Summary: Plant viruses encode movement proteins (MPs) that ensure the transport of viral genomes through plasmodesmata (PD) and use cell endomembranes, mostly the endoplasmic reticulum (ER), for delivery of viral genomes to PD and formation of PD‐anchored virus replication compartments. Here, we demonstrate that the Hibiscus green spot virus BMB2 MP, an integral ER protein, induces constrictions of ER tubules, decreases the mobility of ER luminal content, and exhibits an affinity to highly curved membranes. These properties are similar to those described for reticulons, cellular proteins that induce membrane curvature to shape the ER tubules. Similar to reticulons, BMB2 adopts a W‐like topology within the ER membrane. BMB2 targets PD and increases their size exclusion limit, and these BMB2 activities correlate with the ability to induce constrictions of ER tubules. We propose that the induction of ER constrictions contributes to the BMB2‐dependent increase in PD permeability and formation of the PD‐associated replication compartments, therefore facilitating the virus intercellular spread. Furthermore, we show that the ER tubule constrictions also occur in cells expressing TGB2, one of the three MPs of Potato virus X (PVX), and in PVX‐infected cells, suggesting that reticulon‐like MPs are employed by diverse RNA viruses.

Plant viruses exploit the endomembrane system of infected cells for their replication and cell-to-cell transport. The replication of viral RNA genomes occurs in the cytoplasm in association with reorganized endomembrane compartments induced by virus-encoded proteins and is coupled with the virus intercellular transport via plasmodesmata that connect neighboring cells in plant tissues. The transport of virus genomes to and through plasmodesmata requires virus-encoded movement proteins (MPs). Distantly related plant viruses encode different MP sets, or virus transport systems, which vary in the number of MPs and their properties, suggesting their functional differences. Here, we discuss two distinct virus transport pathways based on either the modification of the endoplasmic reticulum tubules or the formation of motile vesicles detached from the endoplasmic reticulum and targeted to endosomes. The viruses with the movement proteins encoded by the triple gene block exemplify the first, and the potyviral system is the example of the second type. These transport systems use unrelated mechanisms of endomembrane reorganization. We emphasize that the mode of virus interaction with cell endomembranes determines the mechanism of plant virus cell-to-cell transport.

Movement proteins (MPs) of plant viruses enable the translocation of viral genomes from infected to healthy cells through plasmodesmata (PD). The MPs functions involve the increase of the PD permeability and routing of viral genome both to the PD entrance and through the modified PD. Hibiscus green spot virus encodes two MPs, termed BMB1 and BMB2, which act in concert to accomplish virus cell-to-cell transport. BMB1, representing an NTPase/helicase domain-containing RNA-binding protein, localizes to the cytoplasm and the nucleoplasm. BMB2 is a small hydrophobic protein that interacts with the endoplasmic reticulum (ER) membranes and induces local constrictions of the ER tubules. In plant cells, BMB2 localizes to PD-associated membrane bodies (PAMBs) consisting of modified ER tubules and directs BMB1 to PAMBs. Here, we demonstrate that BMB1 and BMB2 interact in vitro and in vivo, and that their specific interaction is essential for BMB2-directed targeting of BMB1 to PAMBs. Using mutagenesis, we show that the interaction involves the C-terminal BMB1 region and the N-terminal region of BMB2.

Retrozymes are nonautonomous retrotransposons with hammerhead ribozymes in their long terminal repeats (LTRs). Retrozyme transcripts can be self-cleaved by the LTR ribozyme, circularized, and can undergo RNA-to-RNA replication. Here, we demonstrate that the Nicotiana benthamiana genome contains hundreds of retrozyme loci, of which nine represent full-length retrozymes. The LTR contains a promoter directing retrozyme transcription. Although retrozyme RNA is easily detected in plants, the LTR region is heavily methylated, pointing to its transcriptional silencing, which can be mediated by 24 nucleotide-long retrozyme-specific RNAs identified in N. benthamiana. A transcriptome analysis revealed that half of the retrozyme-specific RNAs in plant leaves have no exact matches to genomic retrozyme loci, containing up to 13% mismatches with the closest genomic sequences, and could arise as a result of many rounds of RNA-to-RNA replication leading to error accumulation. Using a cloned retrozyme copy, we show that retrozyme RNA is capable of replication and systemic transport in plants. The presented data suggest that retrozyme loci in the N. benthamiana genome are transcriptionally inactive, and that circular retrozyme RNA can persist in cells due to its RNA-to-RNA replication and be transported systemically, emphasizing functional and, possibly, evolutionary links of retrozymes to viroids—noncoding circular RNAs that infect plants.

Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs.

As a continuation of our previous work, in this paper, we examine in greater detail the genome organization and some protein properties of the members of a potential group named Reclovirids and belonging to Benyviridae-related viruses. It can be proposed that the single-component Reclovirid genomes encode previously undiscovered transport genes. Indeed, analysis of the coding potential of these novel viral genomes reveals one or more cistrons ranging in size from 40 to 80 to about 600 codons, located in the 3′-terminal region of the genomic RNA, encoding proteins with predicted hydrophobic segments that are structurally diverse among Reclovirids and have no analogues in other plant RNA viruses. Additionally, in many cases, the possible methyltransferase domain of Reclovirid replicases is preceded by membrane-embedded protein segments that are not present in annotated members of the Benyviridae family. These observations suggest a general association of most Reclovirid proteins with cell membranes.