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. 2015 May 13;2:15017. doi: 10.1038/mtm.2015.17

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Copyright © 2015 American Society of Gene & Cell Therapy

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Positive control proviral DNA constructs used and replication-competent lentivirus (RCL) assay evaluation criteria. (a) Proviral DNA constructs generated in this study. HIVPC encodes knock-out mutations (*) of Vif, Vpr, Vpu, and Nef. HIVPC-ID additionally encodes a D64V Integrase mutation and an extended deletion within the U3 and 3′-PPT regions (ΔU3), rendering it integration defective. HIVΔEnv(GFP) lacks an encoded envelope gene and encodes GFP in the nef transcription unit. MLV-E1001 was generated by mutating the initiator methionine of the 4070A ORF in MLV-4070A, and replacing downstream sequences with the E1001 ORF. (b) RCL assay evaluation criteria. Criterion 1: Ability of E1001-enveloped vector particles to enter the chosen amplification cells. Criterion 2: Release of transduction-competent, E1001-enveloped vector particles from amplification cells. Criterion 3: Replication and amplification of the chosen positive control (PC) virus in the assay cell line from low MOI. GFP, green fluorescence protein; HIVPC, HIV-1 positive control; HIVPC-ID, integration-deficient HIV-1 positive control; MOI, multiplicity of infection.