An integrated protein localization and interaction map for Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus

Abstract

The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species), was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3′-N-X-P-Y-M-G-L-5′, which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3′ leader RNA of 149 nt and a 5′ trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine the subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.

Complete genome sequence and intracellular protein localization of Datura yellow vein nucleorhabdovirus

“…However, DYVV 3 /5 genome complementarity is more extensive and features a short 5 overhang. Such 1 or 2 overhanging bases have been reported previously at either the 3 or 5 genome ends of some nucleo-and cytorhabdoviruses (Heim et al, 2008;Bandyopadhyay et al, 2010;Yan et al, 2015).…”

“…GFP-M accumulation in the nucleus partly excluded RFP-H2B, which was shifted in its localization towards the nuclear periphery (Fig. S1), an effect similar to that seen when GFP-M of potato yellow dwarf nucleorhabdovirus (PYDV) was expressed (Bandyopadhyay et al, 2010). GFP-G was found predominantly in nuclear membranes and to a lesser extent in cytoplasmic ER, colocalized with mCherry-ER marker.…”

“…N protein of SYNV also localizes exclusively in the nucleus and contains an experimentally verified bipartite NLS at the C-terminus (Goodin et al, 2001), similar to that predicted here for DYVV N protein. The N proteins of the nucleorhabdoviruses PYDV and maize fine streak virus (MFSV) also have an exclusive nuclear localization (Bandyopadhyay et al, 2010;Tsai et al, 2005), unlike N protein of the cytorhabdovirus lettuce necrotic yellows virus (LNYV), which is localized exclusively in the cell periphery as component of the cytoplasmic viroplasm (Martin et al, 2012). On the other hand, N proteins of the dichorhaviruses orchid fleck virus (OFV) and coffee ringspot virus (CoRSV) which are related to nucleorhabdoviruses, but have a bipartite genome, are distributed throughout the plant cell and imported into the nucleus through interactions with the exclusively nuclear P proteins (Kondo et al, 2013;Ramalho et al, 2014).…”

Orchid Fleck Virus Structural Proteins N and P Form Intranuclear Viroplasm-Like Structures in the Absence of Viral Infection

“…This was not reproduced when N and P were individually expressed, indicating that both proteins are essential for the formation of VpLSs and, most likely, the viroplasm. In some nucleorhabdoviruses, such as SYNV (19,23) and PYDV (17,18,42), coexpression of N and P results in a localization shift of both proteins into a large nuclear viroplasm-like focus. Similarly, a prototype cytorhabdovirus, lettuce necrotic yellows virus (LNYV), forms cytoplasmic viroplasm-like aggregates through coexpression of its N and P (43).…”

Sensing of biomolecular interactions using fluorescence complementing systems in living cells

“…BiFC technology has also been utilized in study of plant viruses, such as in vitro and in vivo mapping of the Prunus necrotic ring spot virus coat protein C-terminal dimerization domain (Aparicio et al, 2006), visualization of the interaction between the precursors of the viral protein linked to the genome of Turnip mosaic virus and the translation eukaryotic initiation factor iso 4E in plants (Beauchemin et al, 2007), interaction of the helper component-proteinase of Potato virus Y interacts with the chloroplast division-related factor of tobacco (Jin et al, 2007), live-cell imaging of viral RNA genomes of tobacco mosaic virus (TMV) (Tilsner et al, 2009), an integrated protein localization and interaction map for Potato yellow dwarf virus (Bandyopadhyay et al, 2010), nuclear localization of Beet black scorch virus capsid protein and its interaction with importin alpha , interaction of the trans-frame Potyvirus protein with host protein PCaP1 and its role in facilitating Potyvirus movement (Vijayapalani et al, 2012), in vivo membrane association of the movement protein (NSm) of the tospovirus species Bean necrotic mosaic virus, Chrysanthemum stem necrosis virus, Tomato chlorotic spot virus and Tomato spotted wilt virus and the homologous and heterologous interactions among NSm and nucleocapsid protein (Leastro et al, 2015).…”