Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies

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.

Fig. 1. Cryo-EM structures of the 1918 polymerase in three conformations.

Monomeric conformations of the 1918 polymerase in full transcriptase (class 1) (a), partial transcriptase (class 2a) (b), or replicase conformations (class 3) (c).

Fig. 2. Nanobody binding sites of the 1918 polymerase determined by cryo-EM and X-ray crystallography.

a Composite model showing the binding position of each nanobody on the surface of the 1918 polymerase determined using cryo-EM. C-terminal domain of PA, PA-C; N-terminal one third of PB2, PB2-N. b–d Crystal structures of PB2 cap-mid-link and nanobody complexes.

Fig. 3. Effect of nanobodies on 1918 polymerase function and virus growth.

a Effect of nanobodies on the activity of recombinant 1918 RNP in the presence of a source of capped primer. Data are mean ± s.e.m. n = 3 independent RNP purifications and reactions. Ordinary one-way ANOVA was used to compare mRNA and cRNA levels in the absence (no Nanobody) and presence of the indicated nanobody. P < 0.05 is considered significant. b Effect of nanobodies on 1918 influenza polymerase activity using a luciferase-reporter minireplicon assay. Data are mean ± s.e.m. n = 3 independent transfections with n = 2 technical replicates. Ordinary one-way ANOVA was used to compare the relative luciferase intensity in the presence and absence of nanobodies. P < 0.05 is considered significant. c Effect of two sets of nanobodies on the replication of a reassortant A/WSN/1933 virus encoding PB1, PB2, PA and NP of A/Brevig Mission/1/1918 (WSN-1918^RNP) virus in HEK293T cells. Data are mean ± s.e.m. n = 3 independent transfections and infections for each set of nanobodies. PFU plaque-forming units. Ordinary one-way ANOVA was used to compare the viral titres in the presence and absence of nanobodies at a given time point. P values are as follows: for Nb8190 P = 0.0007, P = 0.0067, P = 0.0331 and P = 0.0199, for Nb8207 P = 0.0006, P = 0.0063, P = 0.0301 and P = 0.0185, for Nb8210 P = 0.0084, P = 0.0925, P = 0.3444 and P = 0.0696, for Nb8191 P = 0.1030, P = 0.0009, P = 0.0141 and P < 0.0001, for Nb8192 P = 0.1227, P = 0.0008, P = 0.0136 and P = < 0.0001, for Nb8208 P = 0.1179, P = 0.0011, P = 0.0205 and P = 0.0025, and for Nb8210 P = 0.7246, P = 0.9865, P = 0.2269 and P = 0.0340, at 16, 24, 32 and 48 h post infection, respectively. P < 0.05 is considered significant. For quantification of viral RNA levels during the course of infection, see Supplementary Fig. 8b. Source data are provided as a Source Data file.

Fig. 4. Inhibitory mechanisms of nanobodies.

a Effect of nanobodies on primary transcription by a reassortant A/WSN/1933 virus polymerase and NP of A/Brevig Mission/1/1918 virus in HEK293T cells transfected to express the indicated nanobodies. Data are mean ± s.e.m. n = 3 independent transfections and infections. Ordinary one-way ANOVA was used to compare the relative mRNA levels in the presence and absence of nanobodies. P < 0.05 is considered significant. b Effect of nanobodies on binding of purified recombinant 1918 pandemic influenza virus polymerase to serine-5-phosphorylated Pol II CTD mimic peptide. Top, a representative silver-stained gel. Bottom, quantification of gels from n = 4 independent binding assays. Data are mean ± s.e.m. Ordinary one-way ANOVA was used to compare the relative polymerase binding in the presence and absence of nanobodies. P < 0.05 is considered significant. c, d Effect of nanobodies on cRNA levels produced by a reassortant A/WSN/1933 virus encoding polymerase and NP of A/Brevig Mission/1/1918 virus in HEK293T cells transfected to express the indicated nanobodies and influenza virus NP and polymerase subunits, including a polymerase active site mutant PB1 (PB1a) (c) or wild-type PB1 (d). Plasmid to express PB1a (no PB1a) (c) or PB1 (no PB1) (d) was omitted as negative control. Data are mean ± s.e.m. n = 3 independent transfections and infections. Ordinary one-way ANOVA was used to compare the relative cRNA levels (c) or RNA levels (mRNA, cRNA and vRNA combined) (d) in the presence and absence of nanobodies. P < 0.05 is considered significant. Source data are provided as a Source Data file.

Fig. 5. Inhibitory sites on the 1918 polymerase.

a Binding position of inhibitory nanobodies mapped on to the transcriptase conformation of the polymerase (PDB ID: 7NHX). Note that the binding of Nb8190 and Nb8207 is for illustration purposes only; the binding of these nanobodies is incompatible with the transcriptase conformation shown. Subunits and domains are coloured as follows: PA (yellow), PB1 (blue), PB2 N-terminal domain (grey), cap-binding domain (bright green), mid-link (red), 627 domain/NLS (dark green). 5′ and 3′ vRNA promoters are coloured purple and pink, respectively. b Location of each nanobody interaction site annotated with biological function. C-terminal domain of host RNA polymerase II, Pol II CTD.