Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

Taraporewala, Zenobia F.; Patton, John T.

doi:10.1016/j.virusres.2003.12.006

Cited by 71 publications

(64 citation statements)

References 74 publications

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“…The N terminus of NSP5 may also be masked either by interaction with NSP2, or by the addition of N-terminal epitope tags which may mimic the role of NSP2 (32). However, it is still reported that coexpression of NSP2 is required for NSP5 hyperphosphorylation and the formation of viroplasm-like structures (18,19,42).Two reports have indicated that specific NSP5 residues are required for NSP5 hyperphosphorylation but these reports differ in both the residues and domains required and the cellular kinases involved. Initially it was reported that serines in the 153 to 165 domain of NSP5 were required for NSP5 phosphorylation by casein kinase II (20).…”

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confidence: 99%

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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confidence: 99%

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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“…Viruses of the Cystoviridae and the Reoviridae families encapsidate RdRPs, virally encoded enzymes that have the dual functions of replicating the genome and synthesizing transcripts (1,2). The reoviruses have a complex assembly pathway whereby genome replication and packaging proceed concurrently (2).…”

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confidence: 99%

“…The reoviruses have a complex assembly pathway whereby genome replication and packaging proceed concurrently (2). Cystoviridae, on the other hand, first assemble a closed procapsid (3), into which the three plus-sense singlestranded RNA segments of the viral genome are packaged sequentially via conduits on one or more of the twelve 5-fold vertices.…”

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confidence: 99%

Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

Sen

Heymann

Cheng

et al. 2008

Journal of Biological Chemistry

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The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage ⌽6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as singlestranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage ⌽6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has ϳ10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of ϳ3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.

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“…These are large, nonmembrane bound, electron-dense structures rich in viral RNA, the viral structural proteins VP1, VP2, VP3, and VP6 (13,25) and the nonstructural proteins NSP2, NSP5, and NSP6 (36). Several studies, including recent RNA interference studies, have demonstrated that both NSP2 and NSP5 are critical for the nucleation of viroplasms and for virus replication (5,31).…”

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confidence: 99%

“…Several studies, including recent RNA interference studies, have demonstrated that both NSP2 and NSP5 are critical for the nucleation of viroplasms and for virus replication (5,31). NSP2 is a 35-kDa basic protein that exhibits nucleoside triphosphatase (NTPase) (33), sequence-independent singlestranded RNA (ssRNA)-binding (33), and nucleic acid helixdestabilizing activities (35,36). X-ray crystallographic analysis of NSP2 has shown that, in crystals, NSP2 exists as an octamer (16).…”

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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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Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

Cited by 71 publications

References 74 publications

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

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

Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

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

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