Name: reverse genetics to engineer positive-sense rna virus variants
Uploaded: 2022-06-09T14:00:00
Description: Watch this Scientific Journal Video about Reverse Genetics to Engineer Positive-Sense RNA Virus Variants at JoVE.com

Funding support (grant number BT/PR10906/MED/29/860/2014) for this study was provided by the Department of Biotechnology, Government of India.

NOTE: The JFH1 strain (WT) used here was a kind gift from Takaji Wakita, National Institute of Infectious Diseases. The human hepatoma cell line, Huh7.5, was a kind gift from Charles Rice, The Rockefeller University. A schematic of the method is shown in Figure 1.
1. Genome-wide substitution mutagenesis of JFH1 using error-prone PCR
<ol>
	<li>To perform ep-PCR, prepare the master mix for four sets of experiments with primers J-For 5&#39;-GTTTTCC...

<ol>
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In this study, we have detailed a simple and rapid FL-MRS procedure that integrates ep-PCR18 and reverse genetics for synthesizing HCV full-genome libraries, which can then be used in a cell culture system to generate replication-competent variants for the screening of drug-resistant phenotypes. The use of low-fidelity Taq DNA polymerase is a prerequisite of ep-PCR that allows the incorporation of substitutions during PCR amplification of a full-length viral genome. We tested several low-fidelity ...

Forward genetic screening begins with a viral phenotype of interest and then, through sequencing its genome and comparing it to that of the original strain, attempts to identify the mutation(s) causing that phenotype. In contrast, in reverse genetic screens, random mutations are introduced in a target gene, followed by an examination of the resultant phenotype(s)1. For the reverse genetics approach, in vitro mutagenesis is the most widely used technique to create a pool of variants that are subsequently screened for phenotypes of interest. Various genetic tools have been reported for achieving genome-wide random mutagenesis of RNA viruses, including error-prone PCR (ep-PCR)2,3, circular polymerase extension4, and Mu-transposon insertion mutagenesis 5,6,7. The latter two methods yield libraries harboring limited sequence diversity and are prone to the introduction of large insertions and deletions, which are highly lethal for viruses and severely limit the recovery of infectious viral variants.
ep-PCR is a well-known powerful mutagenesis technique widely used in protein engineering to generate mutant enzymes for the selection of phenotypes with desired properties, such as enhanced thermal stability, substrate specificity, and catalytic activity8,9,10. This technique is easy to perform because it requires simple equipment, does not involve tedious manipulations, uses commercially available reagents, and is quick; moreover, it generates high-quality libraries.
Here, we developed a novel method for full-length mutant RNA synthesis (FL-MRS) to generate complete genomes of hepatitis C virus (HCV) by integrating ep-PCR, which induces random genome-wide substitution mutagenesis and reverse genetics. Even a single nucleotide insertion or deletion is highly deleterious for positive-sense RNA viruses ([+]ssRNA); hence, PCR-based substitution mutagenesis is the preferred method for the iterative generation of large, diverse libraries of full (+)ss RNA virus genomes with good viability.
FL-MRS is a straightforward approach that can be applied to any positive-sense RNA virus with a ~10 kb genome length through the meticulous design of a primer set that binds to the viral cDNA clone. pJFH1 is an infectious cDNA clone that encodes the HCV genotype 2a and can recapitulate all steps of the virus life cycle. By using the FL-MRS approach, we demonstrated the synthesis of randomly mutagenized full-genome libraries (mutant libraries [MLs]) to produce replication-competent JFH1 variants for which there was no prior knowledge of the properties associated with mutations. Upon exposure to an antiviral, some of the viral variants quickly overcame the drug pressure with the desired phenotypic change. Using the protocol described here, a plethora of viral variants can be generated, creating opportunities to study the evolution of (+)ssRNA viruses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 kb plus DNA ladder&#160;</td>
			<td>Thermo Fisher</td>
			<td>10787018</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml centrifuge tube</td>
			<td>Tarsons</td>
			<td>500010</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>Tarsons</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm cell culture dish&#160;</td>
			<td>Tarsons</td>
			<td>960010</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Tarsons</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>HiMedia</td>
			<td>MB080</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrose gel electrophoresis unit</td>
			<td>BioRad</td>
			<td>1704406</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet, ClassII</td>
			<td>ESCO</td>
			<td>AC2 4S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>HiMedia</td>
			<td>MB083</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424-R</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System</td>
			<td>BioRad</td>
			<td>1855201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning plates 90 mm&#160;</td>
			<td>Tarson</td>
			<td>460091</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator&#160;</td>
			<td>New Brunswick</td>
			<td>Galaxy 170R</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri Microvolume Spectrometer</td>
			<td>Titertek-Berthold</td>
			<td>11050140</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB&#160;Substrate&#160;Kit&#160;</td>
			<td>Abcam</td>
			<td>ab94665</td>
			<td></td>
		</tr>
		<tr>
			<td>dATP Solution</td>
			<td>NEB</td>
			<td>N0440S</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Set</td>
			<td>NEB</td>
			<td>N0446</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl Pyrocarbonate (DEPC)&#160;</td>
			<td>SRL chemical</td>
			<td>46791</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>HiMedia</td>
			<td>MB058</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoR1-HF</td>
			<td>NEB</td>
			<td>R3101</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA tetrasodium salt dihydrate</td>
			<td>HiMedia</td>
			<td>GRM4918</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>X328</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde&#160;</td>
			<td>Fishser Scientific</td>
			<td>12755</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>ALPHA IMAGER</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP</td>
			<td>Thermo Fisher</td>
			<td>A16066</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Merck</td>
			<td>107209</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nickon</td>
			<td>&#160;ECLIPSE Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>HiMedia</td>
			<td>M1245</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher</td>
			<td>116680270</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>Mechanical Pipette Set</td>
			<td>Eppendorf&#160;&#160;</td>
			<td>3120000909</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tips 0.2-10 &#181;l&#160;</td>
			<td>Tarsons</td>
			<td>521000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tips 10 - 100 &#181;l&#160;</td>
			<td>Tarsons</td>
			<td>521010</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tips 200-1000 &#181;l&#160;</td>
			<td>Tarsons</td>
			<td>521020</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS buffer&#160;</td>
			<td>GeNei</td>
			<td>3601805001730</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential aminoacids (NEAA)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot TOP10 Chemically Competent&#160;E. coli</td>
			<td>Invitrogen</td>
			<td>C404010</td>
			<td>E.coli DH5&#945;</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>1105-021</td>
			<td>minimal essential medium</td>
		</tr>
		<tr>
			<td>PCR tubes 0.2 ml</td>
			<td>Tarsons</td>
			<td>510051</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencillin/streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEM-T Easy Vector System</td>
			<td>Promega</td>
			<td>A1360</td>
			<td>T-vector DNA</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS)</td>
			<td>HiMedia</td>
			<td>TI1099</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion&#160;High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pibrentasvir</td>
			<td>Cayman Chemical</td>
			<td>27546</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette controller&#160;</td>
			<td>Gilson&#160;</td>
			<td>F110120</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Taq DNA Polymerase&#160;</td>
			<td>Thermo</td>
			<td>10966034</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td>statistical analysis software</td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini kit&#160;</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td>viral isolation kit</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td>cokum purification kit</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit&#160;</td>
			<td>QIAGEN</td>
			<td>74104</td>
			<td>RNA cleanup kit</td>
		</tr>
		<tr>
			<td>Serological Pipettes 25 ml</td>
			<td>Thermo Fisher</td>
			<td>170357N</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes 5 ml</td>
			<td>Thermo Fisher</td>
			<td>170355N</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes10 ml</td>
			<td>Thermo Fisher</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>Single strand RNA Marker 0.2-10 kb</td>
			<td>Merck</td>
			<td>R7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk&#160;</td>
			<td>HiMedia</td>
			<td>M530</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide 0.1 M solution</td>
			<td>Merck</td>
			<td>8591</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase&#160;</td>
			<td>Invitrogen</td>
			<td>18080044</td>
			<td>reverse transcriptase</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 cell culture flask&#160;</td>
			<td>Tarsons</td>
			<td>159910</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 cell culture flask&#160;</td>
			<td>Tarsons</td>
			<td>950040</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RiboMax Express Large Scale RNA Production System&#160;</td>
			<td>Promega</td>
			<td>P1320</td>
			<td>&#160;Large Scale RNA Production System&#160;</td>
		</tr>
		<tr>
			<td>T75 cell culture flask&#160;</td>
			<td>Tarsons</td>
			<td>950050</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>Genetix Biotech (Puregene)</td>
			<td>PGM040</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan RNA-to-CT 1-Step Kit</td>
			<td>Applied Biosystems</td>
			<td>4392653</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan RNA-to-CT&#160;1-Step&#160;Kit</td>
			<td>Thermo Fisher</td>
			<td>4392653</td>
			<td>commercial qRT-PCR kit&#160;</td>
		</tr>
		<tr>
			<td>TOPO-XL--2 Complete PCR Cloning Kit</td>
			<td>Thermo Fisher</td>
			<td>K8050-10</td>
			<td>kit for cloning of long-PCR product&#160;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>HiMedia</td>
			<td>TC072</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>HiMedia</td>
			<td>TCL007</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>HiMedia</td>
			<td>MB067</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Concentrator&#160;</td>
			<td>Eppendorf, Concentrator Plus</td>
			<td>100248</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath&#160;</td>
			<td>GRANT</td>
			<td>JBN-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Xba1</td>
			<td>NEB</td>
			<td>R0145S</td>
			<td></td>
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reverse genetics to engineer positive-sense rna virus variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

A plethora of full-length HCV variants can be generated and screened for drug-resistant phenotypes of interest following the procedures described in Figure 1. Full genome mutant libraries were synthesized using clonal-pJFH1 in decreasing amounts (100-10 ng), as shown in Figure 2. Average yields of ep-PCR products (mutant libraries) ranged from 3.8-12.5 ng/&#181;L. Figure 3 shows the viral transcripts synthesized from the clonal-pJFH...

Watch this Scientific Journal Video about Reverse Genetics to Engineer Positive-Sense RNA Virus Variants at JoVE.com

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

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