RNA-Binding Activity of the Rotavirus Phosphoprotein NSP5 Includes Affinity for Double-Stranded RNA

Vende, Patrice; Taraporewala, Zenobia F.; Patton, John T.

doi:10.1128/jvi.76.10.5291-5299.2002

Cited by 53 publications

(56 citation statements)

References 51 publications

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“…Both NSP2 and NSP5 are involved in rotavirus replication, specifically in the packaging of the 11 double stranded RNA segments into the subviral particle (Kattoura et al 1992, Taraporewala & Patton 2001, Vende et al 2002. These data are consistent with the maximum level of these proteins bound to microtubules is at 12 h after infection.…”

Section: Discussionsupporting

confidence: 78%

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Cabral-Romero

Padilla-Noriega

2006

Mem. Inst. Oswaldo Cruz

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Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.Key words: rotavirus -viroplasms -microtubules -NSP2 -NSP5Group A rotaviruses are the leading etiological agents of severe diarrheal disease in infants and young children worldwide (Parashar 2003). These viruses belong to the genus Rotavirus within the Reoviridae family and their genome consist of 11 double-stranded RNA segments that encode six structural proteins named VP1-VP4, VP6, and VP7, as well as six nonstructural proteins named NSP1 to NSP6 (Estes 2001).Rotavirus replication takes place in the cytoplasm of infected cells in electrodense spherical structures known as "viroplasms" that can be found as early as 4 h after infection . Viroplasms are composed of viral RNA and the proteins VP1, VP2, VP3, VP6, NSP2, and NSP5 (Petrie et al. 1982, Gallegos & Patton 1989, and while virion assembly occur in these structures (Petrie et al. 1982, Gallegos & Patton 1989) the mechanism of viroplasms formation is unknown. Recently, it was reported that the number of rotavirus viroplasms decrease with post-infection time (Eichwald et al. 2004), and while the diameter of single viroplasms increased with time, the total number of viroplasms per cell diminishes, suggesting that growth of these inclusion bodies occur by fusion and probably also by stepwise addition of viral components to the viroplasms surface (Eichwald et al. 2004).In the viroplasms the viral mRNAs are replicated to produce genomic double stranded RNAs (dsRNA) which are simultaneously encapsidated into double layerded viral particles (DLPs), that contain the 11 dsRNA at the core of the particle surrounded by the inner and intermediate protein layers of VP2 and VP6, respectively. The third layer formed by VP4 and VP7 is acquired by budding of the DLP particles into the endoplasmic reticulum (ER), a process that requires assistance of the viral non-structural protein 4 (NSP4), whereby a transient envelope is acquired, that is finally lost within the ER along with NSP4. The fundamental role of NSP...

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Section: Discussionsupporting

confidence: 78%

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Cabral-Romero

Padilla-Noriega

2006

Mem. Inst. Oswaldo Cruz

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“…presented here, other studies on NSP5 have reportedly used conditions which yield only soluble NSP5 by our analysis (6,20,22,32,37,41,43,46). Our studies indicate that the innate insolubility of NSP5 and the primary localization of hyperphosphorylated NSP5 isoforms in insoluble fractions are fundamental aspects of NSP5 function.…”

Section: Vol 80 2006 Hyperphosphorylation Of Rotavirus Nsp5 Proteinmentioning

confidence: 67%

Hyperphosphorylation of the Rotavirus NSP5 Protein Is Independent of Serine 67 or NSP2, and the Intrinsic Insolubility of NSP5 Is Regulated by Cellular Phosphatases

Agresti

Mackow

2006

J Virol

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The NSP5 protein is required for viroplasm formation during rotavirus infection and is hyperphosphorylated into 32-to 35-kDa isoforms. Earlier studies reported that NSP5 is not hyperphosphorylated without NSP2 coexpression or deleting the NSP5 N terminus and that serine 67 is essential for NSP5 hyperphosphorylation. In this report, we show that full-length NSP5 is hyperphosphorylated in the absence of NSP2 or serine 67 and demonstrate that hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% sodium dodecyl sulfate. The last 68 residues of NSP5 are sufficient to direct green fluorescent protein into insoluble fractions and cause green fluorescent protein localization into viroplasm-like structures; however, NSP5 insolubility was intrinsic and did not require NSP5 hyperphosphorylation. When we mutated serine 67 to alanine we found that the NSP5 mutant was both hyperphosphorylated and insoluble, identical to unmodified NSP5, and as a result serine 67 is not required for NSP5 phosphorylation. Interestingly, treating cells with the phosphatase inhibitor calyculin A permitted the accumulation of soluble hyperphosphorylated NSP5 isoforms. This suggests that soluble NSP5 is constitutively dephosphorylated by cellular phosphatases and demonstrates that hyperphosphorylation does not direct NSP5 insolubility. Collectively these findings indicate that NSP5 hyperphosphorylation and insolubility are completely independent parameters and that analyzing insoluble NSP5 is essential for studies assessing NSP5 phosphorylation. Our results also demonstrate the involvement of cellular phosphatases in regulating NSP5 phosphorylation and indicate that in the absence of other rotavirus proteins, domains on soluble and insoluble NSP5 recruit cellular kinases and phosphatases that coordinate NSP5 hyperphosphorylation.Rotavirus is an icosahedral virus belonging to the family Reoviridae and has a genome composed of 11 double-stranded RNA segments (21). One characteristic feature of rotavirus infection is the formation of punctate perinuclear structures called viroplasms 2 to 3 h into the infectious cycle (36). Viroplasms are sites of viral RNA replication and packaging of genome segments into progeny virions. Several rotavirus proteins (VP1, VP2, VP3, VP6, NSP2, NSP5, and NSP6) have been found in viroplasms during infection (25,47). Expression of NSP2 and NSP5 is reportedly required and sufficient for viroplasm formation (19,22). However, it has also been shown that expression of N-terminally tagged NSP5 alone results in the formation of viroplasm-like structures (32).NSP5 contains 198 amino acids with a predicted molecular mass of approximately 21 kDa. NSP5 is highly phosphorylated in infected cells resulting in a series of posttranslationally modified isoforms that range from 26 to 35 kDa (2). The initial modification that results in the shift from 21 to 26 kDa is unknown, but the appearance of 28-and 32-to 35-kDa isoforms from a 26-kDa precursor has been ascribed to O...

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“…It is unclear how NSP5 gets phosphorylated. Protein kinases, such as casein kinase II (11) and NSP2 through its NTPase activity (40), have also been suggested to be involved in NSP5 phosphorylation. In the NSP2 octamer, the NTP binding sites are located in the cleft region of each monomer in close vicinity to the groove and the NSP5 binding region, supporting the possibility of the transfer of phosphate from NSP2 to NSP5.…”

Section: Discussionmentioning

confidence: 99%

“…NSP2 from the SA11 strain of rotavirus was expressed in the Escherichia coli strain SG13009 (QIAGEN) with a six-His tag at the C terminus and purified as described before (16). This His tag construct is the same as that used in previous biochemical characterizations of NSP2, including its NTPase activity, RNA binding (33), and in vitro phosphorylation of NSP5 (40) and crystallographic analysis (16). Based on these studies, the conformationally flexible His tag was considered noninterfering with either RNA or NSP5 binding.…”

Section: Methodsmentioning

confidence: 99%

Cryoelectron Microscopy Structures of Rotavirus NSP2-NSP5 and NSP2-RNA Complexes: Implications for Genome Replication

Jiang¹,

Jayaram²,

Kumar³

et al. 2006

J Virol

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The replication and packaging of the rotavirus genome, comprising 11 segments of double-stranded RNA, take place in specialized compartments called viroplasms, which are formed during infection and involve a coordinated interplay of multiple components. Two rotavirus nonstructural proteins, NSP2 (with nucleoside triphosphatase, single-stranded RNA [ssRNA] binding and helix-destabilizing activities) and NSP5, are essential in these events. Previous structural analysis of NSP2 showed that it is an octamer in crystals, obeying 4-2-2 crystal symmetry, with a large 35-Å central hole along the fourfold axis and deep grooves at one of the twofold axes. To ascertain that the solution structure of NSP2 is the same as that in the crystals and investigate how NSP2 interacts with NSP5 and RNA, we carried out single-particle cryoelectron microscopy (cryo-EM) analysis of NSP2 alone and in complexes with NSP5 and ssRNA at subnanometer resolution. Because full-length NSP5 caused severe aggregation upon mixing with NSP2, the deletion construct NSP5 66-188 was used in these studies. Our studies show that the solution structure of NSP2 is same as the crystallographic octamer and that both NSP5 66-188 and ssRNA bind to the grooves in the octamer, which are lined by positively charged residues. The fitting of the NSP2 crystal structure to cryo-EM reconstructions of the complexes indicates that, in contrast to the binding of NSP5 66-188 , the binding of RNA induces noticeable conformational changes in the NSP2 octamer. Consistent with the observation that both NSP5 and RNA share the same binding site on the NSP2 octamer, filter binding assays showed that NSP5 competes with ssRNA binding, indicating that one of the functions of NSP5 is to regulate NSP2-RNA interactions during genome replication.One of the least understood stages in viral morphogenesis is genome replication and packaging. This is particularly true with complex viruses such as rotavirus, a member of the Reoviridae family and the major cause of infantile gastroenteritis (18). Rotavirus is a large icosahedral virus composed of three concentric capsid layers that enclose the genome. Its genome consists of 11 segments of double-stranded RNA (dsRNA), which encode six structural viral proteins (VPs) and six nonstructural proteins (NSPs) (12). Removal of the outer capsid layer composed of VP4 and VP7 during cell entry triggers the endogenous transcription of the genome in the resulting double-layer particles. The transcription of the dsRNA segments into capped mRNA is assisted by the viral polymerase VP1 (39) and the capping enzyme VP3 (7, 21), which, possibly as heterodimers, are attached to the inside surface of the inner VP2 layer of the virion (27). The transcripts exit from the double-layer particles through the channels at the fivefold axis in the VP6 layer (20). These mRNA molecules function as templates for the progeny RNA and encode all the viral proteins (12).Replication and packaging of the viral genome into the viral capsids takes place in specialized, cytoplasmic ...

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RNA-Binding Activity of the Rotavirus Phosphoprotein NSP5 Includes Affinity for Double-Stranded RNA

Cited by 53 publications

References 51 publications

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Hyperphosphorylation of the Rotavirus NSP5 Protein Is Independent of Serine 67 or NSP2, and the Intrinsic Insolubility of NSP5 Is Regulated by Cellular Phosphatases

Cryoelectron Microscopy Structures of Rotavirus NSP2-NSP5 and NSP2-RNA Complexes: Implications for Genome Replication

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