The Full-Length HIV-1 Molecular Clone Plai 61 Was Used to Produce Wild-Type Virus and To

Supplementary information

Construction of lhRNA-expression constructs

The full-length HIV-1 molecular clone pLAI 1 was used to produce wild-type virus and to study inhibition by lhRNAs directed either against the tat, rev or nef sequences. Nucleotide numbers refer to the position on the genomic HIV-1 RNA transcript, with +1 being the capped G residue. Supplementary Table 1 lists all oligonucleotides used in this study. The tat exon 1 was amplified by PCR on pLAI with primers NotI-WdV005 and WdV002; tat exon 2 with primers WdV007 and NotI-WdV004; rev exon 1 with primers NotI-WdV001 and WdV002; rev exon 2 with primers WdV003 and NotI-WdV004. The tat exon 1 and 2 PCR products were mixed in equimolar amounts and subjected to a second PCR with NotI-WdV005 and NotI-WdV008. Similarly, rev exon 1 and 2 PCR products were reamplified with primers NotI-WdV001 and NotI-WdV004. The 276 bp tat cDNA fragment and 366 bp rev cDNA fragment were ligated into the pGEM-T vector (Promega), yielding pGEM-T-tat and pGEM-T-rev, respectively. The 653 bp HIV-1 nef fragment (position 8390 – 9010) was amplified by PCR on pLAI DNA using primers NotI-WdV011 and NotI-WdV012 and cloned into pGEM-T, creating pGEM-T-nef.

Construction of pEF1α-tat. The human elongation factor 1 α (EF1α) promoter, the Gateway (Invitrogen) cassette and the bovine growth hormone gene polyadenylation signal (BGH-polyA) of plasmid pEF5/FRT/V5-DEST (Invitrogen) were removed by HindIII and SphI digestion. A polylinker DNA fragment containing HindIII, NotI, SacI, AvrII, XhoI, SgrAI, PacI and SphI restriction sites was created by self-annealing of oligos WdV022 and WdV023, WdV024 and WdV025. The resulting DNA fragment was cloned in the HindIII and SphI sites of pEF5/FRT/V5-DEST, yielding pWdV06.3. The EF1α promoter was PCR amplified from pEF5/FRT/V5-DEST with primers HindIII-WdV047 and WdV061. A 300 bp antisense tat (as-tat) DNA fragment was PCR amplified from pGEM-T-tat with primers WdV049 and NotI-WdV050. The EF1α and as-tat fragments were mixed and subjected to a second PCR with primers HindIII-WdV047 and NotI-WdV050, and ligated into the HindIII and NotI sites of pWdV06.3 to yield pWdV17. The BGH-polyA was amplified by PCR on pEF5/FRT/V5-DEST with primersWdV053and PacI-WdV054. A 300 bp sense tat (s-tat) DNA fragment was PCR amplified from pGEM-T-tat with primers AvrII-WdV051 and WdV52. The s-tat and BGH-polyA fragments were mixed and re-amplified by PCR with AvrII-WdV051 and PacI-WdV054 and cloned in the AvrII and PacI sites of pWdV17, yielding pEF1α-tat (Fig. 3).

Construction of pEF1α-rev. The EF1α promoter was amplified by PCR on pEF5/FRT/V5-DEST with primers HindIII-WdV047 and WdV055. A 300 bp anti-sense rev (as-rev) fragment was PCR amplified from pGEM-T-rev with primers WdV056 and NotI-WdV057. Both EF1α and as-rev fragments were mixed and subjected to a second PCR with HindIII-WdV047 and NotI-WdV057. The resulting fragment was ligated into the HindIII and NotI sites of pWdV06.3, yielding pWdV19. The BGH-polyA was amplified by PCR on pEF5/FRT/V5-DEST with primers WdV60 and PacI-WdV054. A 300 bp sense rev (s-rev) was PCR amplified from pGEM-T-rev with primers AvrII-WdV058 and WdV059. The s-rev and BGH-polyA fragments were mixed and re-amplified by PCR with AvrII-WdV058 and PacI-WdV054 and the resulting fragment was ligated in the AvrII and PacI sites of pWdV19 to yield pEF1α-rev (Fig. 3).

Construction of pEF1α-nef1.The EF1α promoter was PCR amplified from pEF5/FRT/V5-DEST with primers HindIII-WdV047 and WdV061. A 300 bp anti-sense nef1 (as-nef1) fragment was amplified by PCR on pGEM-T-nef with primers WdV062 and NotI-WdV063. The EF1α and as-nef1 fragments were fused by PCR with primers HindIII-WdV047 and NotI-WdV063 and cloned into the HindIII/NotI sites of pWdV06.3 to yield pWdV21. The BGH-polyA was amplified by PCR on pEF5/FRT/V5-DEST with primersWdV066 and PacI-WdV054. A 300 bp sense nef1 (s-nef1) fragment was PCR amplified from pGEM-T-nef with primers AvrII-WdV064 and WdV065. Both BGH-polyA and s-nef fragments were mixed and re-amplified by PCR with primers AvrII-WdV064 and PacI-WdV054 and cloned into likewise digested pWdV21, yielding pEF1α-nef1 (Fig. 3).

Construction of pEF1α-GFP. A DNA fragment comprising the hrGFP gene was PCR amplified from PSXrabpA (Stratagene) with primers attB1-WdV020 and attB2-WdV021. The hrGFP was recombined into pEF5/FRT/V5-DEST using the Gateway technology, resulting in the hrGFP gene expression vector pHY01. The EF1α promoter was amplified by PCR on pEF5/FRT/V5-DEST with primers SphI-WdV034and WdV035. A second PCR was performed to amplify the hrGFP fragment from pHY01 with primers WdV036 and BamHI-WdV037. Both EF1α and hrGFP fragments were mixed and subjected to a PCR with SphI-WdV034 and BamHI-WdV037 and the resulting PCR product was ligated into SphI and BamHI sites of pUC19 to yield pWdV09. An antisense fragment from hrGFP (as-hrGFP) gene was amplified by PCR on pHY01 with primers BamHI-WdV038and WdV039. A second PCR was performed to amplify an SV40-polyA (SV40-pA) fragment from plasmid pPUR (Clontech)using primers WdV040 and EcoRI-WdV041. as-hrGFP and SV40-pA fragments were mixed and subjected to PCR with BamHI-WdV038 and EcoRI-WdV041. The resulting fragment was ligated into the BamHI and EcoRI sites of pWdV09, yielding pWdV10. pWdV10 was digested with SphI and the fragment containing the GFP inverted repeat was ligated in the likewise digested pHY01 plasmid to yield pEF1α-GFP (Fig. 3).

Construction of pLTR-tat, pLTR-nef1, p7tetO-tat andp7tetO-nef1.The HIV-1 LTR was PCR amplified from pBlue3’LTR-luc 2with primers SalI-PKF5 and HindIII-PKR5; 7tetO was amplified from the CMV-7tetO promoter/luciferase reporter construct pUHC13-3 3 with primers SalI-PKF6 and HindIII-PKR6. Both LTR and 7tetO amplicons were cloned in the SalI and HindIII sites of pBluescript SK- (Stratagene) to yield pBlue-LTR and pBlue-7tetO, respectively. To create the lhRNA tat and nef1, approximately 300 nt as-tat and as-nef1 fragments were PCR amplified from pEF1α-tat and pEF1α-nef1 with primers HindIII-PKF4 and NotI-PKR2, and cloned into the HindIII and NotI sites of pBlue-LTR and pBlue-7tetO, resulting in pLTRas-tat, pLTRas-nef1, p7tetOas-tat andp7tetOas-nef1. Two oligos PKF8 and PKR13 were self-annealed to create a polylinker NotI-MunI-BsaBI-XbaI-AvrII-SacII-BamHI-EcoRV-BbrPI-SacII that was ligated in the NotI and SacII sites of the above pLTR- and 7tetO-containing plasmids. The intron of the EF1α promoter was amplified from pWdV65 with primers MunI-PKF13 and XbaI-PKR9 and ligated in the MunI and XbaI sites of the polylinker. The s-tat and s-nef1 fragments, together with the BGH-polyA, were amplified from pEF1α-tat and pEF1α-nef1 with primers PKF2 and BamHI-PKR4 and cloned into the AvrII and BamHI sites downstream of the EF1α intron, resulting in pLTR-tat, pLTR-nef1, p7tetO-tat, and p7tetO-nef1 (Fig. 3). Vector pLTR-nef1 (Fig. 3) was created by insertion of the 554 nt HindIII fragment of pLAI (, 76 - 630) into the corresponding site of pLTR-nef1. The Ψ fragment includes the HIV-1 polyA signal, primer-binding site, dimerisation signal, splice donor site and packaging signal. Plasmid pLTR has been described previously 4.

Construction of pT7-nef2 andpT7sh-nef. Plasmid pGEM-T-nef was PCR amplified with primers SacI-T7p-PKF16and asT7p-XbaI-PKR16. The resulting nef2 PCR product that contains convergent T7 promoters and terminators was cloned in pCR2.1-TOPO (Invitrogen) to yield pT7-nef2 (Fig. 3). Plasmid pT7sh-nef was constructed by self-annealing of two complementary oligos NcoI-PKF21 and BamHI-PKR21 that were cloned into the NcoI andBamHI sites of pT7-luc (5,6, a kind gift of Dr. P. Midoux, Centre de Biophysique Moleculaire, Orleans, France), replacing the Photinus pyralis luciferase (firefly luciferse, FL) cDNA. Plasmid pcDNA3-T7pol, expressing bacteriophage T7 polymerase (pT7-pol, a kind gift of Dr. Jean-Marc Jacque, University of Massachusetts Medical School, USA), and pH1sh-nef have been described previously 7,8.

References

1. Peden K, Emerman M, Montagnier L. Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virol 1991; 185: 661-672.

2. Klaver B, Berkhout B. Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus. J Virol 1994; 68: 3830-3840.

3. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992; 89: 5547-5551.

4. Berkhout B, van Wamel JL. Inhibition of human immunodeficiency virus expression by sense transcripts encoding the retroviral leader RNA. Antiviral Res 1995; 26: 101-115.

5. Brisson M, He Y, Li S, Yang JP, Huang L. A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes. Gene Ther 1999; 6: 263-270.

6. Brisson M, Tseng WC, Almonte C, Watkins S, Huang L. Subcellular trafficking of the cytoplasmic expression system. Hum Gene Ther 1999; 10: 2601-2613.

7. Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature 2002; 418: 435-438.

8. Das AT, Brummelkamp TR, Westerhout EM et al. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol 2004; 78: 2601-2605.