a diffuse localization, but retained the protein's ability to suppress RNA silencing, determine infection phenotype and support virus systemic spread. These data indicate that gammab functions are not associated with the protein's localization to peroxisomes.