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Structural analyses reveal the mechanism of inhibition of influenza virus NS1 by two antiviral compounds.

ABSTRACT: The influenza virus is a significant public health concern causing 250,000-500,000 deaths worldwide each year. Its ability to change quickly results in the potential for rapid generation of pandemic strains for which most individuals would have no antibody protection. This pandemic potential highlights the need for the continuous development of new drugs against influenza virus. As an essential component and well established virulence determinant, NS1 (nonstructural protein 1) of influenza virus is a highly prioritized target for the development of anti-influenza compounds. Here, we used NMR to determine that the NS1 effector domain (NS1ED) derived from the A/Brevig Mission/1/1918 (H1N1) strain of influenza (1918H1N1) binds to two previously described anti-influenza compounds A9 (JJ3297) and A22. We then used X-ray crystallography to determine the three-dimensional structure of the 1918H1N1 NS1ED Furthermore, we mapped the A9/A22-binding site onto our 1918H1N1 NS1ED structure and determined that A9 and A22 interact with the NS1ED in the hydrophobic pocket known to facilitate binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), suggesting that the two compounds likely attenuate influenza replication by inhibiting the NS1ED-CPSF30 interaction. Finally, our structure revealed that NS1ED could dimerize via an interface that we termed the ?3-?3 dimer. Taken together, the findings presented here provide strong evidence for the mechanism of action of two anti-influenza compounds that target NS1 and contribute significant structural insights into NS1 that we hope will promote and inform the development and optimization of influenza therapies based on A9/A22.

SUBMITTER: Kleinpeter AB

PROVIDER: S-EPMC6153301 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Structural analyses reveal the mechanism of inhibition of influenza virus NS1 by two antiviral compounds.

Kleinpeter Alex B AB Jureka Alexander S AS Falahat Sally M SM Green Todd J TJ Petit Chad M CM

The Journal of biological chemistry 20180803 38

The influenza virus is a significant public health concern causing 250,000-500,000 deaths worldwide each year. Its ability to change quickly results in the potential for rapid generation of pandemic strains for which most individuals would have no antibody protection. This pandemic potential highlights the need for the continuous development of new drugs against influenza virus. As an essential component and well established virulence determinant, NS1 (nonstructural protein 1) of influenza virus ...[more]

PMID: 30076219

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Project description:The reversible phosphorolysis of purine and pyrimidine nucleosides is an important biochemical reaction in the salvage pathway, which provides an alternative to the de novo purine and pyrimidine biosynthetic pathways. Structural studies in our laboratory and by others have revealed that only two folds exist that catalyse the phosphorolysis of all nucleosides, and provide the basis for defining two families of nucleoside phosphorylases. The first family (nucleoside phosphorylase-I) includes enzymes that share a common single-domain subunit, with either a trimeric or a hexameric quaternary structure, and accept a range of both purine and pyrimidine nucleoside substrates. Despite differences in substrate specificity, amino acid sequence and quaternary structure, all members of this family share a characteristic subunit topology. We have also carried out a sequence motif study that identified regions of the common subunit fold that are functionally significant in differentiating the various members of the nucleoside phosphorylase-I family. Although the substrate-binding sites are arranged similarly for all members of the nucleoside phosphorylase-I family, a comparison of the active sites from the known structures of this family indicates significant differences between the trimeric and hexameric family members. Sequence comparisons also suggest structural identity between the nucleoside phosphorylase-I family and both 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase and AMP nucleosidase. Members of the second family of nucleoside phosphorylases (nucleoside phosphorylase-II) share a common two-domain subunit fold and a dimeric quaternary structure, share a significant level of sequence identity (>30%) and are specific for pyrimidine nucleosides. Members of this second family accept both thymidine and uridine substrates in lower organisms, but are specific for thymidine in mammals and other higher organisms. A possible relationship between nucleoside phosphorylase-II and anthranilate phosphoribosyltransferase has been identified through sequence comparisons. Initial studies in our laboratory suggested that members of the nucleoside phosphorylase-II family require significant domain movements in order for catalysis to proceed. A series of recent structures has confirmed our hypothesis and provided details of these conformational changes. Structural studies of the nucleoside phosphorylases have resulted in a wealth of information that begins to address fundamental biological questions, such as how Nature makes use of the intricate relationships between structure and function, and how biological processes have evolved over time. In addition, the therapeutic potential of suppressing the nucleoside phosphorylase activity in either family of enzymes has motivated efforts to design potent inhibitors. Several research groups have synthesized a variety of nucleoside phosphorylase inhibitors that are at various stages of preclinical and clinical evaluation.

Project description:NS1 of influenza A virus is a potent antagonist of host antiviral interferon responses. This multifunctional protein with two distinctive domains, an RNA-binding domain (RBD) and an effector domain (ED) separated by a linker region (LR), is implicated in replication, pathogenesis, and host range. Although the structures of individual domains of NS1 from different strains of influenza viruses have been reported, the only structure of full-length NS1 available to date is from an H5N1 strain (A/Vietnam/1203/2004). By carrying out crystallographic analyses of full-length H6N6-NS1 (A/blue-winged teal/MN/993/1980) and an LR deletion mutant, combined with mutational analysis, we show here that these full-length NS1 structures provide an exquisite structural sampling of various conformational states of NS1 that based on the orientation of the ED with respect to RBD can be summarized as "open," "semi-open," and "closed" conformations. Our studies show that preference for these states is clearly dictated by determinants such as linker length, residue composition at position 71, and a mechanical hinge, providing a structural basis for strain-dependent functional variations in NS1. Because of the flexibility inherent in the LR, any particular NS1 could sample the conformational space around these states to engage ED in different quaternary interactions so that it may participate in specific protein-protein or protein-RNA interactions to allow for the known multifunctionality of NS1. We propose that such conformational plasticity provides a mechanism for autoregulating NS1 functions, depending on its temporal distribution, posttranslational modifications, and nuclear or cellular localization, during the course of virus infection.NS1 of influenza A virus is a multifunctional protein associated with numerous strain-specific regulatory functions during viral infection, including conferring resistance to antiviral interferon induction, replication, pathogenesis, virulence, and host range. NS1 has two domains, an RNA-binding domain and an effector domain separated by a linker. To date, the only full-length NS1 structure available is that from an H5N1 strain (A/Vietnam/1203/2004). Here, we determined crystal structures of the wild type and a linker region mutant of the H6N6 NS1 (A/blue-winged teal/MN/993/1980), which together with the previously determined H5N1 NS1 structure show that NS1 exhibits significant strain-dependent structural polymorphism due to variations in linker length, residue composition at position 71, and a mechanical hinge. Such a structural polymorphism may be the basis for strain-specific functions associated with NS1.

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Structural analyses reveal the mechanism of inhibition of influenza virus NS1 by two antiviral compounds.

Publications

Structural analyses reveal the mechanism of inhibition of influenza virus NS1 by two antiviral compounds.

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