GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

Abstract

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

Plant virus replication and movement

“…Novel findings indicate that the stable association of the p35 MP with the cortical aggregates is facilitated by binding to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which also binds p27 and thus appears to act as an interstitial agent between the p35 MP and the cortical VRCs. Silencing of GAPDH expression inhibited the accumulation of p35 in the cortical VRCs and also reduced the efficiency of virus movement thus indicating that the cortical VRCs are associated with RCNMV movement whereas the larger, perinuclear VRCs, that occur at a later time, rather function to maximize virus reproduction (Kaido et al, 2014). The role of GAPDH in facilitating the association of the p35 MP with the cortical VRCs adds to the potential role of chloroplastic proteins in virus replication as already noted by other studies (Bhat et al, 2013;Cheng et al, 2013;Lin et al, 2007).…”

A novel block of plant virus movement genes

“…Results of intercellular movement studies employing individual MP genes transiently expressed in the absence of virus infection should be interpreted in view of the recently accepted idea that virus replication complexes (and the replicase itself) are involved in the cell‐to‐cell spread of viral genomic RNA. The data supporting this model have been reported for both single gene‐based transport systems (Amari et al ., ; Guenoune‐Gelbart et al ., ; Heinlein, ; Kaido et al ., ; Kawakami et al ., ) and more complex transport systems (Grangeon et al ., ; Park et al ., ; Tilsner et al ., ). However, viral transport may not involve specific protein–protein binding of MPs to replication complexes (Tilsner et al ., ).…”

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

“…One of the candidate proteins is GAPDH-A. Although GAPDH-A is not required for RCNMV replication, it recruits RCNMV movement protein to viral replication sites and plays an important role in virus cell-to-cell movement [59]. Therefore, PA may also function in bridging viral replication and cell-to-cell movement.…”