Hepatitis B virus core protein allosteric modulators

RESEARCH ARTICLE

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids Christopher John Schlicksup1, Joseph Che-Yen Wang1,2, Samson Francis3, Balasubramanian Venkatakrishnan1, William W Turner3, Michael VanNieuwenhze4, Adam Zlotnick1* 1

Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States; 2Indiana University Electron Microscopy Center, Bloomington, United States; 3Assembly Biosciences, Carmel, United States; 4 Department of Chemistry, Indiana University, Bloomington, United States

Abstract Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (10 mg/ml.

Synthesis of HAP-TAMRA Synthesis of HAP13 (Bourne et al., 2008) and HAP-TAMRA are described in the patent literature (Zlotnick et al., 2014). Analysis of the product by HPLC at the 280 nm and 555 nm wavelengths showed a product with ~99% purity; ESI-TOF MS: calculated for C51H50ClFN8O7, m/z 940.35; M+ + H, m/z 941.36; Found, m/z 941.3; calculated for M+ + Na, m/z 963.4; Found, m/z 963.3.

Negative stain electron microscopy Samples from Figure 2a,b represent Cp149 assembly reactions at 50 mM HEPES, 300 mM NaCl, and 10 mM dimer. The reaction in Figure 2b was initiated in the presence of 40 mM HAP-TAMRA. Both samples were adsorbed to the surface of EMS carbon film 300 mesh coper grids, washed with water, and stained with 2% uranyl acetate. Samples were imaged with a JEOL 3200-FS electron microscope operated at 300kV.

Cryo-electron microscopy and image processing Samples for Cryo-EM were applied to a glow-discharged Quantifoil holey-carbon grids (R2/2). The grids were blotted with filter paper for 4 s before automated plunging into liquid ethane using an FEI vitrobot. The Cp149+HAP TAMRA data from Figure 11 and Figure 4 panel a were imaged using a JEOL 3200-FS electron microscope operated at 300kV with an in-column energy filter. Images were recorded at a nominal magnification of 80,000X, with a pixel size of 1.5 A˚, maintaining less than 35e- A˚2 dose, recorded on a Gatan Ultrascan 4000 4k 4 k CCD detector. The 2D class averages shown in Figure 4 panels b, c, and d were collected on the same JEOL 3200-FS microscope, but with a Direct Electron DE-12 detector, with an image sampling rate of 1.01 A˚/pixel. For the high-resolution 3D structure determination, first presented in Figure 5, samples were imaged on a FEI Titan Krios operated at 300 kV at a nominal magnification of 22,500. Images were recorded on a Gatan K2 Summit detector operating in ‘super-resolution’ mode, resulting in a pixel size of 0.65 A˚ with a dose of ~33 e- A˚2. Each exposure was 8 s long, and was collected as 35 individual frames. The data collection process was semi-automated using the Leginon system.(Suloway et al., 2005) The final maps are deposited in the EMDB database as 7295 for the T = 3 map and 7294 for the T = 4 map (Table 1). Cryo-EM classification and reconstruction was implemented using standard protocols of the EMAN2, Motioncorr2, and Relion software programs (Kimanius et al., 2016; Zheng et al., 2016; Tang et al., 2007). Upon convergence of the 3D structures, each map was sharpened by applying a negative B-factor which was obtained using the Guinier fitting procedure implemented in Relion (Rosenthal and Henderson, 2003). To assess the local variability in the quality of the structure a local resolution analysis was employed, also implemented in Relion. The local resolution procedure determined local FSC with a sampling window of 10 A˚, using the same negative B-factor obtained at the end of refinement.

Schlicksup et al. eLife 2018;7:e31473. DOI: https://doi.org/10.7554/eLife.31473

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Model determination The crystal structures of both apo capsid (1qgt)(Wynne et al., 1999b) and HAP18 bound capsid (5d7y)(Venkatakrishnan et al., 2016) were used as starting points for flexible model refinement imposing icosahedral non-crystallographic symmetry constraints, and using the PHENIX and eLBOW software programs (Afonine et al., 2012; Moriarty et al., 2009; Emsley et al., 2010). The model validation statistics we report were obtained from the final output of the Phenix real space refinement tool. To refine the pixel size of the map, we fit the model of the asymmetric unit into density and measured cross-correlation across pixel sizes. The optimal pixel size was determined to be 1.285 A˚, compared to 1.30 A˚ as determined by the microscope calibration. The final maps and models reflect this change. Structural comparisons and figure creation were carried out in UCSF Chimera. The final structure coordinates are deposited in the protein data bank as 6BVN for the T = 3 structure and 6BVF for the T = 4 structure.

Detection of binding in solution The final buffer conditions for all binding assays were 300 mM NaCl, 20 mM Tris, 1% DMSO, and pH 7.5 with varying protein and HAP-TAMRA. Size exclusion chromatography assays were performed using a superose 6 30/10 column plumbed into a Shimadzu HPLC equipped with a diode array detector. The HAP-TAMRA absorbance eluting in the capsid fractions was attributed to the capsid bound population. All HAP-TAMRA absorbance eluting later was attributed to free ligand. It is important to note that because the HAP-TAMRA probe is synthesized as a racemic mixture, and because only a single enantiomer is known to bind the HAP pocket, we expected to see at least half of the input HAP-TAMRA eluting as free ligand. When plotting the increase in 520 nm signal in the capsid fraction (Figure 2d), the A520 at 7.5 ml was used, corresponding to the pre-established center of the capsid elution volume. Absorbance and fluorescence was measured in a 96-well plate using a 200 ml sample volume by a Biotek Synergy H1 plate reader. Measurements based on fluorescence used an excitation wavelength of 520 nm and monitored emission at 580 nm. For plotting the change in 520/555 nm absorbance ratio, we used the maximum value of the capsid peak at each wavelength. Kinetic assays based on absorbance were sampled in 1 min intervals, where each data point is the value of 520 nm absorbance. To account for the varying presence of excess free probe in the titration time course (Figure 3b), the signal is presented as D520 nm absorbance compared to a probe-only reference. We defined this as DA520 = A520raw - A520inert – A520binding(t=0) where A520inert accounts for the absorbance of dye that does not participate in binding (because it was either the inactive enantiomer or present in superstoichiometric quantities), and A520binding(t=0) is the initial absorbance of the fraction of HAP-TAMRA that will participate in binding. This analysis assumes linear binding until saturation, based on the results in Figure 2d, but could underestimate the signal if binding were weaker.

Acknowledgements We would like to acknowledge critical discussions with Dr. Shefah Qazi regarding TAMRA photophysics. The IU Electron Microscopy Center (IUEMC), notably Dr. David Morgan, and the Purdue cryoEM facility at Purdue University, notably Drs. Thomas Klose and Valerie Bowman, provided invaluable support. HHMI and Assembly Biosciences contributed to upgrading the facilities at IUEMC. This work was supported by NIH R01-AI067417 and a supported research agreement from Assembly BioSciences to AZ.

Additional information Competing interests Christopher John Schlicksup: AZ has an interest in Assembly Biosciences, a company that is pursuing antivirals directed against HBV. Balasubramanian Venkatakrishnan: SF is an employee of Assembly Biosciences, a company that is pursuing antivirals directed against HBV. Michael VanNieuwenhze:


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WW is an employee of Assembly Biosciences, a company that is pursuing antivirals directed against HBV. The other authors declare that no competing interests exist.

Funding Funder

Grant reference number

Author

National Institute of Allergy and Infectious Diseases

R01-AI067417

Adam Zlotnick

Assembly Biosciences

Adam Zlotnick

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions Christopher John Schlicksup, Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing; Joseph Che-Yen Wang, Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing; Samson Francis, Conceptualization, Formal analysis, Supervision, Investigation, Visualization, Writing—review and editing; Balasubramanian Venkatakrishnan, Resources, Investigation, Methodology, Writing—original draft; William W Turner, Formal analysis, Visualization, Methodology; Michael VanNieuwenhze, Conceptualization, Resources, Methodology; Adam Zlotnick, Resources, Funding acquisition Author ORCIDs Adam Zlotnick

http://orcid.org/0000-0001-9945-6267

Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.31473.026 Author response https://doi.org/10.7554/eLife.31473.027

Additional files Supplementary files . Transparent reporting form DOI: https://doi.org/10.7554/eLife.31473.017

Major datasets The following datasets were generated:

Author(s)

Year Dataset title

Dataset URL

Database, license, and accessibility information

Christopher John Schlicksup, Joseph Che-Yen Wang, Adam Zlotnick

2017 Cryo-EM Structure of Hepatitis B virus T=4 capsid in complex with the fluorescent allosteric modulator HAP-TAMRA

http://www.rcsb.org/ pdb/search/structidSearch.do?structureId= 6BVF

Publicly available at the RCSB Protein Data Bank (accession no. 6BVF)


2017 Cryo-EM Structure of Hepatitis B virus T=3 capsid in complex with the fluorescent allosteric modulator HAP-TAMRA

http://www.rcsb.org/ pdb/search/structidSearch.do?structureId= 6BVN

Publicly available at the RCSB Protein Data Bank (accession no. 6BVN)


http://www.ebi.ac.uk/ 2017 Cryo-EM Structure of Hepatitis B pdbe/entry/emdb/EMDvirus T=4 capsid in complex with the fluorescent allosteric modulator 7294 HAP-TAMRA

Publicly available at the Electron Microscopy Data Bank (accession no. EMD-7294)


2017 Cryo-EM Structure of Hepatitis B http://www.ebi.ac.uk/ virus T=3 capsid in complex with pdbe/entry/emdb/EMDthe fluorescent allosteric modulator 7295 HAP-TAMRA

Publicly available at the Electron Microscopy Data Bank (accession no. EMD-7295)


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