Search Results

You searched for all publications:

with publication states 'Published (Approved)'

Search Again...

These results only include publications that have been reviewed and processed by Diamond Light Source. To also view papers that have not yet been processed click here.

You can use the folder icon under Actions to collate papers as required. You can then click on View Folder in the left-hand navigation to review and/or download your folder.

Exact matches
Related results

8 items found (0 items not approved), displaying all items.1

Instruments / Technical Area	Publication Details	Published
	The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome Fangzheng Wang , Carol m. Sheppard , Bhakti Mistry , Ecco Staller , Wendy s Barclay , Jonathan M. Grimes , Ervin Fodor , Haitian Fan Nucleic Acids Research 10 DOI: 10.1093/nar/gkac410 Open Access Show Abstract ▼ Abstract: The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.	May 2022
	Characterization of the SARS-CoV-2 ExoN (nsp14ExoN–nsp10) complex: implications for its role in viral genome stability and inhibitor identification 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 Nucleic Acids Research 73 DOI: 10.1093/nar/gkab1303 Open Access Show Abstract ▼ 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.	Jan 2022
I03-Macromolecular Crystallography	Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies Jeremy R. Keown , Zihan Zhu , Loic Carrique , Haitian Fan , Alexander P. Walker , Itziar Serna Martin , Els Pardon , Jan Steyaert , Ervin Fodor , Jonathan M. Grimes Nature Communications 13 DOI: 10.1038/s41467-021-27950-w Diamond Proposal Number(s): [19946] Open Access Show Abstract ▼ 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.	Jan 2022
B21-High Throughput SAXS	The C-terminal domains of the PB2 subunit of the influenza A virus RNA polymerase directly interact with cellular GTPase Rab11a Hana Veler , Haitian Fan , Jeremy Keown , Jane Sharps , Marjorie Fournier , Jonathan M. Grimes , Ervin Fodor Journal Of Virology DOI: 10.1128/jvi.01979-21 Diamond Proposal Number(s): [28534] Open Access Show Abstract ▼ 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.	Jan 2022
	The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme Alexander P. Walker , Haitian Fan , Jeremy R. Keown , Michael L. Knight , Jonathan M. Grimes , Ervin Fodor Nucleic Acids Research 5 DOI: 10.1093/nar/gkab1160 Open Access Show Abstract ▼ 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.	Nov 2021
I24-Microfocus Macromolecular Crystallography	Structure of an H3N2 influenza virus nucleoprotein Michael L. Knight , Haitian Fan , David L. V. Bauer , Jonathan M. Grimes , Ervin Fodor , Jeremy Keown Acta Crystallographica Section F Structural Biology Communications 77, 208 - 214 DOI: 10.1107/S2053230X2100635X Diamond Proposal Number(s): [19946] Open Access Show Abstract ▼ Abstract: Influenza A viruses of the H1N1 and H3N2 subtypes are responsible for seasonal epidemic events. The influenza nucleoprotein (NP) binds to the viral genomic RNA and is essential for its replication. Efforts are under way to produce therapeutics and vaccines targeting the NP. Despite this, no structure of an NP from an H3N2 virus has previously been determined. Here, the structure of the A/Northern Territory/60/1968 (H3N2) influenza virus NP is presented at 2.2 Å resolution. The structure is highly similar to those of the A/WSN/1933 (H1N1) and A/Hong Kong/483/97 (H5N1) NPs. Nonconserved amino acids are widely dispersed both at the sequence and structural levels. A movement of the 73–90 RNA-binding loop is observed to be the key difference between the structure determined here and previous structures. The data presented here increase the understanding of structural conservation amongst influenza NPs and may aid in the design of universal interventions against influenza.	Jul 2021
Krios IV-Titan Krios IV at Diamond	Host ANP32A mediates the assembly of the influenza virus replicase Loic Carrique , Haitian Fan , Alexander P. Walker , Jeremy R. Keown , Jane Sharps , Ecco Staller , Wendy S. Barclay , Ervin Fodor , Jonathan M. Grimes Nature 4 DOI: 10.1038/s41586-020-2927-z Diamond Proposal Number(s): [20223] Show Abstract ▼ 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.	Nov 2020
B21-High Throughput SAXS I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Structures of influenza A virus RNA polymerase offer insight into viral genome replication Haitian Fan , Alexander P. Walker , Loic Carrique , Jeremy Keown , Itziar Serna Martin , Dimple Karia , Jane Sharps , Narin Hengrung , Els Pardon , Jan Steyaert , Jonathan M. Grimes , Ervin Fodor Nature 573, 287 - 290 DOI: 10.1038/s41586-019-1530-7 Diamond Proposal Number(s): [10627, 14744, 19946, 14856, 20233] Show Abstract ▼ Abstract: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3,4,5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication—which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7,8,9,10—remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0–4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3′ cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs.	Sep 2019

Publications Database

Search Results

You searched for all publications:

Search Again...

Subject Areas (check all that apply) select all

Instruments used to collect data (check all that apply) select all

Collaborations involved (check all that apply) select all

Diamond Offline Facilities used

Relevant Technical Areas (check all that apply) select all

Menu for Anonymous User