293T CRL-3216 cells were purchased from ATCC. All cells are regularly tested and are mycoplasma free. HEK293T and HeLa cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO) at 37°C with 5% CO2). Replication deficient VSV-G pseudotyped HIV-1 virions were produced in HEK293T cells using the packaging plasmid pMDG2, which encodes VSV-G envelope (Addgene plasmid # 12259), pNL4-3-derived pCRV GagPol (HIV-1 clade B)[32 (link)], and pCSGW[33 (link)] as described previously[34 (link)]. Mutagenesis of CA was performed using the QuickChange method (Stratagene) against pCRV GagPol. The HIV-1 clade B infectious molecular clone pNL4-3 was used for all passage and virus release experiments. Mutant constructs were generated with the NEB Q5 site directed mutagenesis kit (NEB E0554).
Protocol detail
Generating Replication-Deficient HIV-1 Virions
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293t cells
Cell lines
Cells
Clone
Cultured medium
Eagle
Glutamine
Hela
Hiv 1
Hiv infectious
Mutagenesis
Mycoplasma
Penicillin
Plasmid
Replication
Site directed mutagenesis
Streptomycin
Virions
Virus release
Corresponding Organization :
Other organizations : MRC Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, EMBL Australia, UNSW Sydney, ARC Centre of Excellence in Advanced Molecular Imaging, MRC Laboratory for Molecular Cell Biology, University College London
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Variable analysis
independent variables
- Mutagenesis of CA (capsid protein) using the QuickChange method (Stratagene) against pCRV GagPol
- Replication deficient VSV-G pseudotyped HIV-1 virions produced in HEK293T cells
- HEK293T and HeLa cell lines cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO) at 37°C with 5% CO2
- 293T CRL-3216 cells from ATCC, regularly tested and mycoplasma free
- Packaging plasmid pMDG2 encoding VSV-G envelope (Addgene plasmid # 12259)
- PNL4-3-derived pCRV GagPol (HIV-1 clade B)
- PCSGW plasmid
- HIV-1 clade B infectious molecular clone pNL4-3 used for all passage and virus release experiments
- Positive control: pMDG2 encoding VSV-G envelope, pNL4-3-derived pCRV GagPol, and pCSGW plasmids used to produce replication deficient VSV-G pseudotyped HIV-1 virions
- Negative control: not explicitly mentioned
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