Cocksfoot mottle sobemovirus coat protein contains two nuclear localization signals

Abstract

Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized to the cytoplasm and to the nucleus in plant cells indicating the involvement of the motif in nuclear localization. In mammalian cells, mutations in the arginine-rich region were sufficient to completely abolish nuclear transport. The analysis of deletions of amino acid residues 1-11, 1-22, and 22-33 of CP demonstrated that there were two separate nuclear localization signals (NLS) within the N-terminus--a strong NLS1 in the arginine-rich region (residues 22-33) and a weaker NLS2 within residues 1-22. Analysis of point mutants revealed that the basic amino acid residues in the region of the two NLSs were individually not sufficient to direct CP to the nucleus. Additional microinjection studies with fluorescently labeled RNA and CP purified from CfMV particles demonstrated that the wild-type CP was capable of transporting the RNA to the nucleus. This feature was not sequence-specific in transient assays since both CfMV and GFP mRNA were transported to the cell nucleus by CfMV CP. Together the results suggest that the nucleus may be involved in CfMV infection.

Cocksfoot mottle virus coat protein is dispensable for the systemic infection

“…Results obtained here, with viruses containing the same mutations in CP, demonstrate that these mutations have no deleterious effect on virus cell-to-cell and systemic movement as well as on mechanical transmission in oats. The majority of CP-EGFP fusion protein localises to the cell nucleus when expressed independently from the rest of the virus genome [13]. Here we observed that when CP-EGFP was expressed together with the rest of the viral proteins, the fluorescence did not accumulate in the nucleus, but remained evenly distributed between cytoplasm and nucleus.…”

“…The CP of mutants R3L and R5X localises to cytoplasm and nucleus whereas the wt CP localises almost exclusively to the nucleus [13]. Results obtained here, with viruses containing the same mutations in CP, demonstrate that these mutations have no deleterious effect on virus cell-to-cell and systemic movement as well as on mechanical transmission in oats.…”

“…The N-terminal part of all sobemoviral CPs is rich in basic amino acids and contains an arginine-rich region, which is necessary for CP-RNA interactions and RNA encapsidation [9-11]. It has also been shown that the N-terminal part of Southern cowpea mosaic virus (SCPMV) CP interacts with membranes [12] and that the N-terminus of Cocksfoot mottle virus (CfMV) CP contains functional nuclear localisation signals [13]. CPs have also been reported to be involved in virus movement.…”

Reverse Genetic Analysis of Ourmiaviruses Reveals the Nucleolar Localization of the Coat Protein in Nicotiana benthamiana and Unusual Requirements for Virion Formation

“…Studies on RNA plant viruses have reported the presence of virus-encoded proteins in the nucleus: in the Cucumber mosaic virus (CMV), the silencing suppressor protein 2b displayed nuclear localization (36); the Tombusvirus p19 is also translocated by plant ALY proteins to the nucleus (13). In other RNA plant virus systems, such as sobemovirus, umbravirus, potyvirus, luteoviruses, and the Satellite panicum mosaic virus (SPMV), the CP itself is localized to the nucleus (20,30,47,49,56). To date, the function of nucleolar localization has been shown in only two systems: in umbraviruses, ORF3 localization to the nucleolus was described as being necessary for long-distance virus spread (31).…”

Virus Operation Control Centers

“…Grapevine rupestris stem pitting-associated virus CP has a signal that brings it into the plant cell nucleus . Similarly, Cocksfoot mottle virus (CfMV) CP has nuclear localization signals (Olspert et al 2010). The role of these localizations is unclear.…”