Reverse Transcriptase Activity as a Marker for Virus-Like Particle Production

Begin with wells containing adherent mammalian cells co-transfected with plasmids encoding viral proteins and either a non-inhibitory control RNA or an inhibitory RNA.

In the control wells, viral proteins, including reverse transcriptase (RT), assemble into mature virus-like particles (VLPs) and are released into the supernatant.

In the test wells, the inhibitory RNA suppresses viral protein expression, reducing VLP production.

Transfer the supernatant to a lysis buffer and incubate to lyse the VLPs, releasing RT.

Add a buffer containing radiolabeled nucleotides, primers, a synthetic RNA template, and cofactors, then incubate.

The RT incorporates the labeled nucleotides into new strands.

Spot the mixture onto a charged filter paper, dry it, and wash with a buffer to remove unbound nucleotides.

Perform an alcohol wash to remove residual salts.

Measure radioactivity to quantify RT activity, indicating VLP production.

Reduced VLP production in test samples indicates suppression by the inhibitory RNA.

To carry out the viral production assay, after removing the 24-well cell culture plates from the incubator, transfer them to the BSL-3 cell culture hood. Gently swirl the plates and then transfer 1