Matrix‐glycoprotein interactions required for budding of a plant nucleorhabdovirus and induction of inner nuclear membrane invagination

Abstract

Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein-nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino-terminal (NT) or carboxyl-terminal (CT) domain. Electron microscopy and sucrose gradient centrifugation analyses showed that the CT domain is essential for virion morphogenesis whereas the NT domain is also required for efficient budding. SYNV infection induces IM invaginations that are thought to provide membrane sites for virus budding. We found that in the context of viral infections, interactions of the M protein with the CT domain of the membrane-anchored G protein mediate M protein translocation and IM invagination. Interestingly, tethering the M protein to endomembranes, either by co-expression with a transmembrane G protein CT domain or by artificial fusion with the G protein membrane targeting sequence, induces IM invagination in uninfected cells. Further evidence to support functions of G-M interactions in virus budding came from dominant negative effects on SYNV-induced IM invagination and viral infections that were elicited by expression of a soluble version of the G protein CT domain. Based on these data, we propose that cooperative G-M interactions promote efficient SYNV budding.

Specificity of Plant Rhabdovirus Cell-to-Cell Movement

“…Transient expression plasmids and BiFC plasmids were introduced into A. tumefaciens strain EHA105 by electroporation, followed by agroinfiltration of wild-type or transgenic RFP-H2B N. benthamiana plants. BiFC assays were carried out essentially as described previously (71). Epifluorescence microscopy was performed with a Zeiss Lumar V12 stereomicroscope.…”

The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

“…To further investigate involvement of the M protein in SYNV SIE, we tested whether an rSYNV-RFP mutant with a deletion in the M gene (rSYNV-RFP-ΔM) affects SIE by analyzing the ability of the mutant virus to prevent superinfection by rSYNV-GFP. As a control, the SYNV G deletion mutant (rSYNV-RFP-ΔG) was included in this experiment because the G and M proteins have a collaborative role in virion budding and maturation (54). Therefore, N. benthamiana leaves were infiltrated with mixtures of agrobacteria harboring plasmids required for simultaneous rescue of rSYNV-GFP and either rSYNV-RFP, rSYNV-RFP-ΔM, or rSYNV-RFP-ΔG.…”

“…Plasmids and recombinant viruses. Plasmids used to recover rSYNV-GFP, rSYNV-RFP, rSYNV-RFPΔM, and rSYNV-RFPΔG have been described in previous studies (51,54). The PVX-GFP plasmid was constructed as described by van Wezel et al (85).…”

“…The recovered PCR products were then inserted into the pGD binary vector (86) by In-Fusion cloning (Clontech). The pGD-CFP-M plasmid used for transient expression of the CFP-M fusion has been described previously (54) and was used to generate the expression plasmids pGD-CFP-M RARA and pGD-CFP-M AAAA .…”

“…During the later stages of replication, accumulating M proteins presumably function to coil gNCs and assemble into viral cores. Through interactions between the M and G protein carboxyl-terminal tail, the condensed NC cores are directed to inner nuclear membrane surfaces from which they bud to form enveloped bullet-shaped or bacilliform particles that accumulate in the perinuclear spaces (53,54).…”