Engineered HIV-1 Variants and Plasmids

Single-cycle HIV-1 NL4-3.GFP.R+ and NL4-3.GFP.R– viruses were previously described (16 (link)). HIV-1(vpr.Q8*) was generated by mutating Vpr Q8 to a stop codon (CAA→TAA) in HIV-1 NL4-3.GFP.R+. Replication-competent HIV-1(vpr.wt) and HIV-1(vpr.Q8*) were constructed by substituting the tag red fluorescent protein (RFP) gene for the GFP gene and restoring the intact NL4-3 env gene. Virus particles were produced from HEK293T cells, concentrated, and normalized by infectivity to Jurkat T cells and by quantitative Gag immunoblotting, as described previously (44 (link), 45 (link)). pLVX-TRE3G and pLVX-Tet-One Puro-inducible expression vectors (Clontech) were produced from HEK 293T cells as described previously (16 (link)). pEasiLv-puro was constructed by replacing the SpeI-SalI fragment comprising the E2-Crimson gene in pEasiLv (46 (link)) with the puromycin N-acetyl-transferase gene. All mutations and constructs were verified by DNA sequencing.

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Yan J., Shun M.C., Hao C., Zhang Y., Qian J., Hrecka K., DeLucia M., Monnie C., Ahn J, & Skowronski J. (2018). HIV-1 Vpr Reprograms CLR4DCAF1 E3 Ubiquitin Ligase to Antagonize Exonuclease 1-Mediated Restriction of HIV-1 Infection. mBio, 9(5), e01732-18.

Publication 2018

pLVX-TRE3G

293t cells Cells Env gene Gene Hiv 1 Jurkat cells Mutations Puromycin n acetyl transferase Red fluorescent protein Replication Stop codon Vectors Virus particles Viruses

Corresponding Organization :

Other organizations : Case Western Reserve University, University of Pittsburgh

Top 5 similar protocols

Variable analysis

independent variables

Vpr mutation (Q8 to stop codon)

dependent variables

Viral replication
Viral infectivity

control variables

Virus production conditions (from HEK293T cells)
Virus normalization (by infectivity and Gag immunoblotting)

controls

Positive control: Replication-competent HIV-1(vpr.wt)
Negative control: NL4-3.GFP.R- virus

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