Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation

Abstract

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus , the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. NMR analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a GFP-fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA co-located during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions and in vitro high molecular weight complexes were formed by both replication-competent and -incompetent viral replicons, and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales . Substitution of the proline residue but not the other residues necessary for replication allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that a perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Plantago asiatica mosaic virus: An emerging plant virus causing necrosis in lilies and a new model RNA virus for molecular research

“…The PMTC is probably subsequently targeted to cellular membranes, possibly the endoplasmic reticulum (ER), to form a mature virus replication complex (VRC). Recently, membrane targeting was shown to be mediated by an amphipathic α‐helix located downstream from the methyltransferase domain (Figure 2a; Komatsu et al, 2021). GFP fusion to the methyltransferase domain forms a large perinuclear complex, possibly representing the VRC of PlAMV, which was disrupted by mutations in the conserved hydrophobic amino acids of this α‐helix.…”

“…GFP fusion to the methyltransferase domain forms a large perinuclear complex, possibly representing the VRC of PlAMV, which was disrupted by mutations in the conserved hydrophobic amino acids of this α‐helix. Interestingly, mutation of a proline residue of this membrane‐associated helix, which is strictly conserved in potexviruses and forms a kink in the helix, hinders virus replication but does not affect the formation of the large complex (Komatsu et al, 2021). This finding implicates the proline residue in the interaction of the amphipathic α‐helix with host factors required for the activation of the VRC.…”

“…Confocal laser scanning microscopy has revealed that the replicase of PVX is associated with ER membranes (Tilsner et al, 2013). Similarly, membrane association is important for replication of PlAMV (Komatsu et al, 2021; Yoshida et al, 2019). Membrane‐associated replication can cause elevated membrane stress.…”

The N-Terminal α-Helix of Potato Virus X-Encoded RNA-Dependent RNA Polymerase Is Required for Membrane Association and Multimerization

“…The replication of potexviruses is also associated with the endomembrane [29]. For instance, the RdRp of potato virus X (PVX), bamboo mosaic virus (BaMV), and plantago asiatica mosaic virus (PlAMV) locates to the ER as granules during virus infection [30][31][32]. A proline-kinked amphipathic α-helix at the C-terminal of the MET domain of PlAMV was recently found to be required for its membrane association [31].…”

“…For instance, the RdRp of potato virus X (PVX), bamboo mosaic virus (BaMV), and plantago asiatica mosaic virus (PlAMV) locates to the ER as granules during virus infection [30][31][32]. A proline-kinked amphipathic α-helix at the C-terminal of the MET domain of PlAMV was recently found to be required for its membrane association [31]. Previously, we found that the N-terminal part of the PVX MET domain, excluding the proline-kinked amphipathic α-helix, also behaved as a membrane protein [28], suggesting that there are additional membrane-association motifs in the MET domain.…”