Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement, Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

Abstract

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-a family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-a paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-a in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-a-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-a interaction in plants, nucleolar targeting, and long-distance movement.

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell‐to‐cell movement of Barley stripe mosaic virus

“…For example, GRV ORF3 contains functional NLS and NoLS sequences; moreover, substitution of the NLS arginine residues with alanine abolishes ORF3 nuclear transit, whereas NoLS leucine mutations interfere with ORF3 nucleolar localization (Kim et al, 2007b). PMTV TGB1 contains two NoLSs responsible for nucleolar localization (Lukhovitskaya et al, 2015). In contrast, the PSLV TGB1 Nterminal domain (NTD) contains a putative NLS (Cluster A, amino acids 116-125) required for nuclear localization and a putative NoLS (Cluster B, amino acids 175-187), and mutations within these regions result in the redistribution of most of the NTD from the nucleolus to the nucleoplasm (Semashko et al, 2012).…”

“…PMTV TGB1 also contains two NoLSs, NoLS-A and NoLS-B. However, the NoLS-A and NoLS-B mutations do not affect viral accumulation in inoculated leaves, and the NoLS-A M mutant and WT virus accumulate to similar levels in the upper leaves, whereas accumulation of the NoLS-B M mutant is only slightly reduced (Lukhovitskaya et al, 2015). Nevertheless, RNA-TGB1 of the double mutant virus, NoLS-AB M , could be detected in the upper leaves, but RNA-CP could not be detected, and PMTV (Fig.…”

“…Beet black scorch virus (BBSV) p7a also localizes to the nucleolus and interacts with Fib2 (Wang et al, 2012). In addition, the Triple Gene Block1 (TGB1) proteins of Potato mop-top virus (PMTV) and Poa semilatent virus (PSLV) also colocalize with Fib2 in the nucleolus, but the effects of these interactions on infectious processes are unknown (Lukhovitskaya et al, 2015;Semashko et al, 2012).…”

In vitro properties of hordeivirus TGB1 protein forming ribonucleoprotein complexes

“…Mutations disrupting the structure or function of the ID abolish virus movement (Donald et al, 1997;Solovyev et al, 1999). The recent data of Lukhovitskaya et al (2015) provide additional evidence in favour of our suggestion. They indicated that ID is required for PMTV TGBp1 self-interaction and cell-to-cell movement.…”

“…NTD deletion is dispensable for these functions. At the same time, PMTV NTD is required for importin-a interaction in plants, nucleolar targeting and long-distance movement (Wright et al, 2010;Lukhovitskaya et al, 2015). In our previous works, we also demonstrated that PSLV NTD is needed for long-distance movement , localization in the nucleus/nucleolus and interaction with two nuclear proteins, i.e.…”