Rotavirus Viroplasm Fusion and Perinuclear Localization Are Dynamic Processes Requiring Stabilized Microtubules

Abstract

Rotavirus viroplasms are cytosolic, electron-dense inclusions corresponding to the viral machinery of replication responsible for viral template transcription, dsRNA genome segments replication and assembly of new viral cores. We have previously observed that, over time, those viroplasms increase in size and decrease in number. Therefore, we hypothesized that this process was dependent on the cellular microtubular network and its associated dynamic components. Here, we present evidence demonstrating that viroplasms are dynamic structures, which, in the course of an ongoing infection, move towards the perinuclear region of the cell, where they fuse among each other, thereby gaining considerably in size and, simultaneouly, explaining the decrease in numbers. On the viral side, this process seems to depend on VP2 for movement and on NSP2 for fusion. On the cellular side, both the temporal transition and the maintenance of the viroplasms are dependent on the microtubular network, its stabilization by acetylation, and, surprisingly, on a kinesin motor of the kinesin-5 family, Eg5. Thus, we provide for the first time deeper insights into the dynamics of rotavirus replication, which can explain the behavior of viroplasms in the infected cell.

A Novel Form of Rotavirus NSP2 and Phosphorylation-Dependent NSP2-NSP5 Interactions Are Associated with Viroplasm Assembly

“…(i) The dNSP2-tubulin interaction requires that NSP2 or another interacting protein in the complex be unphosphorylated. (ii) Tubulin is no longer complexed with vNSP2 despite abundant tubulin surrounding viroplasms in the form of microtubules (19,41,49). We also detected acetylated alpha tubulin associated with dNSP2 from untreated and phosphatase-treated RV-infected cell lysates but not with vNSP2.…”

“…We also detected acetylated alpha tubulin associated with dNSP2 from untreated and phosphatase-treated RV-infected cell lysates but not with vNSP2. Acetylated alpha tubulin is generally associated with stabilized microtubules and is observed surrounding viroplasms (49). Nocodazole treatment of RV-infected cells destabilizes microtubules, and the viroplasms observed in these cells are small and do not fuse into larger viroplasms (41).…”

Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle

“…Additionally, MTs become acetylated in the neighborhood of the viroplasms, a process considered as a requirement for MT-stabilization [18]. Thus, RV replication requires an intact MT-network.…”

Microtubule Regulation and Function during Virus Infection

“…The viral nonstructural NS3 protein can induce formation of motile lipid-derived structures, while reorganization of acetylated MTs that may facilitate replication compartment formation appears to involve the structural viral protein VP1 (52,53). Rotavirus, a double-stranded RNA (dsRNA) virus, forms "viroplasms," which begin as many small structures that coalesce into larger ones in perinuclear regions through interactions between two viral nonstructural proteins, NSP2 and NSP5, with MTs (54,55). Nocodazole blocks the movement and fusion of structures to form viroplasms, which, like norovirus replication complexes, appear to embed in acetylated MTs to maintain structural integrity (55,56).…”

“…Rotavirus, a double-stranded RNA (dsRNA) virus, forms "viroplasms," which begin as many small structures that coalesce into larger ones in perinuclear regions through interactions between two viral nonstructural proteins, NSP2 and NSP5, with MTs (54,55). Nocodazole blocks the movement and fusion of structures to form viroplasms, which, like norovirus replication complexes, appear to embed in acetylated MTs to maintain structural integrity (55,56). Reoviruses replicate in filamentous inclusions formed at perinuclear sites.…”