Jian Lei

Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

Zooming in on the Zika virus protease The lack of a vaccine or antiviral drugs to combat the Zika virus has scientists scrambling to identify and better characterize potential drug targets. One attractive candidate is the NS2B/NS3 viral protease, which, together with host cell proteases, cleaves the viral polyprotein into the individual proteins required for viral replication. Lei et al. report the crystal structure of this protease bound to a peptido-mimetic inhibitor. The structure reveals key interactions that probably contribute to the high catalytic efficiency of this enzyme relative to other flaviviruses, indicating promising starting points for drug design. Science, this issue p. 503 Analysis of Zika virus protease bound to a peptidomimetic inhibitor reveals its catalytic efficiency and promise as a drug target. The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp83 of NS2B; ion-pairing between Asp83 and the P2 residue of the substrate likely accounts for the enzyme’s high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing.

Crystal structure of the papain-like protease of MERS coronavirus reveals unusual, potentially druggable active-site features.

Abstract The Middle-East Respiratory Syndrome coronavirus (MERS-CoV) causes severe acute pneumonia and renal failure. The MERS-CoV papain-like protease (PLpro) is a potential target for the development of antiviral drugs. To facilitate these efforts, we determined the three-dimensional structure of the enzyme by X-ray crystallography. The molecule consists of a ubiquitin-like domain and a catalytic core domain. The catalytic domain displays an extended right-hand fold with a zinc ribbon and embraces a solvent-exposed substrate-binding region. The overall structure of the MERS-CoV PLpro is similar to that of the corresponding SARS-CoV enzyme, but the architecture of the oxyanion hole and of the S3 as well as the S5 specificity sites differ from the latter. These differences are the likely reason for reduced in vitro peptide hydrolysis and deubiquitinating activities of the MERS-CoV PLpro, compared to the homologous enzyme from the SARS coronavirus. Introduction of a side-chain capable of oxyanion stabilization through the Leu106Trp mutation greatly enhances the in vitro catalytic activity of the MERS-CoV PLpro. The unique features observed in the crystal structure of the MERS-CoV PLpro should allow the design of antivirals that would not interfere with host ubiquitin-specific proteases.

Virus–host interactomes — antiviral drug discovery

One of the key questions in virology is how viruses, encoding relatively few genes, gain temporary or constant control over their hosts. To understand pathogenicity of a virus it is important to gain knowledge on the function of the individual viral proteins in the host cell, on their interactions with viral and cellular proteins and on the consequences of these interactions on cellular signaling pathways. A combination of transcriptomics, proteomics, high-throughput technologies and the bioinformatical analysis of the respective data help to elucidate specific cellular antiviral drug target candidates. In addition, viral and human interactome analyses indicate that different viruses target common, central human proteins for entering cellular signaling pathways and machineries which might constitute powerful broad-spectrum antiviral targets.

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin.

The papain-like protease (PLpro) of Middle-East respiratory syndrome coronavirus (MERS-CoV) has proteolytic, deubiquitinating, and deISGylating activities. The latter two are involved in the suppression of the antiviral innate immune response of the host cell. To contribute to an understanding of this process, we present here the X-ray crystal structure of a complex between MERS-CoV PLpro and human ubiquitin (Ub) that is devoid of any covalent linkage between the two proteins. Five regions of the PLpro bind to two areas of the Ub. The C-terminal five residues of Ub, RLRGG, are similar to the P5–P1 residues of the polyprotein substrates of the PLpro and are responsible for the major part of the interaction between the two macromolecules. Through sitedirected mutagenesis, we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PLpro. The enzyme appears not to be optimized for catalytic efficiency; thus, replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities. Ubiquitin binding by MERS-CoV PLpro involves remarkable differences compared to the corresponding complex with SARS-CoV PLpro. The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PLpro.

p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

Significance Severe acute respiratory syndrome coronavirus (SARS-CoV) is one of the most pathogenic human coronaviruses. Virulence is reflected in the molecular interplay between virus and host cells. Here we show a strategy of how SARS-CoV antagonizes the host antiviral factor p53, which impairs viral replication. The papain-like protease of the nonstructural protein 3 of SARS-CoV and other coronaviruses physically interact with and stabilize E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1), thereby augmenting RCHY1-mediated degradation of p53. The SARS-unique domain (SUD) enhances these effects. Knockout of p53 promotes replication of SARS-CoV replicons and of infectious virus. Taken together we identify cellular p53 as antiviral measure of coronavirus-infected cells, which is counteracted via the stabilization of RCHY1 by viral SUD and papain-like protease (PLpro) proteins and via ubiquitination of p53. Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

ABSTRACT The multi‐domain non‐structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome, with an average molecular mass of about 200 kD. Nsp3 is an essential component of the replication/transcription complex. It comprises various domains, the organization of which differs between CoV genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known CoVs: the ubiquitin‐like domain 1 (Ubl1), the Glu‐rich acidic domain (also called “hypervariable region”), a macrodomain (also named “X domain”), the ubiquitin‐like domain 2 (Ubl2), the papain‐like protease 2 (PL2pro), the Nsp3 ectodomain (3Ecto, also called “zinc‐finger domain”), as well as the domains Y1 and CoV‐Y of unknown functions. In addition, the two transmembrane regions, TM1 and TM2, exist in all CoVs. The three‐dimensional structures of domains in the N‐terminal two thirds of Nsp3 have been investigated by X‐ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy since the outbreaks of Severe Acute Respiratory Syndrome coronavirus (SARS‐CoV) in 2003 as well as Middle‐East Respiratory Syndrome coronavirus (MERS‐CoV) in 2012. In this review, the structures and functions of these domains of Nsp3 are discussed in depth. HighlightsNonstructural protein 3 (˜200 kD) is a multifunctional protein comprising up to 16 different domains and regions.Nsp3 binds to viral RNA, nucleocapsid protein, as well as other viral proteins, and participates in polyprotein processing.The papain‐like protease of Nsp3 is an established target for new antivirals.Through its de‐ADP‐ribosylating, de‐ubiquitinating, and de‐ISGylating activities, Nsp3 counteracts host innate immunity.Structural data are available for the N‐terminal two thirds of Nsp3, but domains in the remainder are poorly characterized.

RNA-virus proteases counteracting host innate immunity

Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen‐associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern‐recognition receptors of the host, and innate immune responses are induced. Through production of type‐I and type‐III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive‐sense single‐stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses.

Crystal structure of the middle domain of human poly(A)-binding protein-interacting protein 1.

In eukaryotes, the poly(A)-binding protein (PABP) is one of the important factors for initiation of messenger RNA translation. PABP activity is regulated by the PABP-interacting proteins (Paips), which include Paip1, Paip2A, and Paip2B. Human Paip1 has three different isoforms. Here, we report the crystal structure of the middle domain of Paip1 isoform 2 (Paip1M) as determined by single-wavelength anomalous dispersion phasing. The structure reveals a crescent-shaped domain consisting of 10 α-helices and two antiparallel β-strands forming a β-hairpin. The 10 α-helices are arranged as five HEAT repeats which form a double layer of α helices with a convex and a concave surface. Despite low sequence identity, the overall fold of Paip1M is similar to the middle domain of human eIF4GII and yeast eIF4GI. Moreover, the amino-acid sequence motif and the local structure of eIF4G involved in binding of eIF4A, are conserved in Paip1. The structure reported here is the first of a member of the Paip family, thereby filling a gap in our understanding of initiation of eukaryotic mRNA translation in three dimensions.

Lybatides from Lycium barbarum contain an unusual cystine-stapled helical peptide scaffold

Cysteine-rich peptides (CRPs) of 2–6 kDa are generally thermally and proteolytically stable because of their multiple cross-bracing disulfide bonds. Here, we report the discovery and characterization of two novel cystine-stapled CRPs, designated lybatide 1 and 2 (lyba1 and lyba2), from the cortex of Lycium barbarum root. Lybatides, 32 to 33 amino acids in length, are hyperstable and display a novel disulfide connectivity with a cysteine motif of C-C-C-C-CC-CC which contains two pairs of adjacent cysteines (-CC-CC). X-ray structure analysis revealed the presence of a single cystine-stabilized (α + π)-helix in lyba2, a rare feature of CRPs. Together, our results suggest that lybatides, one of the smallest four-disulfide-constrained plant CRPs, is a new family of CRPs. Additionally, this study provides new insights into the molecular diversity of plant cysteine-rich peptides and the unusual lybatide scaffold could be developed as a useful template for peptide engineering and therapeutic development.

X‐ray structure of the SH3 domain of the phosphoinositide 3‐kinase p85β subunit

Src-homology 3 (SH3) domains are involved in extensive protein-protein interactions and constitute key elements of intracellular signal transduction. Three-dimensional structures have been reported for SH3 domains of various proteins, including the 85 kDa regulatory subunit (p85) of phosphoinositide 3-kinase. However, all of the latter structures are of p85 isoform α and no crystal structure of the SH3 domain of the equally important isoform β has been reported to date. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.0 Å resolution of the SH3 domain of human p85β is described. The structure reveals a compact β-barrel fold very similar to that of p85α. However, binding studies with two classes of proline-rich ligand peptides demonstrate that the ligand-binding specificity differs slightly between the SH3 domains of human p85β and p85α, despite their high structural similarity.