RNA viruses use virally encoded RNA polymerases to replicate their genomes. These polymerases have an intrinsically higher error rate, which allows the virus to quickly adapt and evolve. This video presents an optimized method to screen and identify viruses with mutations in their RNA polymerase that affect the error rate or fidelity during replication.
First mutagens are applied to viral host cells to determine the maximum level of mutagen that can be used without killing the cells or inhibiting the virus. Once the optimal conditions have been determined, the virus is passaged under the selective pressure of RNA mutagens. The mutagen resistant population is then sequenced to identify the mutation responsible.
Next, the mutagen resistant mutant is isolated. The replication kinetics are monitored and the results show increases or decreases in polymerase fidelity. Concurrent with broad resistance to RNA mutagens and alterations in virus population mutation frequency.
The main advantage of this technique over existing methods like biochemical fidelity acids using purified polymerases is that these techniques involved are valid for any RNA virus that can be cultivated in tissue culture. This method can help answer key questions in the virology field, such as the role of polymerase aate in virus adaptation and evolution. Generally, individuals new to this method will struggle because the selective pressures they use are either too strong or too weak, and the sequence sample analyze size of the region sequence and the number of clones has not been optimized for statistical power.
Begin this experiment by determining the range of mutagen concentrations that can be used in the following experiments without resulting in cell death. Aspirate the medium from six well plates containing sub confluent monolayers of hilar cells. Then add two milliliters of mutagen supplemented medium here, the mutagens ribovirin five Flo uracil five azacitidine magnesium chloride and manganese chloride are used.
Incubate the cells at 37 degrees Celsius and 5%carbon dioxide. Then every 24 hours for the next two to seven days, use one plate of cells to check viability by triam blue exclusion. Using a hemo cytometer, calculate the percentage of viable cells for each concentration of mutagen, including the untreated control conditions that result in less than 50%cell death by the infection endpoint.
When maximum titers are reached are ideal, the next step of the procedure is to determine the concentration of mutagen that will exert an optimal selective pressure without over mutagen the population at the appropriate concentrations. Every genome is mutated in at least one or two positions. The mutagen will thus generate the resistance mutation, which will then be selected over passage.
If too many mutations are introduced, the mutants bearing the resistance mutation will themselves be lethally. Mutagen impeding their isolation in six well plates seeded with he. Our cells at two milliliters of mutagen supplemented medium use the same range of concentrations as before, excluding concentrations that resulted in over 50%Cell death.
Incubate for two hours to allow for mutagen uptake following the incubation. Aspirate the medium and infect with virus at low multiplicity of infection in 200 microliters incubate for 15 to 60 minutes to allow the virus to infect the cells, rocking the plate at regular intervals to ensure that the inoculum covers the cell. Monolayer aspirate the inoculated virus and wash twice with two milliliters of PBS to remove as much of the inoculum as possible.
Then add medium with mutagen to each well and incubate cells for the equivalent of three to six replication cycles, usually two to seven days. Harvest virus from each well and perform a standard plaque assay or limiting dilution to determine the antiviral effect on virus titer from the calculated titers. Identify the concentration of mutagen that reduces virus titers compared to the untreated control infection by 0.5 to two logs that is also not highly toxic to cells.
Ideally less than 50%toxicity to isolate mutagen resistant variants begin by passaging virus in healer cells with the optimal concentration of each mutagen, the virus may need to be passaged up to 30 times at each passage, titer the virus and repeat the infection and incubation with mutagen as shown here. Virus titers of mutagen treated samples should drop during the first few passages compared to the original virus titer and the untreated control virus titers. Once the virus titers for a given series reach the same magnitude as the untreated control titers suggesting resistance.
Extract the viral RNA for sequencing using primers that amplify the polymerase or replicate genes of the virus of interest. Generate CDNA of the viral genome by R-T-P-C-R, then sequence the DNA. Once the sequence information is obtained.
Use alignment software to align the resulting sequences with the reference consensus and identify the new point mutations. In addition, check the chromatograms for minority peaks that may have been missed by alignment Software A mutant representing 20 to 30%of the total population will still show as a peak, but too small to be identified as a mixed nucleotide by standard sequence analysis to identify the mutation of interest, look for single nucleotide polymorphisms that appear exclusively in the mutagen treated population compared to untreated control from the same passage as shown here. Once a mutation is identified, the resistant variant must be regenerated from infectious CD NA or isolated in tissue culture and the resistance phenotype must be confirmed.
Here we isolate by plaque assay to isolate the identified mutants. Prepare serial dilution of the nitrogen resistant sample and add them to cell monolayers on six well plates for a plaque assay following an incubation of one hour at 37 degrees Celsius and five sync carbon dioxide. Add an agarose overlay two to five days after infection.
Plaque should be clearly visible. Ideally, one of the plates will produce between 10 and 50 well separated plaques. Mark the location of plaques on the plate with a dot, using a marker, taking in not to dislodge and shift the overlays position, gently plunge a P 200 filter tip through the agros overlay.
To reach the plaque carefully lift the tip out of the overlay and transfer the aros plug to a micro centrifuge tube containing 250 microliters of medium. A sufficient amount of virus will be transferred regardless of whether the aros plug is freed from the tip vortex the tube pick up to TenX per ute treatment. Save a portion of the sample to amplify the live virus later.
Then extract the RNA from the remainder of the sample and perform R-T-P-C-R to identify the plaque sample that contains the desired mutation. Sequence the CDNA. Ensure that there are no additional mutations.
Then make a larger stock of this virus for all downstream studies. Next to confirm the resistance conferred by the identified mutation. Repeat the mutagen assay using several mutagenic conditions.
If the variant is resistant to multiple mutagens, it likely has increased fidelity. Since fidelity altering mutations map to the polymerase, it is important to confirm that replication kinetics are not significantly altered. Before determining mutation frequencies examine replication by at least two complimentary approaches.
For example, an approach that examines virus production such as plaque assay or TCID 50 may be performed in addition to an approach that examines RNA synthesis such as quantitative R-T-P-C-R or northern blot. Next, to confirm that the identified polymerase change conferring resistance to mutagen alters replication for fidelity mutation frequencies are measured. This can be performed on either viable or total virus population.
To determine mutation frequencies of the total virus population, which includes viable and non-viable variants, extract the RNA and R-T-P-C-R amplify an 800 to 1, 200 nucleotide region from a coding sequence that is known to have genetic variants such as that coding for a structural protein. Next topo TA clone the purified product for each virus population to be studied. Select 96 colonies identified as having a positive insert by blue white screening on xga coated plates.
Perform single colony PCR to confirm the validity of blue white screening. Using the fragment size and conditions above about 90%of the colony should be positive. For each positive white colony, use a toothpick to transfer each colony to one milliliter of LB medium in 96.
Well, bacterial culture plates incubate overnight at 37 degrees Celsius with constant shaking. The next day. Isolate the DNA by preparing mini preps in a 96 Well format sequence each plate with enough primers to obtain maximum coverage of the cloned segment.
Two primers of 600 base coverage should be enough. Perform sequence analysis using a reference or consensus sequence for each population and appropriate alignment Software that can readily identify single nucleotide polymorphisms here. Seek man software from DNA star laser.
Gene is used first discard poor quality sequences such as those with bad base calling to many ends or those that are too shorting length. Align the remaining sequences. Identify the nucleotide range that is covered by all sequences in the analysis.
Discard clones that are not fully covered across this region. For comparison reasons, it is essential that the same region is fully covered. Identify and count the single nucleotide polymorphisms that are different from the reference strain.
Calculate the mutation frequency by dividing the number of total single nucleotide polymorphisms by the total number of nucleotides sequenced. Present this number as the average number of mutations per 10, 000 nucleotides sequenced. If the same single nucleotide polymorphism appears on a large number of clones, present two values that include or exclude these repeated mutations.
Determine the mutation distribution. Make a ranked list of the number of clones in each population that presents 0 1 2 3 and so on. Mutations in the region sequenced as an option.
Use the edited sequences to calculate all possible pairwise comparisons within a sample to obtain mean values of population diversity. Several alignment programs are available here. Molecular evolutionary genetics analysis program also called mega, is used to determine the optimal conditions to select for RNA mutagen resistance heal.
Our cells were treated with increasing concentrations of ribovirin and infected with wild-type Cox AKI B virus as a multiplicity of infection of 0.0 1 48 hours post infection. The progeny virus was harvested and Titus were determined by TC ID 50. This figure indicates the percentage of cells surviving treatment below the x axis determined by triam blue staining of 48 hours post infection.
The results show that concentrations of 100 and 200 micromolar reduce virus Titus by one to two log without affecting cell viability. To select for mutagen resistant populations, CHI ganga virus wasd in hilar cells in the presence gray bars or absence black bars of 50 micromolar ribovirin following each passage virus progeny was quantified by classic plaque assay on BHK cells as can be seen here. The mutagenic effect is evident during the first passages where the treated virus Titus dropped by two log.
Gradually Titus returned to normal untreated levels. No significant differences are observed in five mutagen treated populations compared to untreated, suggesting that resistant variants have been selected. Indeed sequencing identified unique mutations in the virus population undergoing ribovirin treatment to confirm broad resistance to RNA mutagens of different structure, the high fidelity A 3 72 V variant of COA virus B three that was initially isolated in the screen was tested for reduced sensitivity to different concentrations of the RNA mutagens RIBOVIRIN five fluoro uracil five azacitidine.
He, our cells were treated with indicated concentrations of ribovirin and infected with wild type Cox AKI virus B three at a multiplicity of infection of 0.0 1 48 hours post infection. The progeny virus was harvested and Titus were determined by TC ID 50 shown here are the titers of wild type and a 3 72 variant as a function of mutagen concentration. A 3 72 V consistently titers higher than wild type.
Under all conditions tested to determine the one step growth kinetics of virus production heal, our cells were infected at a multiplicity of infection of 10 with either wild type high fidelity variant, A 3 72 V or replication deficient variant CX 64 of Cox AKI virus B three at the time points indicated virus progeny was harvested from cells and supernatants by freeze thaw and titered by TC ID 50. The fidelity increase of a 3 72 V does not coincide with an observable replication defect in tissue culture. The variance CX 64 presents a significant delay in replication kinetics and reaches maximum Titus that are 1000 fold lower than wild type virus.
Using the approach described in this video, each clone that is sequenced originates from a unique genome within the total virus population and with thus carry unique mutations. This figure shows a typical alignment following cleanup of poor quality sequences and visualization of single nucleotide polymorphisms. The total single nucleotide polymorphisms within a population accounted and the number of single nucleotide polymorphisms appearing on each clone and noted.
For example, the clone underlined by a bar contains two unique mutations, whereas eight other clones contain a single unique mutation. This data is used to compile a table such as the table shown here. The table shows the ranked distribution of sequenced clones by number of mutations.
Statistical comparison of ranked values can determine whether one distribution is significantly different from another. For easier interpretation, the numerical data obtained from sequence and statistical analysis can be represented either as a chart or histogram. As shown here, the A 3 72 V virus generates fewer mutations than wild type and presents a significantly lower mutation frequency with a P value of less than 0.01.
The same chicken ganga virus population gives similar mutation frequencies. Whether the virus stock or a 10 to the fivefold dilution is used for RNA extraction. So following this procedure, you might wanna perform in vitro biochemical essays or crystallization studies to better understand the mechanistic and structural basis of fidelity in this polymerase variant.
After this development, this technique paved the way for researchers in the field of biology to explore the correlation between genetic diversity and virus fitness in vivo. After watching this video, you should have a good understanding of how to increase your chances of isolating fidelity variants of RNA virus and how to use mutation frequencies to identify these fidelity differences.
