Structure basis for allosteric regulation of lymphocytic choriomeningitis virus polymerase function by Z matrix protein

LCMV-L protein production and purification (strain Armstrong) with an N-terminal 6× His tag and a C-terminal 2× Strep tag was expressed in Sf9 insect cells using the pFASTbac HTB transfer vector (Invitrogen, USA). Sf9 cells expressing polymerase were lysed by sonication in Buffer A containing 50 mM NaH2PO4 (pH 8.0), 600 mM NaCl, 10% (v/v) glycerol, 4 mM MgCl2, and 0.2% NP-40 supplemented with phenylmethylsulfonyl fluoride (PMSF) and 1 U/ml benzonase (Millipore, USA). After clarification by high-speed centrifugation, the cell lysate was loaded onto a Strep column (GenScript, USA). After washing three times with Buffer B (100 mM Tris-HCl pH 8.0, 300 mM NaCl, and 1 mM EDTA) to remove nonspecifically bound protein, the target protein was subsequently eluted using Buffer C containing 100 mM Tris-HCl pH 8.0 and 300 mM NaCl supplemented with 1 mM EDTA and 50 mM biotin. The eluted L protein was pooled and further purified using a heparin column (GE Healthcare, USA) in 20 mM HEPES (pH 7.5) with 2 mM dithiothreitol (DTT). The L protein was eluted from the heparin column using a gradient of NaCl to a final concentration of 600 mM. The fractions near the maximum height of the peak were combined, concentrated with an Amicon Ultra Centrifugal Filter (Millipore, USA), flash-frozen and stored at -80 °C. Full-length LCMV-Z (strain Armstrong) was constructed into pGEX-6p-1 with a GST tag followed by an HRV 3C site in the N-terminus, and LCMV Z mutants were generated using site-directed mutagenesis. The plasmids were transformed into E. coli BL21 (DE3), and the transformed cells were cultured at 37 °C in LB media containing 100 mg/L ampicillin. After the OD600 reached 0.6, the culture was cooled to 16 °C and then induced with 0.5 mM IPTG (supplemented with 100 mM ZnSO4). After overnight induction, the cells were harvested through centrifugation, and the pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl) supplemented with phenylmethylsulfonyl fluoride (PMSF) and homogenized with an ultrahigh-pressure cell disrupter at 4 °C. The insoluble material was removed through centrifugation at 13,000 rpm. The fusion protein was first purified by GST affinity chromatography and eluted through on-column tag cleavage by Prescission protease. The eluate was further purified by a Hitrap Q ion-exchange column (GE Healthcare, USA), where pure LCMV Z was collected in the flow through. Following the ion-exchange purification, the LCMV-Z was purified onto a Superdex 200 10/300 Increase column (GE Healthcare, USA) in a buffer containing 10 mM Tris-HCl, pH 7.5, and 150 mM NaCl. The peak was analyzed by SDS–PAGE and concentrated with an Amicon Ultra centrifugal concentrator (Millipore, USA).

with a molar ratio of L:Z=1:1 at 4 °C for 2 h.The mixture was then loaded onto a Superose 6 10/300 GL (GE Healthcare, USA) column equilibrated with 20 mM HEPES, pH 7.5, 500 mM NaCl, and 1 mM dithiothreitol (DTT).The complex fractions were collected and concentrated to 1 mg/ml.An aliquot of 3 μl of sample at 1 mg/ml was loaded onto a glowdischarged Quantifoil R1.2/1.3Cu grid (400 mesh) (Quantifoil, Germany).The grid was then blotted for 3.5 s with a blot force of 0 in 100% relative humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI, USA).Cryo-EM data were collected with a 300-kV Titan Krios electron microscope (FEI, USA) and a K2 Summit direct electron detector (Gatan, USA).A series of micrographs were recorded as movies (32 frames, 5.76 s) under super-resolution counting mode at a calibrated magnification of 130,000×, resulting in a pixel size of 0.54 Å per pixel.Raw image of LCMV L-Z particles in vitreous ice recorded at -1.5 to -2.5 μm defocus.Statistics for the data collection and refinement are summarized in Supplementary Table 1.

Cryo-EM image processing
Individual frames from each micrograph movie were aligned and averaged using MotionCor2 (Zheng et al., 2017) to produce drift-corrected 2× binned images.Particles were picked and selected in RELION 3.0 (Scheres, 2012) from dose-weighed micrographs with a box size of 220 pixels, and the contrast transfer function (CTF) parameters were estimated using CTFFIND4 (Rohou and Grigorieff, 2015).Subsequent steps for particle picking, 2D and 3D classification and the reconstruction of the final selected particles were performed with THUNDER and CryoSparc (Punjani et al., 2017;Hu et al., 2018).A total of 405,657 particles remained in two classes representing the individual L and the L-Z complex.Homogeneous 3D refinement of these two particle stacks resulted in a 3.4-Å map for the individual L protein and a 3.6-Å map for the L-Z complex.The final resolution was assessed using the gold-standard FSC criterion (FSC = 0.143) with RELION 3.0 (Scheres, 2012).The data quality and the particle distribution were also analyzed by 3DFSC (Tan et al., 2017) and cryo-EF (Naydenova and Russo, 2017).

Model building and refinement
To solve the structure of the LCMV-L protein, the structure of the RdRp region of LASV-L (Peng et al., 2020) and the crystal structure of LACV-Z (Hastie et al., 2016) were individually manually placed, and were rigid-body fitted into the cryo-EM density map with UCSF Chimera (Pettersen et al., 2004).Other parts of the model were manually built in Coot (Emsley et al., 2010) with the guidance of the cryo-EM density, except for several loops or the most C-terminal regions (residues 191-199, 307-314, 356-369, 401-410, 466-471, 507-523, 785-831, 848-856, 883-887, 919-934, 955-1080, 1250-1254, 1549-1566, 1579-1601, 1699-1731, 1757-1771 and 1815-2210) of LCMV-L and the N/C-terminal region (residues 1-26 and 75-90) of LCMV-Z that could not be traced due to a lack of interpretable densities.Manual model building was subsequently performed using Coot in combination with real space refinement with Phenix (Afonine et al., 2012) to replace the corresponding amino acids in the model with those from LCMV L and LCMV L-Z.The density maps were kept constant during the entire fitting process, and only the atomic coordinates were subjected to refinement.The data validation statistics are shown in Supplementary Table 1.

GST pull-down assay
LCMV L (5 μg) was incubated with GST-tagged Z or GST alone (3 μg) in binding buffer (PBS with 250 mM KCl and 0.2% Nonidet P-40) (Volpon et al., 2010) in a total volume of 50 μL at 4 °C for 30 min.Then, the reaction volumes were raised to 500 μl with binding buffer, followed by 20 μl of packed glutathione Sepharose resin (GE Health care, USA) and incubation at 4 °C for 1 h while rotating.Glutathione resin and bound proteins were gently pelleted by centrifugation at 500 ×g for 1 min and washed three times with wash buffer (PBS with 500 mM KCl and 1.0% Nonidet P-40).Proteins were eluted by boiling in 25 μl of SDS loading buffer, and 10 μl was analyzed by 10% denaturing SDS/PAGE and staining with colloidal Coomassie (Sigma, USA).

In vitro polymerase activity assay
An in vitro RNA synthesis assay was performed using a 19-nt template corresponding to the conserved 3' terminus and 20-nt 5' terminus RNA of the viral genome S segment (Supplementary Figure 7).The ds-RNAs were 3' and 5' RNA preannealed at 65 °C before use.1μg of purified L (final reaction concentration of 0.4 μM) was incubated with 2 μM RNA (3'/5'/ds/3' and 5') in transcription buffer (50 mM Tris-HCl pH 7.0, 40 mM NaCl, 5 mM MnCl2, 10 mM KCl, 1 mM dithiothreitol (DTT), and 0.1 mg/mL BSA) at 25 °C for 30 min.After initial incubation, 1 mM cold ATP/CTP/GTP and 1 μL of [α-32P]-UTP (∼0.5 μCi) were added, and the reactions were incubated at 30 °C for 3 h in a final volume of 10 μL.Where indicated, RNA synthesis reactions were supplemented with 2 μM final concentration recombinant Z and Z mutants during L-RNA complex initial incubation.After 3 h incubation, reactions were terminated by 10 μL of 2× stop solution (95% deionized formamide, 20 mM EDTA) and incubation at 95 °C for 2 min.Reactions were analyzed by denaturing gel electrophoresis on a 20% polyacrylamide-urea (7 M) sequencing gel with 0.5× TBE.Gels were exposed for 8 h using a phosphor screen (GE Healthcare, USA), and the radiolabeled RNAs were visualized by a Typhoon FLA 9500 scanner (GE Healthcare, USA).