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

f The P9-1 protein of Rice black-streaked dwarf virus (RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed for in vitro binding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.

Section: Discussionmentioning

confidence: 44%

Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

Wang

et al. 2013

“…7 and Table 2). In light of our crystallographic analysis with the GG nucleotide and the previous cryo-EM studies of NSP2 with decariboadenylnucleotide showing an accumulation of extra density across the groove regions (including the C-terminal helix) (11), one qualitative interpretation is that while both GG and CC can bind to the electropositive grooves (including the C-terminal helix) in a sequence-independent manner (groove binding), the GG nucleotide additionally binds cleft region through sequence-specific interactions. Such a preferential binding of the GG nucleotide within the cleft may contribute to its higher binding affinity.…”

Section: Resultsmentioning

confidence: 67%

“…All previously reported structural studies were carried out with a C-terminal His-tagged NSP2 (10,11,14). To avoid any possible interference of the basic His tag with negatively charged RNA, a new construct was designed such that the His tag could be removed (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

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

Chow

Patton

et al. 2012

Self Cite

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.

“…NSP2 and NSP5 are known to interact with each other. This interaction was described in vitro with purified proteins (24) and in vivo for both infected cells (1,12,40) and uninfected, cotransfected cells, where coexpression of the two proteins leads to up-regulation of NSP5 hyperphosphorylation and to the formation of viroplasm-like structures (VLS) (1,17). The signals promoting the formation of VLS seem to be embedded in the C-and N-terminal regions of NSP5 (12,32).…”

mentioning

confidence: 95%

Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2

Arnoldi

Campagna

Eichwald

et al. 2007

Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.Rotavirus is a major etiologic agent of severe gastroenteritis in infants and young children worldwide (20,21,33). The virion (defined as a triple-layered particle [TLP]) contains a genome consisting of 11 segments of double-stranded RNA (dsRNA) and is made up of three concentric layers of proteins: the outer layer consists of the two proteins VP7 and VP4, the intermediate layer of VP6, and the internal (core) layer of VP2, with VP1 and VP3 attached at its inside as minor components (29, 41). After entry into the host cell, the virion loses the outer layer to become a double-layered particle (DLP), which is active in transcription of viral mRNAs from the dsRNA genome. The viral RNA-dependent RNA polymerase (RdRp) acts as both the transcriptase and the replicase. Several lines of evidence indicate that VP1 is the viral RdRp: (i) VP1 contains sequence motifs that are shared by RdRps of other RNA viruses (31); (ii) VP1 has NTP-binding activity and, when cross-linked with the nucleotide analog 8-azido-ATP, inhibits RNA transcription (50); (iii) VP1 specifically recognizes the 3Ј end of viral mRNAs (35); and (iv) recombinant VP1 can direct template-dependent minus-strand synthesis in vitro in the presence of VP2 (37, 54).Despite partial characterization of rotavirus replication intermediates (3,19,36,37,54), molecular details of viral genome replication and of the different steps of viral morphogenesis still remain to be elucidated. It has been shown that VP1 and VP2, the scaffolding protein of viral cores, are...