TABLE 2.
PCR primers/probes utilized in this studya
| Primer/probe | Direction | Sequence (5′→3′) |
|---|---|---|
| Mutagenesis PCR | ||
| MLV CA mutation | ||
| N QR109TE | F | TTACACCACCACAGAAGGTAGGAACCACCTAGTTCTC |
| N QR109TE | R | TCCCAATCGGGACGTTCA |
| B TE109QR | F | TTACACCACTCAAAGAGGTAGGAACCACCTAGTCCTCTATC |
| B TE109QR | R | TCCCAATCGGGGCGTTCA |
| HIV CA mutation | ||
| Pax E45A | F | GCATTATCAGCTGGAGCCACCC |
| Pax E45A | R | TGAAAACATGGGTATCAC |
| Pax N74D | F | AGAGACCATCGATGAGGAAGC |
| Pax N74D | R | TTTAACATTTGCATGGCTG |
| Pax G89V | F | GTGCATGCAGTGCCTATTGCA |
| Pax G89V | R | TGGATGCACTCTATCCCATT |
| Pax P90A | F | TGCATGCAGGTGCCATTGCACCAG |
| Pax P90A | R | CTGGATGCACTCTATCCC |
| Pax A92E | F | GGGCCTATTGAGCCAGGCCAGATG |
| Pax A92E | R | TGCATGCACTGGATGCAC |
| Pax G94D | F | ATTGCACCAGACCAGATGAGAGAACC |
| Pax G94D | R | AGGCCCTGCATGCACTGG |
| FBKT Mx2 mutation | ||
| FBKT_Mx2 Y21C | F | TCCCAACACTGTCCGAAAAAGGAAATGAATC |
| FBKT_Mx2 Y21C | R | GGGCGCCTGATTGTGCCT |
| FBKT_Mx2 L600P | F | GCGAATTCGCCTGGCAAGACT |
| FBKT_Mx2 L600P | R | GTCTTCTCGGGCCTTTTC |
| FBKT CPSF6 C terminus truncation | ||
| FBKT_CPSF6del | F | ATGGGCAAACCGATTCCGAAC |
| FBKT_CPSF6del | R | GCCTGGAGGTGGAGGTGG |
| pWPT-E2-Crimson and pWPT-tdTomato plasmid construction | ||
| WPT-GFP (Crim) | F | AATTCGATATCAAGCTTATCG |
| WPT-GFP (Crim) | R | TAAGCTTAGGCCTGGATC |
| E2 Crimson | F | CGGATCCAGGCCTAAGCTTAGCCACCATGGATAGCACTG |
| E2 Crimson | R | GATAAGCTTGATATCGAATTTCACTGGAACAGGTGGTGGCG |
| WPT-GFP (tmt) | F | CTAGCTAGTCGAGCTCAACTTCGAATTCGATATC |
| WPT-GFP (tmt) | R | GGCCTGGATCCGCGTCAC |
| tdTomato | F | TCGTGACGCGGATCCAGGCCGCCACCATGGTGAGCAAG |
| tdTomato | R | AGTTGAGCTCGACTAGCTAGTTACTTGTACAGCTCGTCCATG |
| pCFG2-E2-Crimson and pCFG2-tdTomato plasmid construction | ||
| CFG2 F5 | F | ATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGG |
| CFG2 R6 | R | AGATCTGGCCATGGCAGTC |
| tomato F2 | F | AGACTGCCATGGCCAGATCTGCCACCATGGTGAGCAAG |
| tomato R2 | R | CTAAATAAAATCTTTTATTTTATTTACTTGTACAGCTCGTCCATG |
| Crimson F2 | F | AGACTGCCATGGCCAGATCTGCCACCATGGATAGCACTG |
| Crimson R2 | R | CTAAATAAAATCTTTTATTTTATTTACTGGAACAGGTGGTG |
| For constructing delivery vector plasmids containing bat host factors | ||
| pLGatewaySN-vector plasmid | ||
| 583_fwd | F | GATAACTCGAGGATCCGG |
| 583_rev | R | GATAACGAATTCCGGCGC |
| V5-TRIM5-delivery vector plasmid | ||
| TRIM5_fwd | F | TGGAGGTAGTATGGCTACAGGAATCCTG |
| TRIM5_rev | R | AGCCGGATCCTCGAGTTATCTTAAGAGTTTGGAGAAGACAG |
| V5_fwd | F | AGGCGCCGGAATTCGTTATCATGGGCAAACCGATTCCG |
| V5_rev | R | CTGTAGCCATACTACCTCCACCTCCGGTG |
| Mx2-V5-delivery vector plasmid | ||
| FBKT-Mx2_C | F | AGGCGCCGGAATTCGTTATCATGCCAAAGTCCCACAAG |
| FBKT-Mx2_C | R | GTTTGCCCATGCAGGAGAATTTGTAGAGC |
| FBKT-Mx2_C_V5 | F | ATTCTCCTGCATGGGCAAACCGATTCCG |
| V5_C_rev2 | R | AGCCGGATCCTCGAGTTATCTTAACTACCTCCACCTCCG |
| CPSF6-V5-delivery vector plasmid | ||
| FBKT-CPSF6_C | F | AGGCGCCGGAATTCGTTATCATGGCGGACGGCGTGGAC |
| FBKT-CPSF6_C | R | GTTTGCCCATACGATGACGATACTCGCGC |
| FBKT-CPSF6_C_V5 | F | TCGTCATCGTATGGGCAAACCGATTCCG |
| V5_C_rev2 | R | AGCCGGATCCTCGAGTTATCTTAACTACCTCCACCTCCG |
| Full-length cytochrome B | F | CCHCCATAAATAGGNGAAGG |
| R | WAGAAYTTCAGCTTTGGG | |
| EnK Cyt B gene | F | ATGACCAACATTCGAAAATCYCAC |
| R | TCTTCATTTTAGTAGGTGATTTTC | |
| PCR primers to amplify TRIM5, Mx2/MxB, and CPSF6 cDNAs | ||
| Oligo(dT) adapter primer | GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN | |
| 1st-round PCR | ||
| Megabat TRIM5 | F | ATGGCTACAGGAATCCTG |
| Microbat TRIM5 | F | ATGGCTTCRGGAATYSTG |
| Megabat Mx2/MxB | F | ATGCCMAARTCCCACAAG |
| Megabat CPSF6 | F | ATGGCGGACGGYGTGGAC |
| Outer adaptor | R | GCGAGCACAGAATTAATACGACT |
| 2nd-round PCR for Gibson Assembly reaction | ||
| Megabat TRIM5 | R | TTAAGAGTTTGGAGAAGACAG + vector-specific sequence |
| Microbat TRIM5 | R | TTAAGARCYKGGAGAACACAG + vector-specific sequence |
| Megabat Mx2/MxB | R | TTAGCAGGAGAATTTRTAGAG + vector-specific sequence |
| Megabat CPSF6 | R | CTAACGATGACGATACTCGCGC + vector-specific sequence |
| qRT-PCR primers/probes | ||
| Early RT product | F (hRU5-F2) | GCCTCAATAAAGCTTGCCTTGA |
| R (hRU5-R) | TGACTAAAAGGGTCTGAGGGATCT | |
| Probe (hRU5-P) | 6FAM-AGAGTCACACAACAGACGGGCACACACTA-TAMRA | |
| Late RT product | F (MH531) | TGTGTGCCCGTCTGTTGTGT |
| R (MH532) | GAGTCCTGCGTCGAGAGATC | |
| Probe (LRT-P) | 6FAM-CAGTGGCGCCCGAACAGGGA-TAMRA | |
| 2-LTR circle | F (hRU5-F2) | GCCTCAATAAAGCTTGCCTTGA |
| R (MH536) | TCCACAGATCAAGGATATCTTGTC | |
| Probe (P-HUS-SS1) | 6FAM-TAGTGTGTGCCCGTCTGTTGTGTGAC-TAMRA |
a
CA, capsid protein; qRT-PCR, reverse transcription-quantitative PCR; LTR, long terminal repeat.