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 at the N-terminal of the Open Reading Frame (ORF) (Figure 1).
<ol>
	<li>Perform linearization of LIC vectors using SSPI digestion.
	<ol>
		<li>Combine 10 &#956;L of SSPI 10X buffer, 4 &#956;L of SSPI enzyme at a concentration of 5 U/&#956;L, the equivalent volume of 5 &#956;g of vector miniprep DNA, and sterile ddH2O to 100 &#956;L.</li>
		<li>Incubate the digest for 3 hours at 37 &#176;C.</li>
		<li>Run the digest on a 1.0% agarose gel for the extraction of vector DNA.</li>
		<li>Use a gel extraction kit to perform extraction and purification. Suspend the final volume of vector DNA in 30 &#956;L of ddH2O and store it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>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 &#176;C and 60 &#176;C. For the reverse primer, there is sufficient overlap with the reverse complementary strand of the linearized vector for the same reason.
	<ol>
		<li>Perform PCR amplification of the DNA insert using the conditions in Table 2 and Table 3.</li>
		<li>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 &#956;L of ddH2O.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Incubate the mixture for 40 minutes at room temperature. Then, heat-inactivate the polymerase at 75 &#176;C for 20 minutes. Store the reaction mixture at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Anneal the LIC vector and the insert vector.
	<ol>
		<li>Set up a negative control with 2 &#956;L of LIC vector DNA and 2 &#956;L of sterile ddH2O.</li>
		<li>Combine &#160;2 &#956;L of insert DNA and 2 &#956;L of LIC vector DNA from the previous T4 DNA polymerase reactions in a 0.2 mL tube.</li>
		<li>Perform the annealing reaction at room temperature for 10 minutes.</li>
		<li>Quench the reaction with 1.3 &#956;L of EDTA at a concentration of 25 mM.</li>
		<li>Transform the reaction into 100 &#956;L of E. coli Top10 competent cells and plate them on an ampicillin selection plate23,24.</li>
	</ol>
	</li>
	<li>Identify the positive constructs.
	<ol>
		<li>Prepare the plasmid miniprep solution. This can be done through colony picking and inoculation in LB media. Usually, 3 colonies are sufficient.</li>
		<li>Incubate the mixture overnight at 37 &#176;C.</li>
		<li>Centrifuge the mixture at 4,560 x g for 10 minutes and discard the supernatant.</li>
		<li>Resuspend the pellets in 250 &#956;L of P1 buffer and prepare plasmid minipreps using a spin miniprep kit.</li>
		<li>Conduct a digestion analysis of the miniprep product using AseI or other restriction enzymes.</li>
		<li>Load samples on 0.8% agarose gel and run the digested plasmid. Analyze the gel under a UV lamp.</li>
		<li>Use a sequencing service to validate the sequence of the positive product.</li>
	</ol>
	</li>
	<li>Obtain the coexpression DNA insert.
	<ol>
		<li>Perform PCR to obtain N1-391 and P1-126 using the previously constructed 2BT-10 N1-391 and 2BT-10 P1-126 as templates.</li>
		<li>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 &#160;5 &#769;-GTGAAGATCCTGGCTGATGCAATGCGGCGGCG 
		CGCCGCGATCGCGGATCC-3 &#769;.</li>
		<li>Perform the 2nd PCR to obtain P1-126 from the 2BT-10 P1-126 construct using the Forward primer: 5&#8217;-CCGCCGCATTGCATCAGCCAGGATCTTCACTGC 
		AGGACTCGAGTTCTAGA-3 &#769;and the P1-126 Reverse primer (use the PCR conditions in Table 2 and Table 3).</li>
		<li>Finally, perform overlap PCR on the mixed products of the previous 2 PCR reactions to merge N1-391 and P1-126. Use the &#160;N1-391 Forward primer and P1-126 Reverse primer. Use the PCR conditions in Table 5 and Table 6.</li>
	</ol>
	</li>
	<li>Join the vector and the DNA insert.
	<ol>
		<li>Treat the overlap PCR product with T4 DNA polymerase following the protocol in step 1.3.</li>
		<li>Anneal the LIC vector and PCR product following the protocol in step 1.4.</li>
		<li>Identify the positive constructs following the protocol in step 1.5.</li>
	</ol>
	</li>
</ol>
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 &#176;C, but carry out the expression at a reduced temperature (16 &#176;C) overnight. Purify the protein complexes through a combination of cobalt column, ion exchange, and size exclusion chromatography (Figure 2).
<ol>
	<li>Use the E. coli BL21(DE3) strain for protein production. Grow 4 L cell cultures at 37 &#176;C in LB (Luria Broth) medium until OD600 reaches 0.6.</li>
	<li>Lower the temperature to 16 &#176;C. An hour later, induce the expression with 0.5 mM isopropyl 1-thio--D-galactopyranoside (IPTG) overnight.</li>
	<li>Centrifuge the cells at 4,104 x g for 25 min and then discard the supernatant.</li>
	<li>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.</li>
	<li>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 &#176;C freezer.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Dilute the eluted protein 5x with QA buffer (50 mM Tris-HCl pH 8.0, 5% Glycerol) for the Q column.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Run the &#8220;pump wash&#8221; program to wash the machine with QB buffer followed by QA buffer (1-2 CVs/each). Set the system flow to 3 mL/min.</li>
	<li>Set the UV1 to 280 nm and UV2 to 260 nm. Use a 96 deep-well plate to collect the fractions.</li>
	<li>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&#160; protein complex will come out at 15% QB Buffer.</li>
	<li>Once all of the protein is eluted, wash the column with 100% QB Buffer (2 CV).</li>
	<li>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).</li>
	<li>Analyze protein-containing fractions by SDS-PAGE.</li>
</ol>
3.&#160;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).
<ol>
	<li>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&#160;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.</li>
	<li>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).</li>
	<li>Centrifuge the sample with 21,130 x g for 15 min, remove any precipitation and load the supernatant to the SEC column.</li>
	<li>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.</li>
	<li>Collect the peak fractions, run the SDS-PAGE gel, or make grids.</li>
	<li>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.</li>
</ol>
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).
<ol>
	<li>Microwave or heat 5 mL of ddH2O in a small glass beaker&#160;using a Tungsten heater until it is boiling.&#160;</li>
	<li>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.</li>
	<li>Add 4 &#956;L of 10 M NaOH to the staining solution and continue to stir for 15 min, protected from the light.</li>
	<li>Filter the solution through a 0.22 mm filter into a test tube.</li>
	<li>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.</li>
	<li>Cut and fold a parafilm strip. Pipet 2 drops of 40 &#956;L of the buffer on one side of the parafilm, and pipet another 2 drops of 40 &#956;L of staining solution to the other side.</li>
	<li>To make the EM carbon grids, apply 3 &#956;L of protein sample for 1 minute.</li>
	<li>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.</li>
	<li>Hold the grid surface in the second drop of 0.75% uranyl formate staining solution for 30 seconds.</li>
	<li>Blot the grid against a blotting paper to remove excess stain solution and allow the grid to air-dry.</li>
	<li>Store the grids in the grid box before imaging.</li>
</ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+nucleocapsid-like+nucleoprotein-RNA+complex+of+respiratory+syncytial+virus.">Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus.</a> Science. 326 (5957), 1279-1283 (2009).</li><li>Galloux, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+viral+phosphoprotein+binding+site+on+the+surface+of+the+respiratory+syncytial+nucleoprotein.">Characterization of a viral phosphoprotein binding site on the surface of the respiratory syncytial nucleoprotein.</a> Journal of Virology. 86 (16), 8375-8387 (2012).</li><li>Milles, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembly+of+Measles+Virus+Nucleocapsid-like+Particles:+Kinetics+and+RNA+Sequence+Dependence.">Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.</a> Angewandte Chemie Interntional Edition English. 55 (32), 9356-9360 (2016).</li><li>Desfosses, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+cryo-EM+structures+of+RNA-specific+measles+virus+nucleocapsids+provide+mechanistic+insight+into+paramyxoviral+replication.">Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (10), 4256-4264 (2019).</li></ol>

The authors have nothing to disclose.

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.

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

generation-assembly-virus-specific-nucleocapsids-respiratory

Research

JoVE Journal

Biochemistry

3.7K Views.  Emory University School of Medicine. Deze methode maakt gebruik van de verschillende manieren om de recombinante virale eiwitexpressie-uitdagingen aan te pakken met behulp van een viraal eiwit P'als chaperonne voor een ander viraal eiwit N'om RNA-vrije N-eiwitten te verkrijgen. Het RNA-vrije RSV N-eiwit wordt verkregen voordat het virusspecifieke RNA in nucleocapsid in vitro wordt geassemblaged. De methode kan ook worden gebruikt met andere niet-gesegmenteerde negatieve zin RNA virussen.De ligatie onafhankelijke kloonmeth...

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

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Immunology and Infection

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

Medicine

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Biology

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Source: Arevalo, M. T., et al.&#160; <a href="https://app.jove.com/v/54041">Expression and Purification of Virus-like Particles for Vaccination.</a> J. Vis. Exp. (2016).
This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors &#8212; encoding different viral structural proteins &#8212; in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

Source: Arevalo, M. T., et al.&#160; Expression and Purification of Virus-like Particles for Vaccination. J. Vis. Exp. (2016).
This video demonstrates the ...

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

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