825. Product Enhanced Reverse Transcriptase (PERT) Assay for RCL/RCR Detection

Product Enhanced Reverse Transcriptase (PERT) assay has been used extensively to detect reverse transcriptase (RT) activity associated with retroviruses. We have assessed the usefulness of the PERT assay for RCL and RCR testing and compared it with existing assays for RCL (p24gag ELISA/gag PCR) and RCR (S+/L-) detection. As a first step towards evaluating the usefulness of PERT for RCL detection, we determined the sensitivity of detection of purified and virus associated HIV-RT by PERT. The PERT assay was able to detect 100 molecules of purified HIV-1 RT and 1-0.1 IU of a replication competent HIV-1 virus, R7-GFP, in two independent experiments. In a RCL detection assay comprising of a 3-week amplification phase and a 1-week indicator phase, 1 IU of R7-GFP was detected by all three assays (p24 ELISA, gag PCR and PERT) in spite of higher backgrounds associated with the PERT assay. To provide additional support for the use of PERT for RCL testing, we also examined the effect of competing vector particles on RCL detection by p24 ELISA and PERT. Both assays were able to detect 1 IU of R7-GFP mixed with varying concentrations (100, 1000, 5000 and 10,000 ng of p24) of a HIV-1 vector, CS-CGW (provided by Philip Zoltick, Philadelphia, PA), in the RCL detection assay. These results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication competent HIV-1 in a RCL detection assay. For evaluating the ability of the PERT assay to detect RCR, we determined the sensitivity of detection of purified M-MuLV-RT and compared the detection of replication competent retroviruses (GALV-SEATO, RD114, and 4070A) by PERT and S+/L- assays. The PERT assay detected approximately 100 molecules of purified MuLV-RT in three independent experiments. Both assays detected 1 IU of RD114 and 10 IU of 4070A; detection of the GALV-SEATO virus was more sensitive by the S+/L- assay (1-10 IU) than the PERT assay (10-100 IU). These studies indicate that the sensitivity of detection of retroviruses is equivalent by PERT and S+/L- assays. To evaluate the sensitivity of PERT in a RCR detection assay, GALV-SEATO and RD114 viruses were amplified for 3-weeks and serial dilutions of amplified material were tested by S+/L- and PERT assays. Both assays detect 1 IU of RD114 and GALV after the amplification phase suggesting that they have similar sensitivities in an extended RCR assay. In conclusion, the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.