Rotavirus Genome Replication and Morphogenesis: Role of the Viroplasm

Rotavirus nonstructural protein NSP2, a functional octamer, is critical for the formation of viroplasms, which are exclusive sites for replication and packaging of the segmented double-stranded RNA (dsRNA) rotavirus genome. As a component of replication intermediates, NSP2 is also implicated in various replication-related activities. In addition to sequence-independent single-stranded RNA-binding and helix-destabilizing activities, NSP2 exhibits monomer-associated nucleoside and 5′ RNA triphosphatase (NTPase/RTPase) activities that are mediated by a conserved H225 residue within a narrow enzymatic cleft. Lack of a 5′ γ-phosphate is a common feature of the negative-strand RNA [(−)RNA] of the packaged dsRNA segments in rotavirus. Strikingly, all (−)RNAs (of group A rotaviruses) have a 5′ GG dinucleotide sequence. As the only rotavirus protein with 5′ RTPase activity, NSP2 is implicated in the removal of the γ-phosphate from the rotavirus (−)RNA. To understand how NSP2, despite its sequence-independent RNA-binding property, recognizes (−)RNA to hydrolyze the γ-phosphate within the catalytic cleft, we determined a crystal structure of NSP2 in complex with the 5′ consensus sequence of minus-strand rotavirus RNA. Our studies show that the 5′ GG of the bound oligoribonucleotide interacts extensively with highly conserved residues in the NSP2 enzymatic cleft. Although these residues provide GG-specific interactions, surface plasmon resonance studies suggest that the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2. A novel observation from our studies, which may have implications in viroplasm formation, is that the C-terminal helix of NSP2 exhibits two distinct conformations and engages in domain-swapping interactions, which result in the formation of NSP2 octamer chains.

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confidence: 99%

Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5′ GG Sequence for RTPase Activity

Chow

Patton

et al. 2012

“…Associated with each genome segment is a polymerase complex composed of the viral RdRp (VP1) and viral capping enzyme (VP3) (22,36). During infection, VP1 interacts with viral RNA to mediate several important processes, including transcription, RNA packaging, and replication (31,33). Transcription is mediated by VP1 confined within the virion core and results in the synthesis of plus-strand RNAs (ϩRNAs) from the minus strand of a genomic dsRNA segment (32).…”

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confidence: 99%

Residues of the Rotavirus RNA-Dependent RNA Polymerase Template Entry Tunnel That Mediate RNA Recognition and Genome Replication

Ogden

Ramanathan

Patton

2011

To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (؉RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3 end of rotavirus ؉RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral ؉RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with ؉RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of ؉RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with ؉RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates.Prior to catalysis, a viral RNA-dependent RNA polymerase (RdRp) must recognize viral templates, guide them to the catalytic site, and position them appropriately for initiation (25). While interactions between the RdRp and its template may mediate these processes, in many cases the details of these interactions are poorly understood. Rotaviruses (RVs) provide an ideal opportunity to study the functional significance of RdRp-RNA interactions, due to the availability of a highresolution RV RdRp structure and in vitro biochemical assays to analyze polymerase activity (9,18,43). RVs, members of the Reoviridae family, are important etiologic agents of diarrheal disease (27). RV virions are nonenveloped, triple-layered icosahedrons that encapsidate a genome of 11 segments of double-stranded RNA (dsRNA) (36). Associated with each genome segment is a polymerase complex composed of the viral RdRp (VP1) and viral capping enzyme (VP3) (22, 36). During infection, VP1 interacts with viral RNA to mediate several important processes, including transcription, RNA packaging, and replication (31, 33). Transcription is mediated by VP1 confined within the virion core and results in the synthesis of plus-strand RNAs (ϩRNAs) from the minus strand of a genomic dsRNA segment (32). Viral ϩRNAs may be translated by host...

“…The genome (ϳ18 kb) of rotavirus consists of 11 segments of double-stranded RNA (dsRNA) encoding six structural and six nonstructural proteins (NSPs) (13). Several studies have shown that endogenous transcription of dsRNA segments, genome replication/packaging, and assembly of subviral particles take place in specialized compartments called viroplasms, which appear early during infection (30). NSP2, along with NSP5, is essential for the formation of these perinuclear, non-membrane-bound, electron-dense inclusions (14).…”

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confidence: 99%

“…Coexpression of NSP2 and NSP5 alone has been shown to be sufficient for the formation of viroplasm-like structures (14). NSP2 also appears to interact directly or indirectly with other structural proteins present in the viroplasm, such as VP2 (30), which forms the innermost capsid layer of the virion (32), and VP1, which is the viral RNA-dependent RNA polymerase (45).…”

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confidence: 99%

Crystallographic and Biochemical Analysis of Rotavirus NSP2 with Nucleotides Reveals a Nucleoside Diphosphate Kinase-Like Activity

Kumar

Jayaram

Carpió

et al. 2007

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.Rotaviruses are the major cause of life-threatening diarrhea in children worldwide (13). The genome (ϳ18 kb) of rotavirus consists of 11 segments of double-stranded RNA (dsRNA) encoding six structural and six nonstructural proteins (NSPs) (13). Several studies have shown that endogenous transcription of dsRNA segments, genome replication/packaging, and assembly of subviral particles take place in specialized compartments called viroplasms, which appear early during infection (30). NSP2, along with NSP5, is essential for the formation of these perinuclear, non-membrane-bound, electron-dense inclusions (14). Coexpression of NSP2 and NSP5 alone has been shown to be sufficient for the formation of viroplasm-like structures (14). NSP2 also appears to interact directly or indirectly with other structural proteins present in the viroplasm, such as VP2 (30), which forms the innermost capsid layer of the virion (32), and VP1, which is the viral RNA-dependent RNA polymerase (45).Biochemical studies on recombinant NSP2 have shown that in addition to single-stranded-RNA (ssRNA)-binding and helix-destabilizing activities (42), NSP2 exhibits nucleotide triphosphatase (NTPase) activity (40). The protein cleaves...