Lentiviral vectors were derived from pHRSIN-CSGW [31 (link)] by insertion of a second promoter and transgene downstream of the WPRE element. Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped lentivectors were produced by co-transfection of the vector with pCMVR8.91 (Gag/pol) and pMDG (VSV-G envelope) as previously described [32 (link)]. A lentivector co-expressing a shRNA targeting 4-1BB together with NP was constructed as described previously [41 (link)]. Supernatants were concentrated by ultracentrifugation at 24,000 rpm through a 20% sucrose cushion. Titres were measured by reverse transcriptase assay (Roche Diagnostics, UK) and quantitative TaqMan PCR (Applied Biosystems, UK).
Reverse transcriptase assay
Manufactured by Roche
Sourced in United Kingdom, Germany
The Reverse Transcriptase Assay is a laboratory tool used to measure the enzymatic activity of reverse transcriptase, an essential enzyme involved in the replication process of various types of viruses, including retroviruses such as HIV. The assay provides a quantitative assessment of reverse transcriptase activity in a sample, which can be useful in research and diagnostic applications.
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4 protocols using reverse transcriptase assay
1
Lentiviral Vector Production and Characterization
2
Viral Production and Titration Protocol
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Viruses were produced by transfection of 293T cells by using Genejuice transfection reagent (Novagen) as described [4 (link)]. A panel of T/F plasmids were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Panel of full-length transmitted/founder (T/F) HIV-1 Infectious Molecular Clones (Cat #11919) from Dr. John Kappes [8 (link)–10 (link)]. Plasmids were amplified by transformation into STBL3 E.coli bacteria (Invitrogen). For plasmid purification, the nucleobond XtraMidi (Macherey-Nagel) was used and the size of the plasmids was checked by restriction analysis. After transfection, the concentration of the produced viruses was quantified by a p24 antigen ELISA (Perkin Elmer Life Sciences), and the viruses were titrated using the indicator cells TZM-bl (John C. Kappes, Xiaoyun Wu, Birmingham, Alabama, USA and TranzymeInc.), the NIH AIDS Reagent Program [11 (link)]. For infections, the viruses were normalized based on this titration. Infectious molecular clones of five T/F and patient-matched 6-month (6-mo) consensus sequence virus pairs were generated and viral stocks produced as previously described [12 (link),13 (link)]. The sequences for CH164 6-mo and CH042 6-mo have been deposited in the genbank: KC156128 and KC156124 respectively. Viral titers were measured by reverse transcriptase assay (Roche Applied Science).
3
Measuring HIV-2 Reverse Transcriptase Activity
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HIV-2 virions were purified and the level of virion-associated reverse transcriptase was measured according to the manufacturer’s instructions (Reverse Transcriptase Assay, colorimetric, Roche).
4
Quantifying Lentiviral Replication
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Some cell lines were transfected with the luciferase expressing gene using retrovirus or lentivirus vectors. To examine lentivirus replication, the cultured medium of these cell lines was centrifuged at 250g for 10 min at 4°C to remove cell debris. The supernatant was concentrated by centrifugation (22 000g) for 2 h at 4°C, then the pellets were analysed by the Reverse Transcriptase Assay (Roche, Germany, 11468120910) as described by the manufacturer.
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