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

Abstract

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.

Detection of human norovirus in intestinal biopsies from immunocompromised transplant patients

“…IF images were acquired using a Nikon A1-R confocal laser scanning microscope as previously described (Criglar et al, 2014). The images for the biopsy sections along with their corresponding controls were captured using identical parameters to allow comparison between the antibodies and the pre-immune sera.…”

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

“…Its main function is to participate in the formation of viroplasms, which are cytoplasmic structures where genome replication and morphogenesis of new particles occurs (Criglar et al 2014). Among porcine strains, previous data showed low rates of genetic variability NSP5/6, because their structural and functional constraints and lower exposure to immune selection (Barbosa et al 2013), which is a desirable property when selecting targets for nucleic acid detection methods.…”

Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication

“…Binding of NSP2 to these sites could interfere with the assembly of VP2 into core structures. Recently, a new cytoplasmic form of NSP2 has been characterized that interacts with cellular and viral proteins, including VP2 (60). Based on our results and the previous observations, which showed that purified NSP2 impedes viral replication in vitro by interfering with the role of VP2 in forming the minus-strand initiation complex (63), a reasonable hypothesis is that NSP2-VP2 interactions, among other functions, play a role in preventing the premature assembly of VP2 cores in the cytoplasm, prior to VP2 being shuttled into the viroplasms.…”

“…Direct interaction of NSP2 and VP2, although suspected, has not been demonstrated previously. Previous studies showing that high levels of NSP2 are associated with RIs recovered from infected cells that specifically contained VP2 (21) and more recent studies showing that VP2 is pulled down by NSP2 from the lysates of rotavirus-infected cells strongly suggest a possible interaction between the two proteins (60). We hypothesized that in addition to VP1, NSP2 directly binds VP2, possibly coordinating the VP2- The peptide sequences corresponding to the spots denoted 1 to 18 in panel B that showed intensities higher than the 60% cutoff.…”