A Host-Factor Interaction and Localization Map for a Plant-Adapted Rhabdovirus Implicates Cytoplasm-Tethered Transcription Activators in Cell-to-Cell Movement

Abstract

To identify host factors that play critical roles in processes, including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution Nicotiana benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor. Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

“…7b in Martin et al, 2012). In this study, interaction between ADV N and P was also demonstrated by BiFC, where the location of the N:P complex was subnuclear, similar to that reported for SYNV, PYDV, CoRSV and OFV (Bandyopadhyay et al, 2010;Min et al, 2010;Kondo et al, 2013;Ramalho et al, 2014), but different to that observed for LNYV, which was in the cytoplasm . The BiFC results for ADV N and P proteins confirmed the earlier observed co-localization results for these proteins, and showed that the interaction was similar to that reported for these two proteins from nucleorhabdoviruses rather than cytorhabdoviruses.…”

“…The self-interaction of P protein was also reported for SYNV (Min et al, 2010) and LNYV , however was not observed for PYDV (Bandyopadhyay et al, 2010). The subnuclear localization of the ADV P:P complex (Fig.…”

“…The subnuclear localization of the ADV P:P complex (Fig. 4B) resembles that of the sole expression of GFP-P, and is different to that observed for LNYV P at the cell periphery and both nuclear and cytoplasmic localization for SYNV P (Min et al, 2010).…”

“…The construction of such a map is critical for understanding molecular mechanisms that underlie viral infection and transmission processes for negative-sense RNA viruses like ADV, for which reverse genetic systems are not yet available (Min et al, 2010;Mann and Dietzgen, 2014). When direct bilateral protein-protein interactions were tested for ADV, one homotypic and three heterotypic interactions were detected, all involving the P protein (Fig.…”

“…The interaction of N and P proteins is a common feature among not only the already characterized LNYV , PYDV (Bandyopadhyay et al, 2010) and SYNV (Min et al, 2010) but also the recently characterized dichorhaviruses coffee ringspot virus (CoRSV) (Ramalho et al, 2014) and orchid fleck virus (OFV) (Kondo et al, 2013), which have bipartite, negative-strand RNA genomes with clear sequence similarities to those of nucleorhabdoviruses, and the vertebrate rhabdovirus, vesicular stomatitis virus (VSV) (Moerdyk-Schauwecker et al, 2009; Fig. 7b in Martin et al, 2012).…”