Amino Terminus of Reovirus Nonstructural Protein ςNS Is Important for ssRNA Binding and Nucleoprotein Complex Formation

Gillian, Anne Lynn; Nibert, Max L.

doi:10.1006/viro.1997.8905

Cited by 51 publications

(62 citation statements)

References 30 publications

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“…Moreover, the stack images suggested that viroplasms are well-defined spherical structures. In reoviruses, it has also been reported that sNS, the non-structural protein homologous to rotavirus NSP2 with ssRNA binding activity and capacity to form higher-order homo-oligomeric structures (Huismans & Joklik, 1976;Richardson & Furuichi, 1985;Gillian & Nibert, 1998;Gillian et al, 2000), can also form inclusionbody-like structures when co-expressed with reovirus protein mNS (Becker et al, 2003). NS2 of bluetongue virus and rotavirus NSP2 have been considered to be proteins of similar function, since they share NTPase activity, non-specific ssRNA binding and localization to inclusions bodies Fillmore et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

Eichwald

Rodrı́guez

Burrone

2004

Journal of General Virology

124

196

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Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an in vivo binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131-198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.

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Section: Discussionmentioning

confidence: 99%

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

Eichwald

Rodrı́guez

Burrone

2004

Journal of General Virology

124

196

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“…Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gels as described previously (18). Proteins were visualized by staining with Coomassie brilliant blue R-250 (Coomassie blue).…”

Section: Sds-page and Immunoblot Analysismentioning

confidence: 99%

“…NS-expressing recombinant baculovirus and protein expression. The recombinant baculovirus expressing the T1L NS protein was previously described (18). T. ni cells were infected with recombinant baculovirus as described previously (18).…”

Section: Sds-page and Immunoblot Analysismentioning

confidence: 99%

Reovirus Nonstructural Protein μNS Binds to Core Particles but Does Not Inhibit Their Transcription and Capping Activities

Broering

McCutcheon

Centonze

et al. 2000

J Virol

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Previous studies provided evidence that nonstructural protein NS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68: [455][456][457][458][459][460][461][462][463][464][465][466] 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of NS. Given the possible role of NS in particle assembly and/or transcription implied by those findings, we tested whether recombinant NS can bind to cores in vitro. The NS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of NS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of NS were bound. Thus, the NS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the NS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to NS. The NS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5 ends of the transcripts. In vitro competition assays showed that mixing NS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins 1 and 3. These findings are consistent with the presence of NS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, NS may block or delay outer-capsid assembly and allow continued transcription by these particles.

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“…Inmammalian genera of the family reoviridae, μNS is sufficient to form VFs (viral factories) [24], and it recruits σNS to VFs [27]. The μNS protein acts as a scaffold to recruit viral core surface proteins [25], while σNS binds preferentially to ss-RNA and ss-DNA [26,27]. In members of the genus fijivirus such as RBSDV, nonstructural protein RBSDV P9-1 is important for the functioning of the relative proteins in Vps during viral morphogenesis [28], RBSDV P9-1 self-associates into aggregate formations and preferentially binds ss-RNA molecules [29,30].…”

Section: Introductionmentioning

confidence: 99%

Analysis and application of the SRBSDV P9-1 octamer crystal structure for the target of α-amino phosphonate derivatives

Ding¹,

Li²,

Chen³

et al. 2018

Oncotarget

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Southern rice black-streaked dwarf virus (SRBSDV) P9-1 octameric protein accumulates viroplasms in SRBSDV-infected plant and insect cells, our previous studies found α-amino phosphonate drug-dufulin had a micromole affinity with SRBSDV P9-1. Now we focus our studies on the SRBSDV P9-1 crystal structure and use it as the target for α-amino phosphonate derivatives. The structure of the SRBSDV P9-1 cylindrical octamer was determined to a 2.2 Å resolution using X-ray crystallography, this structure was composed of nine α-helices, nine β-sheets and a series of interconnecting loops. The structures of all eight subunits were nearly identical, except for some differences in the β-sheet core. There were six different sites (R20K, V109D, F164T, V123L, C124L and V128T) present between the SRBSDV P9-1 and the previously reported RBSDV P9-1 crystal structure. Fluorescence titration, isothermal calorimetry and microscale thermophoresis experiments showed that α-amino phosphonate derivatives GUFCC-013 and GUFCC-023 bound to SRBSDV P9-1 with micromole binding affinities. These results will provide important help for the further improvement of the lead compound and develop the potential anti-SRBSDV drugs.

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Amino Terminus of Reovirus Nonstructural Protein ςNS Is Important for ssRNA Binding and Nucleoprotein Complex Formation

Cited by 51 publications

References 30 publications

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

Reovirus Nonstructural Protein μNS Binds to Core Particles but Does Not Inhibit Their Transcription and Capping Activities

Analysis and application of the SRBSDV P9-1 octamer crystal structure for the target of α-amino phosphonate derivatives

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