Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

Abstract

Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCEIntracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.T he actomyosin system is essential for the intracellular transport of endomembranes and other macromolecular complexes and for the spatial arrangement of organelles (1). Trafficking along actin filaments is primarily mediated by molecular motor myosins. Myosins of Arabidopsis thaliana are divided into the two subfamilies, class VIII and class XI myosins. Class XI myosins provide the motive force for the movement of org...

The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus

“…If NSm trafficks along the ER membranes, disruption of the ER network should negatively affect this trafficking. We thus tested the effect of brefeldin A (BFA), a pharmacological drug that at high concentrations can disrupt the integrity of the ER network [ 79 , 80 ], on the structural integrity of the ER network in N . benthamiana leaves expressing mCherry-HDEL.…”

“…Tospovirus NSm is predominantly membrane associated [ 49 , 51 , 58 ]. The VRC of Fig mosaic virus , another multipartite negative-strand plant RNA virus, was also found to traffic intracellularly along the ER and actin microfilaments [ 80 ]; however, little has been known about the mechanism of intercellular movement of multipartite negative-strand RNA plant viruses. Our findings have important new implications to guide future mechanistic studies on intercellular trafficking of tospoviruses and other multipartite negative-strand plant RNA viruses.…”

Pigeonpea sterility mosaic virus: a legume‐infecting Emaravirus from South Asia

“…Recent studies on the macromolecular trafficking of the virus particles of FMV, employing live imaging and ultrastructural analysis, have revealed that the agglomerates of the N protein are passively dragged by the actomyosin‐mediated streaming of the ER, resulting in their rapid movement in plant cells (Ishikawa et al . , ). In virus‐infected cells, the N protein agglomerates are surrounded by ER membranes, which may indicate that they form the basis of enveloped virus particles (Ishikawa et al .…”

“…In virus‐infected cells, the N protein agglomerates are surrounded by ER membranes, which may indicate that they form the basis of enveloped virus particles (Ishikawa et al . , ). The PPSMV N protein may perform similar functions in viral protein trafficking through the ER.…”

Passive virus movements with organelle dynamics

“…We studied intracellular trafficking of a plant RNA virus, fig mosaic virus (FMV), which has enveloped particles similar to animal viruses [ 5 ]. Using confocal scanning laser microscopy (CSLM), we found that the FMV nucleocapsid protein (NP), a major structural protein, forms cytoplasmic agglomerates (hereafter called NP bodies or NBs) that rapidly move along the actin-ER network when ectopically expressed in plant cells.…”