Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | TRIM5α | NCBI | NM_033034.2 | |
| Gene (Macaca mulatta) | TRIM5α | NCBI | NM_001032910.1 | |
| Strain, strain background (Escherichia coli) | DH5α | NEB | C2987H | Chemically competent cells |
| Cell line (Homo sapiens) | HEK-293T/17 | ATCC | CRL-11268; RRID:CVCL_1926 | Purchased fresh stock from ATCC |
| Cell line (Felis catus) | CRFK | ATCC | CCL-94; RRID:CVCL_2426 | Purchased fresh stock from ATCC |
| Cell line (Felis catus) | CRFK + Human TRIM5α | This study | Cell line stably expressing human TRIM5α (from ~ single random integration event per cell); see Materials and methods | |
| Cell line (Felis catus) | CRFK + Human-v1DMS TRIM5α | This study | Cell line stably expressing library of human TRIM5α single amino acid variants; see Materials and methods | |
| Cell line (Felis catus) | CRFK + Human-R332P TRIM5α | This study | Cell line stably expressing human TRIM5α-R332P; see Materials and methods | |
| Cell line (Felis catus) | CRFK + Human-R332P-v1DMS TRIM5α | This study | Cell line stably expressing library of human TRIM5α-R332P single amino acid variants; see Materials and methods | |
| Cell line (Felis catus) | CRFK + Rhesus TRIM5α | This study | Cell line stably expressing rhesus TRIM5α; see Materials and methods | |
| Cell line (Felis catus) | CRFK + Rhesus-v1DMS TRIM5α | This study | Cell line stably expressing library of rhesus TRIM5α single amino acid variants; see Materials and methods | |
| Antibody | anti-HA.11 (mouse monoclonal) | Biolegend | 901516; RRID:AB_2565335 | (1:1000) |
| Antibody | anti-β-actin (rabbit polyclonal) | Abcam | ab8227; RRID:AB_2305186 | (1:5000) |
| Antibody | IRDye 680RD anti-mouse (donkey polyclonal) | LI-COR | 926–68072; RRID:AB_10953628 | (1:10,000) (secondary) |
| Antibody | IRDye 800CW anti-rabbit (donkey polyclonal) | LI-COR | 926–32213; RRID:AB_621848 | (1:10,000) (secondary) |
| Recombinant DNA reagent | MD2.G | Addgene | 12259 | VSV-G expression, CMV promoter |
| Recombinant DNA reagent | L-VSV-G | PMID:9245614 | VSV-G expression, Tat-driven | |
| Recombinant DNA reagent | CMV-Tat | PMID:9245614 | Tat expression, CMV promoter | |
| Recombinant DNA reagent | pHIV-ZsGreen | PMID:18371425 | HIV-1 transfer vector | |
| Recombinant DNA reagent | pALPS-eGFP | PMID:30546110 | HIV-1 transfer vector | |
| Recombinant DNA reagent | p8.9NdSB bGH Blp1 BstEII | PMID:15479815 | NL4.3 HIV-1 gag/pol | |
| Recombinant DNA reagent | p8.9NdSB bGH Blp1 BstEII HIV-2 CA | PMID:26181333 | NL4.3 HIV-1 gag/pol with HIV-2ROD CA residues 1–202 | |
| Recombinant DNA reagent | pHIV-MAC | PMID:18417575 | HIV-1 gag/pol with SIVmac239 CA residues 1–146 (A77V) | |
| Recombinant DNA reagent | pHIV-Gb2 | PMID:18417575 | HIV-1 gag/pol with SIVcpzGab2 CA residues 1–146 | |
| Recombinant DNA reagent | pCIG3N | PMID:10906195 | N-MLV gag/pol | |
| Recombinant DNA reagent | pQCXIP | Takara Bio | 631516 | Puromycin-selectable retroviral expression cassette |
| Recombinant DNA reagent | pQCXIP-eGFP | This study | N-MLV transfer vector; see Materials and methods | |
| Recombinant DNA reagent | pQCXIP-Human TRIM5α-HA | This study | Stable expression of human TRIM5α; see Materials and methods | |
| Recombinant DNA reagent | pQCXIP-Human-R332P TRIM5α-HA | This study | Stable expression of human TRIM5α-R332P; see Materials and methods | |
| Recombinant DNA reagent | pQCXIP-Human-v1rhesus TRIM5α-HA | This study | Stable expression of human TRIM5α containing rhesus v1 loop; see Materials and methods | |
| Recombinant DNA reagent | pQCXIP-Rhesus TRIM5α-HA | This study | Stable expression of rhesus TRIM5α; see Materials and methods | |
| Peptide, recombinant protein | Q5 high-fidelity DNA polymerase | NEB | M4091L | Used for Quikchange and DMS library construction |
| Commercial assay or kit | NEBuilder HiFi DNA assembly cloning kit | NEB | E5520S | Gibson assembly kit |
| Commercial assay or kit | PureYield plasmid miniprep kit | Promega | A2495 | Transfection-quality plasmid miniprep (low LPS) |
| Commercial assay or kit | NuceloBond Xtra midiprep kit | Takara Bio | 740410.50 | Transfection-quality plasmid midiprep (low LPS) |
| Commercial assay or kit | DNeasy Blood and Tissue kit | Qiagen | 69504 | Genomic DNA purification |
| Commercial assay or kit | QIAquick PCR purification kit | Qiagen | 28106 | PCR cleanup kit |
| Commercial assay or kit | Agencourt Ampure XP beads | Beckman Coulter | A63880 | Double-sided size selection of PCR products |
| Chemical compound, drug | Trans-IT 293T transfection reagent | Mirus Bio | MIR 2700 | Transfect plasmids into HEK-293T |
| Chemical compound, drug | polybrene | Sigma | TR-1003-G | Transduction reagent |
| Cchemical compound, drug | puromycin | Fisher | 50488918 | Antibiotic selection for stably transduced cells |
| Software, algorithm | DMS data analysis | This study | https://github.com/jtenthor/T5DMS_data_analysis | R scripts for DMS data analysis; see Materials and methods |
| Software, algorithm | PAML | PMID:967129 | For analysis of rapid evolution |