Name: generation and assembly of virus-specific nucleocapsids of the respiratory syncytial virus
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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.

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&#8217;-5&#8217; Exo activity of the T4 DNA&#160; 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 a...

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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 d...

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. &#160;
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.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agarose</td>
			<td style="width:302px;">SIgma</td>
			<td style="width:301px;">A9539-500G</td>
			<td style="width:319px;">making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td>Millipore</td>
			<td>UFC901024</td>
			<td>concentrate the protein sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Ampicillin sodium</td>
			<td style="width:302px;">GOLD BIOTECHNOLOGY</td>
			<td style="width:301px;">5118.111317A</td>
			<td>antibiotic for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">AseI</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">R0526S</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cobalt (High Density) Agarose Beads</td>
			<td style="width:302px;">Gold Bio</td>
			<td>H-310-500</td>
			<td>For purification of His-tag protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Corning LSE Digital Dry Bath Heater</td>
			<td style="width:302px;">CORNING</td>
			<td style="width:301px;">6885-DB</td>
			<td>Heate the sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">dCTP</td>
			<td style="width:302px;">Invitrogen</td>
			<td style="width:301px;">10217016</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">dGTP</td>
			<td style="width:302px;">Invitrogen</td>
			<td style="width:301px;">10218014</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Glycerol</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">G5516-4L</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HEPES</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">H3375-100G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HiTrap Q HP</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">GE29-0513-25</td>
			<td>Protein purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Imidazole</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">I5513-100G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">IPTG (Isopropyl-beta-D-thiogalactopyranoside)</td>
			<td style="width:302px;">GOLD BIOTECHNOLOGY</td>
			<td style="width:301px;">1116.071717A</td>
			<td>induce the expression of protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microcentrifuge Tubes</td>
			<td style="width:302px;">VWR</td>
			<td style="width:301px;">47730-598</td>
			<td>for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Misonix Sonicator XL2020 Ultrasonic Liquid Processor</td>
			<td style="width:302px;">SpectraLab</td>
			<td style="width:301px;">MSX-XL-2020</td>
			<td>sonicator for lysing cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Negative stain grids</td>
			<td style="width:302px;">Electron Microscopy Sciences</td>
			<td style="width:301px;">CF400-Cu-TH</td>
			<td>For making negative stain grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">New Brunswick Innova 44/44R</td>
			<td style="width:302px;">eppendorf</td>
			<td style="width:301px;">M1282-0000</td>
			<td>Shaker for culturing the cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Nonidet P 40 Substitute</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">74385-1L</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">OneTaq DNA Polymerase</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">M0480L</td>
			<td>PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">QIAquick Gel Extraction Kit</td>
			<td style="width:302px;">QIAGEN</td>
			<td style="width:301px;">28706</td>
			<td>Purify DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">SSPI-HF</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">R3132S</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Superose 6 Increase 10/300 GL</td>
			<td style="width:302px;">Sigma</td>
			<td>GE29-0915-96</td>
			<td>Protein purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">T4 DNA polymerase</td>
			<td style="width:302px;">Sigma</td>
			<td>70099-3</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Thermo Scientific Sorvall RC 6 Plus Centrifuge</td>
			<td style="width:302px;">Fisher Scientific</td>
			<td style="width:301px;">36-101-0816</td>
			<td>Centrifuge, highest speed 20,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Trizma hydrochloride</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">T3253-250G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Uranyl Formate</td>
			<td style="width:302px;">Electron Microscopy Sciences</td>
			<td style="width:301px;">22451</td>
			<td>making negative stain solution</td>
		</tr>
	</tbody>
</table>

generation and assembly of virus-specific nucleocapsids of the respiratory syncytial virus

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.

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...

Watch this Scientific Journal Video about Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus at JoVE.com

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

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 ...

This method utilize the various way to tackle the recombinant viral protein expression challenges using one viral protein P'as a chaperone for another viral protein N'to obtain RNA-free N proteins. The RNA-free RSV N protein is obtained before assembling the virus specific RNA into nucleocapsid in vitro. The method can also be used with other non-segmented negative sense RNA viruses.The ligation independent cloning method is a quick technique for acquiring co-expression constructs. Why or electives stain electron microscope is a quick method for checking large assemble in raw cell. We want to give you two tips.First, get high yield and solid results for each step before moving to the next step. Second, set up proper programs for chromatography and practice picking up grids with forceps. For bi-cistronic construction of the N and P co-expression, grow four one liter cultures of E coli BL21DE3 strain cells in LB medium at 37 degrees Celsius.When the optical density at 600 nanometers reaches 0.6, lower the temperature to 16 degrees Celsius. After one hour, induce protein expression by treatment with 0.5 millimolar IPTG overnight. The next morning, collect the cells by centrifugation and resuspend the pellets in 200 milliliters of lysis buffer.lyse the cells by sonication for 15 minutes, with three seconds on, three seconds off pulses. And collect the lysates by centrifugation. Load the supernatant into a 2.5 by 10 centimeter cobalt gravity column, with approximately 10 milliliters of beads.Pre-equilibrated with 5 to 10 column volumes of lysis buffer. And wash the column with five column volumes of buffer B and five volumes of buffer C.Use two volumes of buffer D to elute the protein from the beads. And equilibrate the Q column with five one milliliter volumes of QA buffer.Use a peristaltic pump to load the diluted sample onto the column. Melt the loaded Q column onto the HPLC machine, along with fresh QA and QB buffers. Run the pump wash to successfully wash the machine with one to two volumes of QB and QA buffers.After the last wash, set the flow rate to one milliliter per minute, and set UV1 to 280 nanometers and UV2 to 260 nanometers, using a 96 deep well plate to collect the fractions. Then use a stepwise gradient application of three to four volumes of each concentration of elution agent to elute the proteins, starting at a 0%QB concentration and increasing the percentage by 5%each time. The N-0 P protein complex will elute at the 15%QB buffer concentration.Isolate the protein by gel filtration, in a one by 30 centimeters small scale purification column, equilibrated with buffer E.Then analyze the protein containing fractions by SDS page. For virus specific nucleocapsid assembly, mix and incubate the purified N-0 P complex with RNA oligo at a 1:1.5 molecular ratio at room temperature for one hour. Pre-equilibrate a small scale purification gel filtration column with buffer E.And remove any precipitation by centrifugation.Load the supernatant, to the size exclusion chromatography column. And compare the size exclusion chromatography images of the protein to RNA assembly, and protein alone control samples, combining the nucleic acid purity ratios, to identify which peaks are the assembled N RNA, N-0 P and free RNA. To assemble an N RNA complex, collect all of the N RNA peak fractions, perform an RNA extraction, and run a urea page gel to double-check the length of the specific RNA.To prepare a negative stain grid for negative stain electron microscopy, Use a heat plate to heat 5 mL of double distilled water to boiling and add 37.5 milligrams of uranyl formate to the water to obtain a 0.75%uranyl formate staining solution. Transfer the solution to an aluminum foil covered beaker. And add four microliters of 10 molar sodium hydroxide.After 15 minutes of stirring protected from light, filter the solution through a 0.22 micron filter into a test tube. To make the continuous carbon coated electron microscopy grids hydrophilic, place the grids in a chamber connected to a power supply and apply negatively charged ions to the grids. Cut and fold a two by two inch parafilm strip.Add two 40 microliter drops of distilled water to one end of the strip. And two 40 microliter drops of 0.75%uranyl formate staining solution to the other end. Add three microliters of protein sample to each grid.After one minute, block the grids against blotting paper and dip the grids two times in the distilled water droplets. And once in the first staining solution droplet. Then immerse the grid in the second staining solution droplet for 30 seconds, before blotting the grids against blotting paper to remove any excess stain solution and allowing the grids to air-dry before imaging.Using this protocol, a large-scale soluble hetero dimeric respiratory syncytial virus N0 P complex can be obtained. In E coli, the full length of N and N-terminal portions of the P proteins are co-expressed with a 10 X His tag on the N protein. Based on the UV absorbance nucleic acid purity ratio, N0 P contained both the full length N and N-terminal P, but did not contain cellular RNA.The purified N0 P could then be stimulated and assembled into nucleocapsid like particles on electron microscopy grids via incubation with specific RNA oligos as demonstrated. When purifying the protein avoid air bubbles during the column purification and dilute the sample with QA buffer to adjust the pH and salt concentration before loading the sample onto the column. Take care to hold the electron microscopy grids at the outer edge to avoid contaminating the grids.This will help us to determine the non-positive RSV genome packing questions, whether it is left handed or right handed helical structure. Following those procedures and authentic an RNA template for the RSV polymerase activity assay can be performed. And this RSV viral specific nucleocapsid can be used for the high resolution cryo EM analysis.

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