248 Nucleic Acids Research, 2007, Vol. 35, No. 1 Table 1. Summary of targeted sites in HIV-1 provirus and their effect on viral p24 expression U1 snRNA name Targeted sequence Location Reading frame Suppression of p24 expression Conservationa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 CTTCAGCTAC ATAGCAGTAG TTTGCTATAA CAGGTACCTT CATCCGGAGT TGGGGTACCT GCCTGTGTAC GAAAAGGGGG GACTGGAAGG ACACACAAGG AACTACACAC GGGGGACTGG TTTTAAAAGA GGCAGGGATA TTAGGCAGGG 8519 8682 8786 9013 9391 6344 6441 9073 9083 9143 9172 9079 9065 8350 8347 Rev exon 2 Env Env Nef LTR Env Env Nef LTR LTR LTR LTR Nef Env Env ++ ++ + + + 66% 67% 56% 37% 71% 92% 56% 78% 74% 95% 91% 85% 87% Position of targeted sequences is stated relative to the reference HIV-1 provirus HXB2 (K03455). a% of time the complete target sequence is found in a database of 1005 viral genomes belonging to the M and O clades of HIV-1. ++-High activity, suppression of viral p24 expression to background level. +-Moderate activity, >50% reduction in p24 production expression. -No significant change in viral p24 expression observed. viral mRNAs. Furthermore, the conserved nature of the sequences targeted suggests that this approach may provide broad resistance to a range of HIV-1 strains. MATERIALS AND METHODS Bioinformatics A computation-based selection algorithm was devised for the selection of potential anti-HIV target sites in the 30-terminal exon of HIV transcripts. Consensus sequences of the env and nef regions of all M and O clades of HIV-1 were retrieved from the Los Alamos HIV Sequence Database (http://www. hiv.lanl.gov/) and were aligned using ClustalW (http:// www.ebi.ac.uk/clustalw/) followed by manual adjustment of the multiple sequence alignment (MSA) using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) or JalView (http://www.ebi.ac.uk/~michele/jalview/). A set of well- conserved continuous 10 nt sites was identiﬁed by inspection of the MSA. The number of BLAST hits returned for each potential target site against the HIV-1 or human subset of GenBank sequences was recorded. The SR-value of a 10 bp site is deﬁned as the number of BLAST hits returned for the oligo against GenBank HIV-1 sequences. The number of returned BLAST hits for the same oligo against GenBank human sequences was assigned a value N, the number of hits for a non-HIV directed oligo [anti-CAT, (21)] was designated M. The SF-value of an oligo was calculated using its associated SR, N and M values according to the formula SF ¼ SR/(N/M). Multiple sites that satisfy the condition SF > SR were selected for the construction of U1 targeting constructs described. Plasmid constructs HIV-1 Hxb2 R-/RI- was provided by E. Cohen (Institut de recherche cliniques de Montreal). Generation of U1 targeting vectors has been previously described (21). The mutant U1 anti-HIV snRNA expression constructs pU1 aHIV-1#1-15 were derived from pUCB U1 [provided by H. Schaal (Institute of Virology, Heinrich Heine University Dusseldorf, Germany)] by PCR (21) or using quickchange mutagenesis (Stratagene). Targeted sequences are indicated in Table 1. Figure 1 illustrates the relative positions of ﬁve of the target sites in the HIV-1 30 LTR proximal region. The follow- ing system of nomenclature is employed in this report regard- ing U1 targeting constructs: pU1 aHIV-1 #1 or pU1 aCAT refers to the plasmid encoding the 50 end mutated U1 snRNA sequence while U1 aHIV-1 #1 or U1 aCAT refers to the inhibiting U1 snRNAs expressed from those plasmids. To investigate the requirement for the various U1 snRNP protein components to achieve suppression of HIV-1 expres- sion, a number of mutants of the aHIV-1 #1 and #5 expres- sion vector were generated by quickchange (Stratagene) using the following primer pairs: DSL1, 50-CGAAGATCTCAAC- TCCGGATGGCAGGGCCCAGGGCGAGGCTTATC-30, 50- GATAAGCCTCGCCCTGGGCCCTGCCATCCGGAGTTG- AGATCTTCG-30; SL1mut, 50-GCAGGGGAGATACCAA- CTAGTGCTTCCTGGTTTTCCCAGGGC-30, 50-GCCCTG- GGAAAACCAGGAAGCACTAGTTGGTATCTCCCCTGC- 30; DSL2, 50-GGGAAATCGATGGAAAACCACCTTCGTG- A-30, 50-GGTGGTTTTCCATCGATTTCCCCAAATGT-30; SL2mut, 50-GGGCGCGGCTTATCCATTACGCGGATGT- GCTGACCCC-30, 50-GGGGTCAGCACATCCGCGTAATG- GATAAGCCTCGCCC-30; DSm, 50-CACTACCACCTCGA- GTGCAGTCGAGTTTG-30, 50-TCGACTGCACTCGAGGT- GGTAGTG-30. All constructs were conﬁrmed by sequencing. Transfection and western blots 293T cells were transfected by calcium phosphate precipita- tion with 1.0 mg of HxBruR /RI and a maximum of 4 mg of pUCB U1 or its derivatives (24). At 48 h post-transfection, cells were harvested and lysed in whole cell lysis buffer (10 mM Na2HPO4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% Na azide, 0.5% Na deoxycholate and 1 mM Na orthovanadate) for 20 min. For p24 blots, protein lysates were fractionated on 12% SDS–PAGE then transferred onto