Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure

Abstract

Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this process.Infection with positive-stranded RNA viruses often causes extensive membrane rearrangements in the host cell, establishing a distinct compartment where viral RNA synthesis occurs. The viral replication complexes are associated with these membranes, which can originate from different intracellular membranes including the late and early endomembrane system (21,22,27,29,30,33). Despite the central role of such a virusinduced membranous compartment in the replicative cycle, the cellular components involved in the formation of this compartment are largely unknown.Cowpea mosaic virus (CPMV), a bipartite positive-stranded RNA virus, is the type member of the comoviruses, which bear strong resemblance to animal picornaviruses in both the gene organization and the amino acid sequence of replication proteins (1, 14). Both RNA1 and RNA2 are translated into large polyproteins, which are proteolytically cleaved into the different cleavage products by the 24-kDa proteinase (24K) (Fig. 1). The proteins encoded by RNA1 are necessary and sufficient for replication, whereas RNA2 codes for the capsid proteins and the movement protein. The RNA1-encoded 87-kDa protein (87K) contains a domain specific to RNA-dependent RNA polymerases; however, a 110-kDa protein (110K; 87K plus 24K) is the only viral protein present in highly purified, RNAdependent RNA polymerase preparations capable of elongating nascent RNA chains, suggesting that fusion to 24K is required for replicase activity (13).Upon infection of cowpea plants with CPMV, a typical cytopathic structure is formed, often adjacent to the nucleus, consisting of an amorphous matrix of electron-dense material that is traversed by rays of small membranous vesicles (12). Autoradiography in conjunction with electron microscopy on sections of CPMV-infect...

Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes

“…Replication-associated proteins were also shown to colocalize at this site (21,42). Infection of ER-GFP plant cells by other picornavirus-like viruses (including CPMV and TEV) is also characterized by the induction of GFP aggregates similar to the ones described here, and the replication proteins of these viruses colocalize with the modified ER membranes (6,7,47). Several viral proteins expressed outside the context of the virus genome have been shown to localize to intracellular membranes and to induce the alteration of the morphology of these membranes.…”

“…Many positive-strand RNA viruses induce extensive proliferation and modification of intracellular membranes in their hosts, which often results in the accumulation of numerous membranous vesicles. Doublestranded RNA (dsRNA) replication intermediates and viral replication factors are associated with the membranous vesicles, which are thought to be the site of the replication (6,7,18,25,48,49,52). The requirement for intact membranes for successful virus replication has also been demonstrated using cell-free replication systems (2,35,59).…”

The Bean Pod Mottle Virus RNA2-Encoded 58-Kilodalton Protein P58 Is Required in cis for RNA2 Accumulation

“…Exactly how RNA2 recruits RNA1-encoded proteins to replicate itself is not well understood for BPMV or comoviruses in general. Earlier investigations using CPMV, another comovirus, showed that mutations disrupting the coding region of the N-terminally located P58 open reading frame (ORF) abolished RNA2 accumulation in protoplast cells, although it was not further examined whether the P58 protein itself or its coding RNA played a more critical role or how this genome region facilitated RNA2 accumulation (11,20,21). It was also found that RNA2 of GFLV, a virus of the genus Nepovirus of the Secoviridae family, encodes a 5=-proximal 2A protein that is needed for RNA2 accumulation and colocalizes with some of the RNA1-encoded proteins (12).…”

Characterization of Membrane Association Domains within the Tomato Ringspot Nepovirus X2 Protein, an Endoplasmic Reticulum-Targeted Polytopic MembraneProtein

“…Various ER-derived structures, including spherules (Brome mosaic bromovirus) and membranous vesicles or rosettes (picornavirus superfamily), are found in infected cells (3,4,6,32,33,41). Viral nonstructural proteins and RNA synthesis localize to these modified ER structures, suggesting that they are the sites of viral replication.…”