Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design

Ng, Adolf K.Y.; Zhang, Hongmin; Tan, Kemin; Li, Zongli; Liu, Jin-huan; Chan, Paul K.S.; Li, Sui-Mui; Chan, Wai Lun; Au, Shannon Wing-Ngor; Joachimiak, Andrzej; Walz, Thomas; Wang, Jia-Huai; Shaw, Pang‐Chui

doi:10.1096/fj.08-112110

Cited by 201 publications

(301 citation statements)

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“…The other mutant NPs examined (NP-G2, NP-G3, NP-G4, and NP-⌬74-88) yielded detectable levels of cRNA accumulation and replication ( Fig. 2A and B), with efficiencies broadly corresponding to the reported RNA binding affinities of the respective NP mutants (14). The exception was NP-G3, which inhibited cRNA accumulation and replication compared to the cRNA accumulation and replication with wild-type NP despite having only a negligible effect on RNA binding.…”

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confidence: 82%

“…The rest of the vRNA interacts with multiple copies of NP, each molecule covering approximately 24 nucleotides (reviewed in reference 17). NP binds ssRNA with high affinity but little or no sequence specificity (2, 9, 22) and has been shown to homo-oligomerize and interact with the PB1 and PB2 subunits of the viral RdRp (4,8,14,15,23). Although cryoelectron microscopy reconstitution images of a mini-RNP containing nine NP molecules have been obtained (6), a detailed high-resolution structure of the vRNP is still lacking.…”

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confidence: 99%

“…RanBP5 detected with a rabbit polyclonal antibody (Santa Cruz) was used as a loading control. (H and I) Surface plasmon resonance results for wild-type (H) or G1(4) mutant (I) H1N1 NP against immobilized RNA suitable for binding one NP molecule (24 nucleotides in length) (14). Different concentrations of purified proteins were injected as shown, and the signal (resonance units [RU]) was plotted over time.…”

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confidence: 99%

“…The high-resolution structures of H1N1 and H5N1 NP revealed potential RNA-binding sites involving a large number of basic amino acid residues (14,23). We initially analyzed the effect of previously characterized RNA-binding mutants of H5N1 NP (14) on the accumulation and replication of cRNA in the presence of catalytically inactive mutant or wild-type H1N1 RdRp ( Fig.…”

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confidence: 99%

“…2F and G). Finally, the RNAbinding properties of wild-type NP and NP-G1(4) proteins expressed in Escherichia coli and purified to homogeneity were studied by surface plasmon resonance (14). Wild-type NP showed a high RNA-binding affinity, with a dissociation constant (K D ) of 16.5 nM (Fig.…”

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Stabilization of Influenza Virus Replication Intermediates Is Dependent on the RNA-Binding but Not the Homo-Oligomerization Activity of the Viral Nucleoprotein

Vreede

Shaw

et al. 2011

J Virol

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The influenza virus nucleoprotein (NP) is believed to play a central role in directing a switch from RNA genome transcription to replication by the viral RNA polymerase. However, this role has recently been disputed with the proposal of alternative regulatory mechanisms. It has been suggested that the expression of viral polymerase and NP allows genome replication by stabilization of cRNA replication intermediates and complementary ribonucleoprotein (cRNP) assembly. Here, we demonstrate that the RNA-binding activity of NP is necessary for stabilization of cRNA, whereas, surprisingly, homo-oligomerization of NP is not essential. However, both RNA binding and homo-oligomerization activities are essential for genome replication.Influenza virus, a member of the Orthomyxoviridae family, contains a segmented, negative-sense, single-stranded RNA (ssRNA) genome. Each viral RNA (vRNA) segment is bound by the viral RNA-dependent RNA polymerase (RdRp) and nucleoprotein (NP) to form viral ribonucleoprotein (vRNP) complexes. Within the vRNP, the RdRp binds to the corkscrew structure formed by the partially complementary conserved 5Ј and 3Ј ends of the vRNA representing the vRNA promoter. The rest of the vRNA interacts with multiple copies of NP, each molecule covering approximately 24 nucleotides (reviewed in reference 17). NP binds ssRNA with high affinity but little or no sequence specificity (2, 9, 22) and has been shown to homo-oligomerize and interact with the PB1 and PB2 subunits of the viral RdRp (4,8,14,15,23). Although cryoelectron microscopy reconstitution images of a mini-RNP containing nine NP molecules have been obtained (6), a detailed high-resolution structure of the vRNP is still lacking.During the viral life cycle, the vRNA is transcribed into mRNA and replicated through a cRNA intermediate into more copies of vRNA by the viral RdRp (reviewed in references 10, 17, and 18). Viral mRNAs associate with cellular factors normally associating with host mRNAs, e.g., nuclear and cytoplasmic cap-binding proteins, leading to the stabilization of viral mRNAs and their processing, nuclear export, and translation (3,16,21). In contrast, cRNAs are stabilized by the association of viral factors, i.e., the viral RdRp and NP, which together form a cRNP structure similar to that of vRNPs. During infection, viral mRNAs can be detected initially, while cRNAs become detectable only after the onset of viral protein synthesis (12). This has been interpreted to mean that a switch of polymerase function from a transcriptase to a replicase is required for cRNA synthesis, and various models have been proposed that implicate viral and host factors, as well as small viral RNAs (svRNAs), in the switching (reviewed in reference 17). However, an alternative interpretation is that both mRNA and cRNA are synthesized from early on in a stochastic manner but cRNA is degraded by host nucleases until sufficient amounts of viral RdRp and NP accumulate to stabilize it (20). In agreement with this interpretation, the overexpression of catalytically...

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