Lentiviral Expression of TRIM5α Variants

pEasiLV plasmids expressing luciferase, CD8, MX2 or TRIMCYP have been described6 (link). cDNAs encoding TRIM5α and TRIM5α isoform X7 lacking the SPRY domain were amplified using the SuperScript III One-Step RT–PCR System with Platinum Taq polymerase (Invitrogen) from 20 ng RNA obtained from IFNα-treated (500 U ml^-1) U87-MG CD4⁺ CXCR4⁺ cells and inserted into pEasiLV-MCS6 (link) between the AgeI and XhoI restriction sites. The TRIM5α R332G/R335G and H43Y mutants were obtained by site directed mutagenesis (SDM). CRISPR-resistant TRIM5α constructs (rTRIM5α) were produced by SDM using primers designed to silently mutate the guide RNA target sequence. pFLAG-TRIM5α was generated by subcloning a cDNA encoding TRIM5α with an N-terminal FLAG tag into pCAGGS (Addgene) using the EcoRI and XhoI sites. pCAGGS expressing FLAG-tagged GFP, pFLAG-GFP, was derived from a plasmid encoding HA-tagged GFP36 (link). The vectors encoding HA-tagged ubiquitin and the K48R and K63R mutant derivatives have been described37 (link)
38 (link).

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Jimenez-Guardeño J.M., Apolonia L., Betancor G, & Malim M.H. (2019). Immunoproteasome activation enables human TRIM5α restriction of HIV-1. Nature microbiology, 4(6), 933-940.

Publication 2019

SuperScript III One-Step RT–PCR System with Platinum Taq polymerase pCAGGS

Cd4 cells Cdna Crispr Cxcr4 Derivatives Ecori Ifnα Isoform Luciferase Plasmid Platinum Primers Rna sequence Rt pcr Site directed mutagenesis Spry domain Taq polymerase Ubiquitin Vectors

Corresponding Organization :

Other organizations : King's College London

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Variable analysis

independent variables

TRIM5α and TRIM5α isoform X7 lacking the SPRY domain
TRIM5α R332G/R335G and H43Y mutants
CRISPR-resistant TRIM5α constructs (rTRIM5α)
PFLAG-TRIM5α
PFLAG-GFP
HA-tagged ubiquitin and the K48R and K63R mutant derivatives

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

PEasiLV plasmids expressing luciferase, CD8, MX2 or TRIMCYP

negative controls

Not explicitly mentioned

Annotations

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