Assessing Viral Replication Capacity Using a Radiolabeled Reverse Transcriptase Assay

Abstract

Source: Claiborne, D. T. et al. A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses. J. Vis. Exp. (2014)

This video demonstrates the assessment of viral replication capacity using a radiolabeled reverse transcriptase assay. Culture supernatants containing progeny virions are added to a reverse transcription mixture containing a radiolabeled nucleotide. The incorporated signal reflects viral output and replication efficiency. Phosphor imaging is used to detect and compare signals between the test and wild-type samples.

Protocol

1. Analysis of Reverse Transcriptase (RT) in Cell Culture Supernatants

1. Set up a biological safety hood in a biosafety level 3 (BSL-3) facility for use with radioactive materials.
   1. Place absorbent paper in the hood and replace the aspirator with an aspirator designated for radioactive use. 
      NOTE: All radioactive waste must be properly disposed of in accordance with safety regulations.
2. Add 1-2 µl of 10 mCi/ml of [α-³³P] deoxythymidine triphosphate (dTTP) and 4 µl of 1 M dithiothreitol (DTT) to each 1 ml aliquot of RT master mix (see Table 1).
3. Carefully mix each 1.5 ml microcentrifuge tube of RT master mix, DTT, and [α-³³P] dTTP with a 1,000 µl pipette and transfer to a sterile trough.
4. Dispense 25 µl of labeled RT mix into each well of a 96-well thin-walled polymerase chain reaction (PCR) plate. 
   NOTE: Include space for a positive control (MJ4 wild-type (WT) infection) and a negative control (Mock infection).
5. Add 5 µl of each supernatant to the PCR plate containing the RT master mix.
6. Seal the PCR plate with an adhesive foil cover and incubate for 2 hr at 37 °C in a thermocycler. For safety reasons, rinse pipette tips with Amphyl and dispose of tips in a small container containing Amphyl waste, which will later be disposed of in radioactive waste.
7. After incubation, make small holes in the foil cover using a 200 µl multi-channel pipette. 
   NOTE: If pipette tips touch the liquid in the well, replace tips before moving to the next column or row.
8. Mix samples 5x and transfer 5 µl of each sample to the DE-81 paper. Air dry 10 min.
9. Wash blots 5x with 1x saline-sodium citrate (SSC), and then 2x with 90% ethanol at 5 min per wash. Allow to air dry.
   1. Place each blot in a separate washing container, such as a sandwich storage box, add enough wash buffer (1x SSC or 90% ethanol (EtOH)) to cover the blot, and shake for 5 min.
   2. Pour off the wash buffer into a separate container and repeat. NOTE: The first 3 washes are considered radioactive and should be disposed of properly. The last 2 washes can be disposed of regularly.
10. Once dry, carefully wrap the blot in Saran wrap and expose to a phosphoscreen in a tightly sealed cassette overnight at room temperature.
11. Analyze phosphoscreens with a phosphorimager and quantify radioactive transcripts.
12. Draw a circle around each radioactive signal using OptiQuant software. Graph the Digital Light Unit (DLU) values to generate replication curves.
13. In order to generate replication capacity scores, DLU values were log₁₀-transformed, and slopes were calculated using the day 2, 4, and 6 time points. Replication slopes were then divided by the slope of WT MJ4 in order to generate replication capacity scores. It is important to normalize based on the MJ4 WT values obtained from the same RT plate to reduce intra-assay variability.

Table 1: Recipe for radiolabeled reverse-transcriptase assay master mix.
NOTE: Store as 1 ml of aliquots at -20 °C.

Reagent	Volume (ml)
Nuclease-free H2O	419.5
Tris-Cl, pH 7.8 (1 M)	30
Potassium chloride (1 M)	37.5
Magnesium chloride (1 M)	2.5
Nonidet P-40 (10%)	5
EDTA (0.5 M)	1.02
Polyadenylic acid, potassium salt (2 mg/ml)	1.25
Oligo-dT primer (25 μg/ml)	3.25
Total volume	500

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell Culture Reagents
Amphyl cleaner/disinfectant	Fisher	22-030-394
Reservoir Trays for Multichannel, 55 ml	Fisher	13-681-501
1 M Magnesium chloride solution	Sigma-Aldrich	M1028-100ML
Reverse-Transcriptase Quantification Assay Reagents
dTTP, [α-³³P]- 3000 Ci/mmol, 10 mCi/ml, 1 mCi	Perkin-Elmer	NEG605H001MC
1 M Tris-Cl, pH 8.0	Life Technologies/Invitrogen	15568025	Must be adjusted to pH 7.8 with KOH
2 M Potassium chloride (KCl)	Life Technologies/Invitrogen	AM9640G	Adjust to 1 M solution
0.5 M EDTA	Life Technologies/Invitrogen	15575-020
Nonidet P40	Roche	11333941103
Polyadenylic acid (Poly rA) potassium salt	Midland Reagent Co.	P-3001
Oligo d(T) primer	Life Technologies/Invitrogen	18418-012
Dithiothreitol (DTT)	Sigma-Aldrich	43815-1G
SR, Super Resolution Phosphor Screen, Small	Perkin-Elmer	7001485
Corning Costar Thermowell 96-well plate model (M) Polycarbonate	Fisher	07-200-245	Manufactured by Corning Life
Corning 96-well Microplate Aluminum Sealing Tape, Nonsterile	Fisher	07-200-684	Manufactured by Corning Life
DE-81 anion exchange paper	Whatman	3658-915
Trisodium citrate dihydrate	Sigma-Aldrich	S1804-1KG
Sodium Chloride	Fisher	S671-3
Autoradiography cassette	Fisher	FB-CA-810
Cyclone storage phoshpor screen	Packard
Thermocycler, PTC-200	MJ Research