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Hannah T.
Baddock
,
Sanja
Brolih
,
Yuliana
Yosaatmadja
,
Malitha
Ratnaweera
,
Marcin
Bielinski
,
Lonnie p.
Swift
,
Abimael
Cruz-Migoni
,
Haitian
Fan
,
Jeremy R.
Keown
,
Alexander P.
Walker
,
Garrett m.
Morris
,
Jonathan M.
Grimes
,
Ervin
Fodor
,
Christopher J.
Schofield
,
Opher
Gileadi
,
Peter J.
Mchugh
Open Access
Abstract: The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.
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Jan 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Influenza A virus (IAV) contains a segmented RNA genome that is transcribed and replicated by the viral RNA polymerase in the cell nucleus. Replicated RNA segments are assembled with viral polymerase and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packaged into progeny virions. Host GTPase Rab11a associated with recycling endosomes is believed to contribute to this process by mediating the cytoplasmic transport of vRNPs. However, how vRNPs interact with Rab11a remains poorly understood. In this study, we utilised a combination of biochemical, proteomic, and biophysical approaches to characterise the interaction between the viral polymerase and Rab11a. Using pull-down assays we show that vRNPs but not cRNPs from infected cell lysates bind to Rab11a. We also show that the viral polymerase directly interacts with Rab11a and that the C-terminal two thirds of the PB2 polymerase subunit (PB2-C) comprising the cap-binding, mid-link, 627 and nuclear localization signal (NLS) domains mediate this interaction. Small-angle X-ray scattering (SAXS) experiments confirmed that PB2-C associates with Rab11a in solution forming a compact folded complex with a 1:1 stoichiometry. Furthermore, we demonstrate that the switch I region of Rab11a, that has been shown to be important for binding Rab11 family interacting proteins (Rab11-FIPs), is also important for PB2-C binding suggesting that IAV polymerase and Rab11-FIPs compete for the same binding site. Our findings expand our understanding of the interaction between the IAV polymerase and Rab11a in the cytoplasmic transport of vRNPs.
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Jan 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Open Access
Abstract: Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
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Jan 2022
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Open Access
Abstract: SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5′ capping of viral RNAs. The formation of the 5′ 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5′ triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5′ end of viral RNA via a 5′ to 5′ triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.
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Nov 2021
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Krios IV-Titan Krios IV at Diamond
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Max
Renner
,
Wanwisa
Dejnirattisai
,
Loic
Carrique
,
Itziar
Serna Martin
,
Dimple
Karia
,
Serban L.
Ilca
,
Shu F.
Ho
,
Abhay
Kotecha
,
Jeremy R.
Keown
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Jonathan M.
Grimes
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.
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Feb 2021
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[20223]
Abstract: Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.
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Nov 2020
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Open Access
Abstract: The impact of COVID-19 on public health and the global economy has led to an unprecedented research response, with a major emphasis on the development of safe vaccines and drugs. However, effective, safe treatments typically take over a decade to develop and there are still no clinically approved therapies to treat highly pathogenic coronaviruses. Repurposing of known drugs can speed up development and this strategy, along with the use of biologicals (notably monoclonal antibody therapy) and vaccine development programmes remain the principal routes to dealing with the immediate impact of COVID-19. Nevertheless, the development of broadly-effective highly potent antivirals should be a major longer term goal. Structural biology has been applied with enormous effect, with key proteins structurally characterised only weeks after the SARS-CoV-2 sequence was released. Open-access to advanced infrastructure for structural biology techniques at synchrotrons and high-end cryo-EM and NMR centres has brought these technologies centre-stage in drug discovery. We summarise the role of Diamond Light Source in responses to the pandemic and note the impact of the immediate release of results in fuelling an open-science approach to early-stage drug discovery.
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Nov 2020
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Yanchun
Peng
,
Alexander J.
Mentzer
,
Guihai
Liu
,
Xuan
Yao
,
Zixi
Yin
,
Danning
Dong
,
Wanwisa
Dejnirattisai
,
Timothy
Rostron
,
Piyada
Supasa
,
Chang
Liu
,
César
López-Camacho
,
Jose
Slon-Campos
,
Yuguang
Zhao
,
David I.
Stuart
,
Guido C.
Paesen
,
Jonathan M.
Grimes
,
Alfred A.
Antson
,
Oliver W.
Bayfield
,
Dorothy E. D. P.
Hawkins
,
De-Sheng
Ker
,
Beibei
Wang
,
Lance
Turtle
,
Krishanthi
Subramaniam
,
Paul
Thomson
,
Ping
Zhang
,
Christina
Dold
,
Jeremy
Ratcliff
,
Peter
Simmonds
,
Thushan
De Silva
,
Paul
Sopp
,
Dannielle
Wellington
,
Ushani
Rajapaksa
,
Yi-Ling
Chen
,
Mariolina
Salio
,
Giorgio
Napolitani
,
Wayne
Paes
,
Persephone
Borrow
,
Benedikt M.
Kessler
,
Jeremy W.
Fry
,
Nikolai F.
Schwabe
,
Malcolm G.
Semple
,
J. Kenneth
Baillie
,
Shona C.
Moore
,
Peter J. M.
Openshaw
,
M. Azim
Ansari
,
Susanna
Dunachie
,
Eleanor
Barnes
,
John
Frater
,
Georgina
Kerr
,
Oliver
Gould
,
Teresa
Lockett
,
Robert
Levin
,
Yonghong
Zhang
,
Ronghua
Jing
,
Ling-Pei
Ho
,
Richard J.
Cornall
,
Christopher P.
Conlon
,
Paul
Klenerman
,
Gavin R.
Screaton
,
Juthathip
Mongkolsapaya
,
Andrew
Mcmichael
,
Julian C.
Knight
,
Graham
Ogg
,
Tao
Dong
Open Access
Abstract: The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
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Sep 2020
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B24-Cryo Soft X-ray Tomography
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Ilias
Kounatidis
,
Megan L.
Stanifer
,
Michael A.
Phillips
,
Perrine
Paul-Gilloteaux
,
Xavier
Heiligenstein
,
Hongchang
Wang
,
Chidinma
Okolo
,
Thomas M.
Fish
,
Matthew C.
Spink
,
David I.
Stuart
,
Ilan
Davis
,
Steeveh
Boulant
,
Jonathan M.
Grimes
,
Ian M.
Dobbie
,
Maria
Harkiolaki
Diamond Proposal Number(s):
[21046, 18314]
Open Access
Abstract: Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.
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Jun 2020
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I03-Macromolecular Crystallography
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David Craig
Mcgowan
,
Wendy
Balemans
,
Werner
Embrechts
,
Magali
Motte
,
Jeremy
Keown
,
Christophe
Buyck
,
Jordi
Corbera
,
Mario
Funes
,
Laura
Moreno
,
Ludwig
Cooymans
,
Abdellah
Tahri
,
Julien
Eymard
,
Bart
Stoops
,
Rudy
Strijbos
,
Joke
Van Den Berg
,
Ervin
Fodor
,
Jonathan
Grimes
,
Anil
Koul
,
Tim H. M.
Jonckers
,
Pierre
Raboisson
,
Jerome
Guillemont
Diamond Proposal Number(s):
[14744]
Abstract: In the search for novel influenza inhibitors we evaluated 7-fluoro-substituted indoles as bioisosteric replacements for the 7-azaindole scaffold of Pimodivir, a PB2 (polymerase basic protein 2) inhibitor currently in clinical development. Specifically, a 5,7-difluoroindole derivative 11a was identified as a potent and metabolically stable influenza inhibitor. 11a demonstrated a favorable oral pharmacokinetic profile and in vivo efficacy in mice. In addition, it was found that 11a was not at risk of metabolism via aldehyde oxidase, an advantage over previously described inhibitors of this class. The crystal structure of 11a bound to influenza A PB2 cap region is disclosed here and deposited to the PDB.
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Oct 2019
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