tetano
Editor, Senior Moderator
Nature. 2019 Sep 4. doi: 10.1038/s41586-019-1530-7. [Epub ahead of print]
[h=1]Structures of influenza A virus RNA polymerase offer insight into viral genome replication.[/h] Fan H[SUP]1[/SUP], Walker AP[SUP]1[/SUP], Carrique L[SUP]2[/SUP], Keown JR[SUP]2[/SUP], Serna Martin I[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3[/SUP], Karia D[SUP]2[/SUP], Sharps J[SUP]1[/SUP], Hengrung N[SUP]1,[/SUP][SUP]2,[/SUP][SUP]4[/SUP], Pardon E[SUP]5[/SUP], Steyaert J[SUP]6[/SUP], Grimes JM[SUP]7,[/SUP][SUP]8[/SUP], Fodor E[SUP]9[/SUP].
[h=3]Author information[/h] 1 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK. 2 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. 3 Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands. 4 Francis Crick Institute, London, UK. 5 VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium. 6 Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium. 7 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. jonathan@strubi.ox.ac.uk. 8 Diamond Light Source, Didcot, UK. jonathan@strubi.ox.ac.uk. 9 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK. ervin.fodor@path.ox.ac.uk.
[h=3]Abstract[/h] Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans[SUP]1,2[/SUP]. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol[SUB]A[/SUB]) composed of PB1, PB2 and PA subunits[SUP]3-5[/SUP]. Although the high-resolution crystal structure of FluPol[SUB]A[/SUB] of bat influenza A virus has previously been reported[SUP]6[/SUP], there are no complete structures available for human and avian FluPol[SUB]A[/SUB]. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase[SUP]7-10[/SUP]-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol[SUB]A[/SUB] 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 ?, in the presence or absence of a cRNA or vRNA template. In solution, FluPol[SUB]A[/SUB] 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 FluPol[SUB]A[/SUB] 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 FluPol[SUB]A[/SUB] 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 FluPol[SUB]A[/SUB] dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol[SUB]A[/SUB], as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol[SUB]A[/SUB] that could be targeted in the development of antiviral drugs.
PMID: 31485076 DOI: 10.1038/s41586-019-1530-7
[h=1]Structures of influenza A virus RNA polymerase offer insight into viral genome replication.[/h] Fan H[SUP]1[/SUP], Walker AP[SUP]1[/SUP], Carrique L[SUP]2[/SUP], Keown JR[SUP]2[/SUP], Serna Martin I[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3[/SUP], Karia D[SUP]2[/SUP], Sharps J[SUP]1[/SUP], Hengrung N[SUP]1,[/SUP][SUP]2,[/SUP][SUP]4[/SUP], Pardon E[SUP]5[/SUP], Steyaert J[SUP]6[/SUP], Grimes JM[SUP]7,[/SUP][SUP]8[/SUP], Fodor E[SUP]9[/SUP].
[h=3]Author information[/h] 1 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK. 2 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. 3 Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands. 4 Francis Crick Institute, London, UK. 5 VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium. 6 Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium. 7 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. jonathan@strubi.ox.ac.uk. 8 Diamond Light Source, Didcot, UK. jonathan@strubi.ox.ac.uk. 9 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK. ervin.fodor@path.ox.ac.uk.
[h=3]Abstract[/h] Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans[SUP]1,2[/SUP]. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol[SUB]A[/SUB]) composed of PB1, PB2 and PA subunits[SUP]3-5[/SUP]. Although the high-resolution crystal structure of FluPol[SUB]A[/SUB] of bat influenza A virus has previously been reported[SUP]6[/SUP], there are no complete structures available for human and avian FluPol[SUB]A[/SUB]. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase[SUP]7-10[/SUP]-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol[SUB]A[/SUB] 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 ?, in the presence or absence of a cRNA or vRNA template. In solution, FluPol[SUB]A[/SUB] 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 FluPol[SUB]A[/SUB] 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 FluPol[SUB]A[/SUB] 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 FluPol[SUB]A[/SUB] dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol[SUB]A[/SUB], as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol[SUB]A[/SUB] that could be targeted in the development of antiviral drugs.
PMID: 31485076 DOI: 10.1038/s41586-019-1530-7