The overall goal of this protocol is to quantitatively measure arenavirus particle attachment to host cells. The main advantage to this technique is its high sensitivity. It can also be adapted to measure virion endocytosis, as well as the release of genome into the cytoplasm following fusion.
To begin, to make complete DMEM, to a 500 milliliter bottle of DMEM, add 50 milliliters of heat inactivated fetal bovine serum, and five milliliters each of 100X penicillin-streptomycin, and 100X HEPES buffer. At the end of the day, seed 2.5 times 10 to the 4th Vero E6 cells per well, in triplicate for each virus, in a 48-well tissue culture plate in a final volume of 500 microliters of complete DMEM. Incubate the plate at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 10 to 18 hours.
Under a Class 2 biosafety cabinet, using ice-cold complete DMEM, dilute the Junin Candid 1 virus samples to be tested to 200 to 2, 000 focus forming units or plaque forming units. Plaque forming units will be referred to as PFUs in the remainder of the protocol. For example, to inoculate with 50 microliters of 2, 000 PFUs in duplicate, add 4, 800 PFUs into a total volume of 120 microliters of medium.
Store on ice until ready to add to the Vero cultures. Remove the plates from the 37 degree Celsius incubator and place on ice or at 4 degrees Celsius for 15 minutes to inhibit the ability of the plated cells to carry out endocytosis. Next, aspirate the complete DMEM from each well, and use 100 microliters of ice-cold PBS to rapidly wash each well twice.
Then add 50 microliters of the pre-diluted virus samples to the appropriate wells. Wrap the plate in plastic and immediately place it back on ice or at 4 degrees Celsius for 60 to 90 minutes to allow virus attachment to occur. Keep the wash buffer and virus samples at 4 degrees Celsius throughout this step.
After the incubation, aspirate the virus inoculum and use 200 microliters of ice-cold PBS to wash each well three times. To extract viral RNA from Junin Candid 1 particles, use a commercial RNA isolation kit according to the manufacturer's protocol for purification of RNA from animal cells using spin technology as follows. Lyse the cells as directed by adding 350 microliters of lysis buffer, RLT, directly to each well.
Mix the buffer several times to ensure cell lysis. Then use an inverted microscope to ensure lysis by the absence of cells. At this point, the virus has been neutralized, so the remaining steps can be performed outside of the Class 2 biosafety cabinet, provided kit tubes were not contaminated.
Transfer the lysate to the kit column, and follow the rest of the protocol as described. Include the optional Step 9 from the manufacturer's protocol, which is an additional centrifugation of the spin column to eliminate any possible carryover of buffer RPE. For the final step, use 30 microliters of nuclease-free double-distilled water to elute the purified RNA from the spin column.
Store the RNA at 80 degrees Celsius, or use directly in the qRT-PCR assay. To convert Junin Candid 1 S-segment RNA into cDNA for qPCR, set up a 50 microliter RT Reaction Master Mix using the reagents listed in the text protocol. Gently mix the RT Master Mix, and then aliquot 40 microliters into individual 0.2 milliliter PCR tubes.
Next, add 10 microliters of viral RNA to each tube. Include a no template control tube and a no RT enzyme control tube to screen for contamination of RT reagents. Include RNA extracted from uninfected cells with a full RT Master Mix to control for assay specificity in the presence of cellular RNA.
Also include a known positive viral RNA sample with a full Master Mix to verify the quality of the RT reagents. Use the cycling conditions listed below for RT.When the program is complete, leave the samples in the thermocycler at 4 degrees Celsius or store them at 20 degrees Celsius. Next, prepare 25 microliter qPCR reactions by combining the qPCR Master Mix, PCR primers, and the qPCR probe as listed in the text protocol.
Gently mix the solution, and then add 20 microliters to each qPCR well. Add five microliters of each RT reaction containing plasmin numbers that fall within the standard curve to the appropriate qPCR wells in triplicate. Load the qPCR plate into the thermocycler and run the reaction conditions shown below.
Finally, collect and analyze the data to determine the quantity of viral RNA present in each sample, according the manufacturer's protocol. As shown here, known quantities of a plasmid encoding the NP gene from Junin virus Candid 1 were subjected to qPCR to demonstrate the sensitivity and dynamic range of the assay. In this experiment, various quantities of Junin virus Candid 1 were prebound to Vero E6 cells and quantified by qRT-PCR.
As illustrated in this figure, the assay can detect viral attachment events using as few as 20, or as many as 2 times 10 to the 6th PFU, which translates to multiplicities of infection of 0.0004 and 40, respectively. In this example, the protocol was modified to also measure the rate of amplification of the viral genome inside of cells over time. Interestingly, the quantity of viral genome appears to decrease during the first six hours following attachment.
This suggests that a portion of the viral RNA is degraded. This could happen to particles that fail to be taken into the cell, and degrade in the extracellular space, or perhaps due to degradation within the endolysosomal network. The data shown demonstrates that 12 or 18 hours post-infection would be optimal times to screen for active genome replication.
Once mastered, this technique can be done in a single day. After watching this video, you should have a very good idea of how to accurately quantitate arenavirus attachment to host cells.
