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Podsumowanie

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N⁰) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

Abstract

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N⁰) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N⁰ with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

Introduction

Nonsegmented negative-sense (NNS) RNA viruses include many significant human pathogens, such as rabies, Ebola, and respiratory syncytial virus (RSV)^1,2. RSV is the leading cause of respiratory illness such as bronchiolitis and pneumonia in young children and older adults worldwide³. Currently, no effective vaccines or antiviral therapies are available to prevent or treat RSV⁴. As part of the life cycle, the RSV genome serves as the template for replication by the RSV RNA dependent RNA polymerase to produce an antigenome, which in turn acts as the template to generate a progeny genome. Both genome and antigenome RNAs are entirely encapsidated by the nucleoprotein (N) to form the nucleocapsids (NCs)³. Because the NCs serve as the templates for both replication and transcription by the RSV polymerase, proper NC assembly is crucial for the polymerase to gain access to the templates for RNA synthesis⁵. Interestingly, based on the structural analyses of the NNS viral polymerases, it is hypothesized that several N proteins transiently dissociate from the NCs to allow the access of the polymerase and rebind to RNA after the RNA synthesis^{6,7,8,9,10,11,12}.  

Currently, the RSV RNA polymerization assay has been established using purified RSV polymerase on short naked RNA templates^13,14. However, the activities of the RSV polymerase do not reach optimal, as observed in the non-processive and abortive products generated by the RSV polymerase when using naked RNA templates. The lack of NC with virus-specific RNA is a primary barrier for the further mechanistic understanding of the RSV RNA synthesis. Therefore, using an authentic RNA template becomes a critical need to advance the fundamental knowledge of RSV RNA synthesis. The known structures of the nucleocapsid-like particles (NCLPs) from RSV and other NNS RNA viruses reveal that the RNAs in the NCLPs are either random cellular RNAs or average viral genomic RNAs^{15,16,17,18,19}. Together, the main hurdle is that N binds non-specifically to cellular RNAs to form NCLPs when N is overexpressed in the host cells.

To overcome this hurdle, we established a protocol to obtain RNA-free (N⁰) first and assemble N⁰ with authentic viral genomic RNA into NCLPs²⁰. The principle of this protocol is to obtain a large quantity of recombinant RNA-free N (N⁰) by co-expressing N with a chaperone, the N-terminal domain of RSV phosphoprotein (P_NTD). The purified N⁰P could be stimulated and assembled into NCLPs by adding RSV-specific RNA oligos, and during the assembly process, the chaperone P_NTD is displaced upon the addition of RNA oligos.

Here, we detail a protocol for the generation and assembly of RSV RNA-specific NCs. In this protocol, we describe the molecular cloning, protein preparation, in vitro assembly, and validation of the complex assembly. We highlight the cloning strategy to generate bi-cistronic constructs for protein coexpression for molecular cloning. For protein preparation, we describe the procedures of cell culture, protein extraction, and the purification of the protein complex. Then we discuss the method for in vitro assembly of the RSV RNA-specific NCs. Finally, we use size exclusion chromatography (SEC) and negative stain electron microscopy (EM) to characterize and visualize the assembled NCLPs.

Protokół

1. Molecular cloning NOTE: Ligase Independent Cloning (LIC) was used to make an RSV bi-cistronic coexpression construct plasmid. LIC is a method developed in the early 1990s, which uses the 3’-5’ Exo activity of the T4 DNA  polymerase to create overhangs with complementarity between the vector and the DNA insert21,22. The constructs were made using the 2BT-10 vector DNA, which consists of a 10x His tag at the N-terminal of the Open Reading Frame (ORF) (Figure 1). Perform linearization of LIC vectors using SSPI digestion. Combine 10 μL of SSPI 10X buffer, 4 μL of SSPI enzyme at a concentration of 5 U/μL, the equivalent volume of 5 μg of vector miniprep DNA, and sterile ddH2O to 100 μL. Incubate the digest for 3 hours at 37 °C. Run the digest on a 1.0% agarose gel for the extraction of vector DNA. Use a gel extraction kit to perform extraction and purification. Suspend the final volume of vector DNA in 30 μL of ddH2O and store it at -20 °C. Prepare the DNA inserts for N1-391 and P1-126 using the N1-391 Forward Primer, N1-391 Reverse Primer, P1-126 Forward Primer, and P1-126 Reverse Primer (Table 1). NOTE: There is sufficient overlap with the linearized vector to ensure a melting temperature of between 55 °C and 60 °C. For the reverse primer, there is sufficient overlap with the reverse complementary strand of the linearized vector for the same reason. Perform PCR amplification of the DNA insert using the conditions in Table 2 and Table 3. Extract the amplified DNA insert. Run the PCR products from the previous step on a 1.0% agarose gel, then extract and purify the bands by gel extraction. Suspend the final volume of extracted DNA in 15 μL of ddH2O. T4 DNA polymerase treatment of vector and insert DNA (Table 4). NOTE: Treatment must be performed separately for the vector DNA and the insert DNA. Incubate the mixture for 40 minutes at room temperature. Then, heat-inactivate the polymerase at 75 °C for 20 minutes. Store the reaction mixture at -20 °C. Anneal the LIC vector and the insert vector. Set up a negative control with 2 μL of LIC vector DNA and 2 μL of sterile ddH2O. Combine  2 μL of insert DNA and 2 μL of LIC vector DNA from the previous T4 DNA polymerase reactions in a 0.2 mL tube. Perform the annealing reaction at room temperature for 10 minutes. Quench the reaction with 1.3 μL of EDTA at a concentration of 25 mM. Transform the reaction into 100 μL of E. coli Top10 competent cells and plate them on an ampicillin selection plate23,24. Identify the positive constructs. Prepare the plasmid miniprep solution. This can be done through colony picking and inoculation in LB media. Usually, 3 colonies are sufficient. Incubate the mixture overnight at 37 °C. Centrifuge the mixture at 4,560 x g for 10 minutes and discard the supernatant. Resuspend the pellets in 250 μL of P1 buffer and prepare plasmid minipreps using a spin miniprep kit. Conduct a digestion analysis of the miniprep product using AseI or other restriction enzymes. Load samples on 0.8% agarose gel and run the digested plasmid. Analyze the gel under a UV lamp. Use a sequencing service to validate the sequence of the positive product. Obtain the coexpression DNA insert. Perform PCR to obtain N1-391 and P1-126 using the previously constructed 2BT-10 N1-391 and 2BT-10 P1-126 as templates. Perform the 1st PCR to obtain N1-391 from the 2BT-10 N1-391 construct using the PCR conditions in Table 2 and Table 3 with the N1-391 Forward primer and Reverse Primer  5 ́-GTGAAGATCCTGGCTGATGCAATGCGGCGGCG CGCCGCGATCGCGGATCC-3 ́. Perform the 2nd PCR to obtain P1-126 from the 2BT-10 P1-126 construct using the Forward primer: 5’-CCGCCGCATTGCATCAGCCAGGATCTTCACTGC AGGACTCGAGTTCTAGA-3 ́and the P1-126 Reverse primer (use the PCR conditions in Table 2 and Table 3). Finally, perform overlap PCR on the mixed products of the previous 2 PCR reactions to merge N1-391 and P1-126. Use the  N1-391 Forward primer and P1-126 Reverse primer. Use the PCR conditions in Table 5 and Table 6. Join the vector and the DNA insert. Treat the overlap PCR product with T4 DNA polymerase following the protocol in step 1.3. Anneal the LIC vector and PCR product following the protocol in step 1.4. Identify the positive constructs following the protocol in step 1.5. 2. Protein expression and purification NOTE: Use E. coli for the bi-cistronic construct of the coexpression of both N and P. Culture the cells at 37 °C, but carry out the expression at a reduced temperature (16 °C) overnight. Purify the protein complexes through a combination of cobalt column, ion exchange, and size exclusion chromatography (Figure 2). Use the E. coli BL21(DE3) strain for protein production. Grow 4 L cell cultures at 37 °C in LB (Luria Broth) medium until OD600 reaches 0.6. Lower the temperature to 16 °C. An hour later, induce the expression with 0.5 mM isopropyl 1-thio–D-galactopyranoside (IPTG) overnight. Centrifuge the cells at 4,104 x g for 25 min and then discard the supernatant. Resuspend the cell pellets in 200 mL of lysis buffer A (50 mM sodium phosphate, pH 7.4, 500 mM NaCl, 5 mM imidazole, 10% glycerol, and 0.2% NP40). Use 50 mL of lysis buffer to resuspend the cell pellets from 1 L of cell culture. Lyse the cells by sonication for 15 min, 3 seconds on, and 3 seconds off. Then centrifuge cells at 37,888 x g for 40 min. NOTE: The protocol can be paused by freezing the cells before sonication in a -80 °C freezer. Load the supernatant into a cobalt gravity column (diameter x length: 2.5 cm x 10 cm) with ~10mL of beads pre-equilibrated with 5-10 column volumes (CV) of lysis buffer. Wash the column with 5 CV of buffer B (50 mM Tris-HCl pH 7.4, 1 M NaCl, 10% glycerol, and 5 mM imidazole) and 5 CV of buffer C (50 mM Tris-HCl pH 7.4, 500 mM NaCl, 10% glycerol, and 5 mM imidazole). Elute the protein from the beads using 2 CV of buffer D (50 mM Tris-HCl pH 7.4, 500 mM NaCl, and 250 mM imidazole). Dilute the eluted protein 5x with QA buffer (50 mM Tris-HCl pH 8.0, 5% Glycerol) for the Q column. Wash the 5 mL of Q column with QA buffer to equilibrate the column, then load the diluted sample into the Q column using the peristaltic pump (e.g., Rabbit). Load the Q column into the HPLC machine along with QA buffer and QB buffer(50 mM Tris-HCl pH7.4, 1.5 M NaCl, 5% glycerol). Set up the flow rate as 1 mL/min. Run the “pump wash” program to wash the machine with QB buffer followed by QA buffer (1-2 CVs/each). Set the system flow to 3 mL/min. Set the UV1 to 280 nm and UV2 to 260 nm. Use a 96 deep-well plate to collect the fractions. Elute proteins using a stepwise gradient of elution agent (QB Buffer) applying 3-4 CV of each concentration, increasing the percentage by 5% each time starting at 0% QB. N0P  protein complex will come out at 15% QB Buffer. Once all of the protein is eluted, wash the column with 100% QB Buffer (2 CV). Isolate the protein by gel filtration Superdex 200 Increase 10/300 GL column (diameter x length: 1.0 cm x 30 cm) and equilibrate with buffer E (20 mM HEPES pH 7.4 and 200 mM NaCl). Analyze protein-containing fractions by SDS-PAGE. 3. In vitro assembly of the virus-specific NC NOTE: The in vitro assembly of the RSV-specific NC (N:RNA) was performed by incubating the prepared N0P complex with RNA oligos. Then, SEC chromatography was used to separate the assembly complex from the N0P and excess RNA (Figure 2). Mix and incubate the purified N0P complex with RNA oligo with the molecular ratio of 1:1.5 at room temperature for 1 hour, usually 1 mL of the protein N0P with a concentration of 1 mg/mL is enough for the next step. Set up the control sample, which only contains the same amount of N0P protein. Pre-equilibrate the gel filtration Superdex 200 Increase 10/300 GL column with the buffer E (20 mM HEPES, pH 7.4, 200 mM NaCl). Centrifuge the sample with 21,130 x g for 15 min, remove any precipitation and load the supernatant to the SEC column. Compare the SEC chromatography images of N:RNA assembly sample and N0P control sample, combine the A260/A280 ratio to identify which peaks are the assembled N-RNA, N0P, and free RNA. Collect the peak fractions, run the SDS-PAGE gel, or make grids. For the assembly N-RNA complex, collect all the fractions of N-RNA peak, do the RNA extraction and run the Urea-PAGE gel to double-check the length of specific RNA, which is the same as mixed and incubated at the first step. 4. Making negative stain grids NOTE: Negative stain electron microscopy (EM) is a method in which the molecules are adsorbed to a carbon film and then embedded in a layer of heavy metal atoms. Negative stain EM produces a high image contrast, making it easy to see and computationally align the particles. Another advantage is that the adsorption of the particles to a carbon film usually induces the molecules to adhere to the grid with few preferred orientations. When the molecules are in a similar orientation, it is easy to separate them into structurally distinct classes. Negative stain EM is thus the appropriate technique to guide sample preparation25,26 (Figure 3). Microwave or heat 5 mL of ddH2O in a small glass beaker using a Tungsten heater until it is boiling.  Weigh 37.5 mg of uranyl formate and add to 5 mL of heated ddH2O to make a 0.75% uranyl formate staining solution. Stir under an aluminum foil covered beaker to protect from light. Add 4 μL of 10 M NaOH to the staining solution and continue to stir for 15 min, protected from the light. Filter the solution through a 0.22 mm filter into a test tube. Use glow discharge to make the continuous carbon-coated EM grids hydrophilic27. NOTE: The grids are placed inside a chamber connected to a power supply. When high voltage is applied, the gas within the chamber ionizes, and the negatively charged ions deposit on the carbon grids to make them hydrophilic. Cut and fold a parafilm strip. Pipet 2 drops of 40 μL of the buffer on one side of the parafilm, and pipet another 2 drops of 40 μL of staining solution to the other side. To make the EM carbon grids, apply 3 μL of protein sample for 1 minute. Blot the grids against a blotting paper. NOTE: The grids are washed 2x with buffer, and 1x with the 0.75% uranyl formate staining solution blotted after each step. Hold the grid surface in the second drop of 0.75% uranyl formate staining solution for 30 seconds. Blot the grid against a blotting paper to remove excess stain solution and allow the grid to air-dry. Store the grids in the grid box before imaging.

Representative Results

Purification of RNA-free N0P protein With this protocol, a large-scale soluble heterodimeric RSV N0P complex can be obtained. The full length of N and N terminal part of P proteins were co-expressed with 10X His-Tag on the N protein in E. coli. N0P was purified using a cobalt column, ion exchange, and size exclusion chromatography. N0P contains both the full-length N and N terminal P but did not contain cellular RNA based on the UV absorbance A<s…

Discussion

The known nucleocapsid-like particle (NCLP) structures of the nonsegmented negative-sense (NNS) RNA viruses show that the assembled NCLPs are the complex N with host cellular RNAs when overexpressed in bacterial or eukaryotic expression systems^{15,16,17,18,19}. Previous studies have attempted to get the RNA free N with a variety of methods, such as the RNase A d…

Materials

Agarose	SIgma	A9539-500G	making construct using LIC method
Amicon Ultra-15 Centrifugal Filter Unit	Millipore	UFC901024	concentrate the protein sample
Ampicillin sodium	GOLD BIOTECHNOLOGY	5118.111317A	antibiotic for cell culture
AseI	NEB	R0526S	making construct using LIC method
Cobalt (High Density) Agarose Beads	Gold Bio	H-310-500	For purification of His-tag protein
Corning LSE Digital Dry Bath Heater	CORNING	6885-DB	Heate the sample
dCTP	Invitrogen	10217016	making construct using LIC method
dGTP	Invitrogen	10218014	making construct using LIC method
Glycerol	Sigma	G5516-4L	making solution
HEPES	Sigma	H3375-100G	making solution
HiTrap Q HP	Sigma	GE29-0513-25	Protein purification
Imidazole	Sigma	I5513-100G	making solution
IPTG (Isopropyl-beta-D-thiogalactopyranoside)	GOLD BIOTECHNOLOGY	1116.071717A	induce the expression of protein
Microcentrifuge Tubes	VWR	47730-598	for PCR
Misonix Sonicator XL2020 Ultrasonic Liquid Processor	SpectraLab	MSX-XL-2020	sonicator for lysing cell
Negative stain grids	Electron Microscopy Sciences	CF400-Cu-TH	For making negative stain grids
New Brunswick Innova 44/44R	eppendorf	M1282-0000	Shaker for culturing the cell
Nonidet P 40 Substitute	Sigma	74385-1L	making solution
OneTaq DNA Polymerase	NEB	M0480L	PCR
QIAquick Gel Extraction Kit	QIAGEN	28706	Purify DNA
SSPI-HF	NEB	R3132S	making construct using LIC method
Superose 6 Increase 10/300 GL	Sigma	GE29-0915-96	Protein purification
T4 DNA polymerase	Sigma	70099-3	making construct using LIC method
Thermo Scientific Sorvall RC 6 Plus Centrifuge	Fisher Scientific	36-101-0816	Centrifuge, highest speed 20,000 rpm
Trizma hydrochloride	Sigma	T3253-250G	making solution
Uranyl Formate	Electron Microscopy Sciences	22451	making negative stain solution