LEI 10882 DE 2004 PDF

Departamento de Física, Universidade de Aveiro, Aveiro, Portugal, CICECO, Universidade de Aveiro, Publication Date (Web): September 4, .. The Journal of Physical Chemistry C (29), .. Lei Zhang, Linlin Fu, Xingxing Yang, Zuoling Fu, Xiangdong Qi, Zhijian Wu. Chem., , (26), pp – Qian Zhou, Kendall Fitzgerald, Paul D. Boyle and Wesley A. Henderson Shu Li, Zhen Cao, Yuxing Peng, Lei Liu, Yonglong Wang, Shu Wang, Ji-Qiang Wang, Tianying Yan, Xue-Ping Gao, De- Ying Song and Pan-Wen .. Journal of Fluorine Chemistry , Nature Communications volume 7, Article number: () | Download Citation . (d) Data sets cover a range of detector types, including Area Welberry, T. Diffuse X-Ray Scattering and Models of Disorder OUP Oxford () . . Chinese Academy of Sciences, Shanghai , China. Ming Lei.

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Influenza viruses cause a highly contagious respiratory disease in humans. 200 we report the 2. We demonstrate that single-site alanine replacements of basic residues in this site lead to reduced RNA-binding activity, and that recombinant influenza B viruses expressing these mutant NS1B proteins are severely attenuated in replication. Most importantly, this study reveals an unexpected RNA-binding function in the C-terminal domain of NS1B, a novel function that distinguishes influenza B viruses from influenza A viruses.

Critical questions in the field of influenza virus research involve elucidating the features of influenza A and influenza B viruses that distinguish their infection mechanism s and virulence. The NS1 protein of influenza A virus NS1A protein has been extensively studied and shown to have multiple functions that counter host antiviral responses and regulate other cellular and viral functions Krug and Garcia-Sastre, The host binding partners of the NS1B protein of influenza B viruses are less well studied.

A somewhat larger residue N-terminal segment of NS1B binds the interferon-induced antiviral human ISG15 protein, and the structure of this complex has been determined Guan et al.

This basic surface leo to bind RNA. Disruption of this RNA binding surface in the NS1B CTD by single site mutations of key basic residues substantially attenuates the replication of influenza B viruses in human cells, demonstrating that that this RNA-binding activity is required for optimal influenza B virus replication.

Several constructs, differing by varying the length of the N-terminal segment of the CTD, were produced and assessed by small-scale expression and NMR studies. Such conserved basic surfaces are typical of nucleic acid binding sites, a prediction that we verify below. Residues for which CSPs could ds be determined, including Pro residues, are shown in white. The molecular orientations in panels abcand d-left are the same. Panel d also shows an orientation rotated by deg, revealing no significant CSPs on the opposite side of the molecule.

Group publications – Inorganic Chemistry and Catalysis

Amino-acid residues in the basic surface and in an adjacent acid patch, identified in the X-ray crystal structure exhibit backbone 15 N- 1 H chemical shift perturbations upon binding of this mer dsRNA Figure 1ddemonstrating that the RNA-binding site in the NS1B CTD involves the conserved basic surface observed in the X-ray crystal structure Figure 1b. Less extensive chemical shift perturbations are also observed on this same face of the NS1 CTD molecule upon binding a single-strand nt RNA substrate data not shown.

The yellow histogram bar in each plot designates data for dimer-disrupting mutant RA at the protein-protein interface formed at high protein concentrations and observed in the X-ray crystal structure. CSPs may arise simply by proximity to bound nucleic acid, rather kei due to specific interactions. 108882 addition, some leei shown in white in Figure 1b within df apparent binding site did not provide reliable CSP data, or are prolines which lack 15 N- 1 H resonances. Dimeric interactions observed in dw structures may or may not occur in solution, particularly 20004 dilute protein conditions.

Residues are colored as follows: The sidechains of residue Arg, mutated to Ala to form a monomer, are shown in yellow. All data were leii on a Bruker MHz spectrometer at K. In the crystal structure, residues R and R are involved in intermolecular salt bridges with residues N and E, respectively, at the dimer interface Figure 3c. These biophysical studies demonstrate that the NS1B CTD has a weak propensity to form a homodimeric structure with the same interface observed in the X-ray crystal structure.


In order to verify this conclusion, and to assess the possibility that dimer formation is driven by RNA binding, we also measured ssRNA and dsRNA binding activity of the ArgAla mutation at the homodimer interface, which reduces the homodimer self association Figure 3a.

From these measurements we conclude that homodimer formation of the NS1B CTD, while potentially important in cooperative binding to large RNA substrates in vivois not required for binding these short RNA substrates. Re structural and biophysical results outlined above reveal a novel, unanticipated RNA binding function in the C-terminal domain of the NS1 protein of influenza B viruses.

The key basic residues responsible for RNA-binding activity are shown in green in Figs. The replication of ed mutant viruses was assayed lwi human A cells using a multiplicity of infection of 0. All three mutant viruses were highly attenuated in replication Figure 4c. We were not able to generate a recombinant virus expressing an NS1B protein with a KA mutation, which has the weakest RNA binding activity in the FP assay Figure 2c,dsuggesting that this mutation renders the virus even more attenuated.

Consistent with the RNA binding results obtained in the biophysical studies, the KA dee virus is not attenuated and replicates like the wt virus Figure 4d. The data of Fig. Infection by each le these influenza B viruses caused activation of IRF3 at early times 2—4 hours postinfectiontriggered directly by the incoming virus Makela et al. As soon as significant levels of the full-length wt or mutant NS1B proteins were synthesized, starting at 6 hours postinfection, activated IRF3 disappeared.

These results indicate that the function of this RNA-binding activity does not involve suppression of IRF3 activation. To determine whether the homodimerization interface of the CTD plays any role in viral replication, we generated a recombinant virus expressing a NS1B protein with the RA mutation that inhibits CTD dimerization.

This mutant virus is attenuated in replication approximately fold Figure 6a.

Unexpected RNA-binding site in the NS1B Protein from Influenza B Virus not present in NS1A

Although we cannot exclude alternative interactions with residue R that have other functions in viral replication, these data suggest NS1B dimerization mediated by its CTD plays some role in providing optimal viral replication in influenza B virus-infected cells.

They form a largely basic patch on the surface of the protein structure, and are highly conserved across influenza B NS1B proteins Supplemental Figure S1b.

However, using a structure-based sequence alignment, the corresponding residues in influenza A NS1A proteins are neither basic nor 20044 cf. Our combined biophysical and virology studies of the CTD of 20004 influenza B NS1B protein have led to an unexpected and novel finding regarding differences in the mechanisms of infection by A and B strains of influenza viruses.

This result highlights differences in the replication strategies of influenza A and B 0204. This is a classic example in which the three-dimensional protein structure provides evidence for an unexpected and important biological function, which was then validated first by biophysical studies with purified protein samples, followed by the 10828 of viruses bearing specific mutations.

The NS1 proteins of influenza A and B strains bind some common dw factors; e. However, they also interact with some different host factors. These distinct binding-partner networks underlie the multiple differences in the mechanisms used by A and B viral strains in host infection. Figure 1b and Figure 7c. The novel RNA-binding function in the C-terminal domain of NS1B proteins described in this study, arising from its conserved, broad, basic surface, is a unique property of NS1B proteins of influenza B viruses, that is not shared by NS1A proteins of influenza A viruses.

However, this dimer structure differs significantly in the relative orientations of protomers from those reported for the influenza A virus NS1A CTD Aramini et al. The details of interchain packing at this interface varies across these published crystal structures of NS1A CTD, and the interface exhibits intrinsic dynamics 1882 have been studied by 19 F NMR nuclear relaxation measurements Lie et al.

While dimerization mediated leei the CTD of NS1B is not required for binding small RNA substrates, our observation that virus replication is attenuated fold by a dimer-disrupting RA mutation suggests that dimerization of full-length NS1B mediated by its Lek may none-the-less contribute to the efficiency of viral replication. Alternatively, this mutation may be affecting other functions of NS1B 1082 influenza B virus-infected cells.


We showed that 108822 is not the case. To determine the function of the NS1B CTD RNA-binding activity, it will be necessary to next identify the RNA species that bind to this domain in infected cells, followed by a determination of the mechanism s by which this RNA inhibits specific steps in virus replication.

Details of sample preparation for crystallization, nucleic acid binding, and NMR studies, including procedures for production of isotope-enriched samples, are presented as Supplemental Information.

Further crystallization optimization was carried out using hanging drop evaporation method. At the home X-ray source wavelength 1. Crystallographic statistics and final structure refinement and geometry statistics are summarized in Table 1. Sequence-specific resonance assignments and chemical shift perturbation studies were carried out using standard methods, outlined in Supplemental Information.

A threshold of 20 ppb 0.

Unexpected RNA-binding site in the NS1B Protein from Influenza B Virus not present in NS1A

Sedimentation velocity and equilibrium ultracentrifugation analyses were carried out by the University of Connecticut Analytical Ultracentrafugation Facility, directed by Drs. Details of sample preparation and methods used in these biophysical studies are presented as Supplemental Information. Fluorescence polarization was measured on a Tecan GENios-Pro plate reader with excitation at nm and emission at nm.

FP values were reported in millipolarization units mP. Details of these FP measurements are presented as Supplemental Information. For all FP-binding assays, fluorescence intensity FI and FP ldi measured after 50 minutes of incubation at room temperature to ensure equilibrium measurements. Each measurement was made in triplicate. Virus stocks were grown in day-old lfi eggs. For multiple cycle growth, A cells were infected with 0.

Assays for the activation phosphorylation of IRF3 were carried out as described previously Kuo et al. Where indicated, infected cell extracts were analyzed by immunoblots using antibodies against the viral M1 protein Southern Biotech and NS1B protein.

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Lary of the University of Connecticut Analytical Ultracentrafugation Facility for providing analytical ultracentrifugation analysis, and Dr. We also thank Drs. Xiao for helpful discussions and comments on this manuscript. This is a PDF file of an unedited manuscript that has been accepted for publication.

As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Mao carried out sample preparation, crystallization, and biophysical studies.

National Center for Biotechnology InformationU. Author manuscript; available in PMC Sep 6. Find articles by Shanshan Wang. Find articles by Robert M. Author information Copyright and License information Disclaimer. The publisher’s final edited version of this article is available at Structure. See other articles in PMC that cite the published article.

Associated Data Supplementary Materials. Abstract Influenza viruses cause a highly contagious respiratory disease in humans. Open in a separate window. Table 1 Data collection and refinement statistics.

The monomeric form of the NS1B CTD is sufficient to bind RNA Dimeric interactions observed in crystal structures may or may not occur pei solution, particularly under dilute protein conditions.

Mutation of key basic residues in the RNA-binding surface on the Cterminal domain of full-length NS1B protein results in attenuated influenza B viruses The structural and biophysical results outlined above reveal a novel, unanticipated RNA binding function in the C-terminal domain of d NS1 protein of influenza B viruses.

Structure determination and refinement At the home X-ray source wavelength 1. Supplementary Material Click 1882 to view. Acknowledgments We thank Drs. SAD phasing using iodide ions in a high-throughput structural genomics environment.