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).
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.
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 synthesis6,7,8,9,10,11,12.
Currently, the RSV RNA polymerization assay has been established using purified RSV polymerase on short naked RNA templates13,14. 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 RNAs15,16,17,18,19. 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 (PNTD). The purified N0P could be stimulated and assembled into NCLPs by adding RSV-specific RNA oligos, and during the assembly process, the chaperone PNTD 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.
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).
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).
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).
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).
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 A260/A280 ratio20 (Figure 4).
Assembly of N-RNA and checking with negative stain
We then demonstrated that the purified N0P could be stimulated and assembled into Nuceloplasmid-like particles (NCLPs) by incubating with specific RNA oligos. The NCLPs were assembled by incubating the N0P with RNA oligos with the ratio of 1:1.5 at room temperature for 1 hour and then run through the gel filtration column. When the N:RNA complex forms, it shows three peaks: the 1st peak is N:RNA, the 2nd peak is N0P, and the 3rd peak is excess free RNA. The highest fraction of the N:RNA peak creates the negative stain grids for checking with EM20 (Figure 5).
Figure 1. The illustration of the plasmid constructions. A. The construct of the RSV N1-391; B. The construct of the RSV P1-126; C. The bi-cistronic construct for the coexpression of N1-391 and P1-126. The first gene RSV N1-391, the second gene RSV P1-126, the antibiotic-resistant gene (AmpR), and the promoters are highlighted in yellow, cyan, pink, and orange boxes, respectively. In summary, the gene inserts of the RSV N1-391 and the RSV P1-126 are constructed separately and assembled. Please click here to view a larger version of this figure.
Figure 2. The flowchart of the purification of the protein complex N1-391P1-126. It outlines the inoculation and large scale grow-ups of the E. coli cell culture and harvesting the cell by centrifugation. Followed by cell lysis, the protein samples are purified by the affinity chromatography (i.e., Co2+ column), ion-exchange chromatography (i.e., Q column), and gel filtration size exclusion (SEC) chromatography. The protein samples are further analyzed by the SDS-PAGE gel. Please click here to view a larger version of this figure.
Figure 3. Preparation of negative stain EM grids for imaging. A. Glow discharges the grids. B. The procedure for negative staining grids is shown. The tweezers are used to pick up a grid, followed by applying the protein sample for 1 minute. The grids are blotted with blotting paper. The grid is washed twice with buffer and twice with the 0.75% uranyl formate staining solution. The grids are held in the second staining solution drop for 30 seconds. The grids are blotted after each wash and air-dried after the final blotting. C. The grids are stored in the grid box for imaging. Please click here to view a larger version of this figure.
Figure 4. Representative results of the copurification of the N1-391P1-126 complex. A. The SEC profile of N1-391P1-126. B. The SEC profile of the assembly N1-391P1-126 with RNA. C. The SDS-PAGE gel shows the N protein only for the N-RNA complex and the bands for both N and P proteins of the N1-391P1-126 complex. Please click here to view a larger version of this figure.
Figure 5. Representative images of N-RNA. A and B are representative negative stain EM images of N-RNA from the N1-391-RNA peak in Figure 4. The N-RNA complexes are stained using the procedure described in Figure 3. Please click here to view a larger version of this figure.
Primers | |||
N1-391 Forward | 5’-TACTTCCAATCCAATGCAATGGCCCTGAGCAAAGTGAAG-3’ | ||
N1-391 Reverse | 5’-TTATCCACTTCCAATGTTATTACAGTTCCACGTCGTTGTCCTTGG-3' | ||
P1-126 Forward | 5’-TACTTCCAATCCAATGCAATGGAAAAGTTCGCCCCCGAG-3' | ||
P1-126 Reverse | 5’-TTATCCACTTCCAATGTTATTACTGGTCGTTGATTTCCTCGTAGC-3’ |
Table 1. Primer sequences.
PCR amplification of DNA insert | |
15 μL | 10x Pfu polymerase reaction buffer |
3 μL | Forward primer (100 μM concentration) |
3 μL | Reverse primer (100 μM concentration) |
15 μL | dNTP mix at 2.5 mM concentration |
6 μL | Plasmid DNA contains the gene of N or P (100ng/ μL) |
7 μL | DMSO |
3 μL | Pfu polymerase at 2.5U/μL |
Volume to fill to 150 μL | Sterile ddH2O |
Table 2. PCR amplification of DNA insert reagents.
PCR amplication of DNA insert | |||
Step | Time | Temperature | Cycles |
Denaturation | 4 min. | 95 ºC | 1 |
Denaturation | 45 sec. | 95 ºC | 30 |
Annealing | 30 sec. | 62 ºC | |
Extension* | 90 sec. | 72 ºC | |
Extension | 10 min. | 72 ºC | 1 |
Hold | ∞ | 4 ºC | 1 |
The 150 μL mixture can be run in three separate PCR reactions (3 x 50μL). | |||
*For the Pfu DNA polymerase, 1 kb/min is the recommended speed for the extension phase. Here, both the lengths of the N1-391 gene or the P1-126 gene are shorter than 1.5 Kb. | |||
Thus, 90 seconds was used for the extension step. |
Table 3. PCR amplification of DNA insert thermocycling program.
T4 DNA polymerase treatment | |
10 x Buffer | 2 μL |
Vector/Insert DNA (0.1 pmol vector or 0.2 pmol insert) | 5 μL |
dNTP* at 25 mM | 2 μL |
DTT at 100 mM | 1 μL |
T4 DNA polymerase (LIC qualified) | 0.4 μL (1.25 U) |
Sterile ddH2O | 9.6 μL |
*dGTP was used for the vector, and dCTP was used for the insert DNA. |
Table 4. T4 DNA polymerase treatment.
Overlap PCR | |
15 μL | 10 X Pfu polymerase reaction buffer |
3 μL | Forward primer (100 μM) |
3 μL | Reverse primer (100 μM) |
15 μL | dNTP Mix (2.5 mM) |
3 μL | DNA from 1st round PCR which contain the gene N1-391 (100 ng/μL ) |
3 μL | DNA from 1st round PCR which contain the gene P1-126 (100 ng/μL ) |
7 μL | DMSO |
3 μL | Pfu polymerase (2.5 U/μL) |
Volume to fill to 150 μL | Sterile ddH2O |
Table 5. Overlap PCR reagents.
Overlap PCR | |||
Step | Time | Temperature | Cycles |
Denaturation | 4 min. | 95 °C | 1 |
Denaturation | 45 sec. | 95 °C | 30 |
Annealing | 30 sec. | 62 °C | |
Extension* | 2 min. | 72 °C | |
Extension | 10 min. | 72 °C | 1 |
Hold | ∞ | 4 °C | 1 |
The 150 μL mixture can be run in three separate PCR reactions (3 x 50 μL). | |||
*For the Pfu DNA polymerase, 1 kb/min is the recommended speed for the extension phase. Here, the total length of the gene N1-391 and P1-126 is shorter than 2.0 Kb. Thus, 2 minutes were used for the extension step. |
Table 6. Overlap PCR thermocycling program.
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 systems15,16,17,18,19. Previous studies have attempted to get the RNA free N with a variety of methods, such as the RNase A digestion, high salt washing, or adjusting different pH buffers to remove the nonspecific cellular RNAs28,29. However, none of the above methods can be successfully used for the assembly of the virus-specific NCs. To obtain the RNA-free RSV N, we also tried a combination of methods, including RNase A digestion, high salt (1.5M NaCl) wash, adjusting the buffer pH from pH 5.0 to pH 9.0, protein denaturation and renaturation. After many failed trials, we still could not get RNA-free N with the above methods. We will briefly discuss the attempts and potential reasons.
One method to obtain RNA-free N is to digest host cellular RNA in assembled NCLPs with RNase A. In VSV, the incubation of the purified NCLP with RNase A at a final concentration of 1mg/ml at 37 °C for 1 h completely removed RNA from the NC28. Purified empty oligomeric N was then incubated with poly-A (250-nt or longer) in a molar ratio of 1:5 in the presence of RNase inhibitors. Analysis of the RNA isolated from reconstituted N:poly-A showed that the RNA was approximately 90 nt in length. This suggested that the RNA outside the nucleoprotein is susceptible to nonspecific digestion by the contaminated RNase A from the previous step. The strategy of RNase A digestion to remove RNA was not successful when applied to RSV. This may be due to two reasons. First, contaminated RNase A will digest the RSV-specific RNA, which will subsequently be incubated and assembled with N. Second, the efficiency of the RNase A digestion is much lower in RSV. This is because the RNAs assemble differently in different NNS viruses. The known crystal structures of N:RNA show that RNA binds outside of the NC in VSV, but inside of the NC in RSV30,31. The configuration of RNA binding inwards of the NC may lead to low efficiency for RNase A to access and digest.
Another method to get RNA-free N is to make the truncations that cut both the N-terminal motif (N-arm) and the C-terminal motif (C-arm) of N. However, this truncated RNA-free N cannot be used to assemble with RNA into a stable NC because the N-arm of N is folded into its neighboring subunits by interacting with the C-terminal domain (CTD) of the precedent N subunit. The extended C-arm is positioned to the CTD of the next N subunit31.
An additional method to get RNA-free N is to prepare mutant N. For example, the result obtained by Galloux et al. showed that RSV RNA free N0P complex could be obtained by coexpression of a K170A/R185A double N mutant with the N-terminus of P in bacteria32. However, it has two potential issues for further structural characterizations. One issue is the low stability of this mutant complex at high concentrations. The other issue is that the mutant complex lost the RNA binding ability, which cannot be used in the next step of assembly.
Despite the tremendous challenges, we have established and optimized the protocol to obtain virus-specific NCs using the coexpression of N with a chaperone P. Recently, another successful method is to make a chimeric fusion construction encoding for P1-50-TEV-N1-405-8xHis for MeV33,34. N0P can be obtained after the TEV protease cleavage. The purity of N0P depends on the efficiency of TEV cleavages, which cut the chimeric fusion between N and P protein.
Instead of using the chimeric fusion method, we designed a bi-cistronic coexpression construct. Specifically, the coexpression constructs of the N0P complex have been designed and engineered in two open reading frames, comprising the full length of N (1-391) with a 10x His-tag at N-terminal in the first ORF, and the N-terminal peptides (1-126) from P in the second ORF20. Briefly, the overall procedure of the N0P purification is to purify His-tagged N0P and N-RNA from cellular lysis samples with cobalt beads, remove the nonspecific RNA and N:cellular RNA complex with Q column, and get a pure N0P complex with the SEC column. In the SEC step, the ratio of A260/A280 can be monitored and double-checked with RNA extraction from the N0P peak fractions.
Collectively, in this protocol, the most critical steps are the strategy to design the construction of the coexpression of N0P complex and using a series affinity and ion-exchange column to separate the RNA free N0P complex from the other N-cellular RNA complexes. The efficiency of the coexpression strategy to get N0P complex is relatively low; around 50% N protein is still N-cellular RNA complex. The protocol may also be applied for getting RNA free N0P and assembly with specific RNA with N to get N:RNA complex of other NNS viruses.
The authors have nothing to disclose.
The research programs in the Liang laboratory at Emory are supported by the US National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH) under award number R01GM130950, and the Research Start-Up Fund at Emory University School of Medicine. The author acknowledges the members of the Liang laboratory for helpful support and critical discussion.
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 |