Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Rotavirus (RV) replication occurs in cytoplasmic inclusions called viroplasms whose formation requires the interactions of RV proteins NSP2 and NSP5; however, the specific role(s) of NSP2 in viroplasm assembly remains largely unknown. To study viroplasm formation in the context of infection, we characterized two new monoclonal antibodies (MAbs) specific for NSP2. These MAbs show high-affinity binding to NSP2 and differentially recognize distinct pools of NSP2 in RV-infected cells; a previously unrecognized cytoplasmically dispersed NSP2 (dNSP2) is detected by an N-terminal binding MAb, and previously known viroplasmic NSP2 (vNSP2) is detected by a C-terminal binding MAb. Kinetic experiments in RV-infected cells demonstrate that dNSP2 is associated with NSP5 in nascent viroplasms that lack vNSP2. As viroplasms mature, dNSP2 remains in viroplasms, and the amount of diffuse cytoplasmic dNSP2 increases. vNSP2 is detected in increasing amounts later in infection in the maturing viroplasm, suggesting a conversion of dNSP2 into vNSP2. Immunoprecipitation experiments and reciprocal Western blot analysis confirm that there are two different forms of NSP2 that assemble in complexes with NSP5, VP1, VP2, and tubulin. dNSP2 associates with hypophosphorylated NSP5 and acetylated tubulin, which is correlated with stabilized microtubules, while vNSP2 associates with hyperphosphorylated NSP5. Mass spectroscopy analysis of NSP2 complexes immunoprecipitated from RV-infected cell lysates show both forms of NSP2 are phosphorylated, with a greater proportion of vNSP2 being phosphorylated compared to dNSP2. Together, these data suggest that dNSP2 interacts with viral proteins, including hypophosphorylated NSP5, to initiate viroplasm formation, while viroplasm maturation includes phosphorylation of NSP5 and vNSP2.

Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

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

Criglar

Crawford

et al. 2014

“…This finding suggests that VF movement on microtubules plays a role in VF coalescence. Similarly, a recent study showed that both NSP2 and NSP5 associate with cellular microtubules and that nocodazole treatment of rotavirus-infected cells results in small, diffuse viroplasms that do not develop into larger, more perinuclear structures (10). Nonetheless, while some FLS and VLS localize near each other in the cytoplasm (perhaps suggesting the movement of both of them on the same microtubule track), this association with microtubules does not result in coalescence events between NS and EGFP/NSP5.…”

Section: Vol 84 2010 Localization Of Mrv Proteins To Cytoplasmic Scmentioning

confidence: 79%

Localization of Mammalian Orthoreovirus Proteins to Cytoplasmic Factory-Like Structures via Nonoverlapping Regions of μNS

Miller

Arnold

Broering

et al. 2010

106

Virally induced structures called viral factories form throughout the cytoplasm of cells infected with mammalian orthoreoviruses (MRV). When expressed alone in cells, MRV nonstructural protein NS forms factory-like structures very similar in appearance to viral factories, suggesting that it is involved in forming the structural matrix of these structures. NS also associates with MRV core particles; the core proteins 2, 1, 2, 3, and 2; and the RNA-binding nonstructural protein NS. These multiple associations result in the recruitment or retention of these viral proteins or particles at factory-like structures. In this study, we identified the regions of NS necessary and sufficient for these associations and additionally examined the localization of viral RNA synthesis in infected cells. We found that short regions within the amino-terminal 220 residues of NS are necessary for associations with core particles and necessary and sufficient for associations with the proteins 2, 1, 2, 2, and NS. We also found that only the 3 protein associates with the carboxyl-terminal one-third of NS and that viral RNA is synthesized within viral factories. These results suggest that NS may act as a cytoplasmic scaffolding protein involved in localizing and coordinating viral replication or assembly intermediates for the efficient production of progeny core particles during MRV 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).…”

Section: Cytoplasmic Replication: Specialized Compartments Need Mts Toomentioning

confidence: 99%

Microtubule Regulation and Function during Virus Infection

Naghavi

Walsh

2017

105

Microtubules (MTs) form a rapidly adaptable network of filaments that radiate throughout the cell. These dynamic arrays facilitate a wide range of cellular processes, including the capture, transport, and spatial organization of cargos and organelles, as well as changes in cell shape, polarity, and motility. Nucleating from MT-organizing centers, including but by no means limited to the centrosome, MTs undergo rapid transitions through phases of growth, pause, and catastrophe, continuously exploring and adapting to the intracellular environment. Subsets of MTs can become stabilized in response to environmental cues, acquiring distinguishing posttranslational modifications and performing discrete functions as specialized tracks for cargo trafficking. The dynamic behavior and organization of the MT array is regulated by MT-associated proteins (MAPs), which include a subset of highly specialized plus-end-tracking proteins (ϩTIPs) that respond to signaling cues to alter MT behavior. As pathogenic cargos, viruses require MTs to transport to and from their intracellular sites of replication. While interactions with and functions for MT motor proteins are well characterized and extensively reviewed for many viruses, this review focuses on MT filaments themselves. Changes in the spatial organization and dynamics of the MT array, mediated by virus-or host-induced changes to MT regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection.KEYWORDS virus, cytoskeleton, microtubules, nucleation, microtubule-associated proteins, plus-end-tracking proteins T he dense cytosolic environment poses a major barrier to the free movement of macromolecules, making microtubule (MT)-based transport a critical aspect of virus replication. Indeed, almost as soon as these filaments were identified and characterized, virus particles were observed proximal to "microtubuli," with evidence that MTassociated proteins (MAPs) might mediate this association. Chemicals that disrupt MT networks, still commonly used today, were reported to suppress virus infection. Moreover, either infection or expression of viral proteins was observed to alter the organization of these networks, suggesting that MTs not only facilitated infection but were also likely to be actively manipulated by viruses. In subsequent decades an enormous body of work helped unravel how virus particles exploit MT motors to traffic within infected cells, and we direct readers to a recent review of this subject (1). Here, we discuss recent advances in our understanding of how MTs themselves are regulated and function during infection, limiting this minireview to some illustrative examples in each case. TWO ENDS TO THE STORY: THE BASICS OF MT POLARITY AND ORGANIZATIONMTs form through the polymerization of ␣/␤-tubulin heterodimers into polarized filaments that have minus and plus ends (Fig. 1A and B). MTs nucleate through minus-end seeding at MT-organizing cen...