250 Nucleic Acids Research, 2007, Vol. 35, No. 1 blots were stripped and reprobed with mouse anti-tubulin antibody (Sigma). Northern blot analysis 293T cells (3 · 106) were transfected with 4 mg of HxBruR /RI and a total of 12 mg of pUCB U1 or its deriva- tives. At 48 h post-transfection cells were harvested and lysed in GT (4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7), 0.5% sarcosyl, 0.1M b-mercaptoethanol). Total RNA was isolated (25) and analyzed by northern blotting. Blots were probed with 32P labeled DNA to HIV-1 LTR (generated by PCR using the primers 50-CTAATTCACTCCCAACG- AAGA-30 and 50-TGCTAGAGATTTTCCACACTG-30), end labeled oligo to GAPDH (50-AAAGGTGGAGGAGTGGGT- GTCGCTGTTGAA-30), or end labeled oligos speciﬁc for each of the U1 snRNA variants (anti-HIV #1 50-CCTGCC- TTCAGCTAC-30; anti-HIV #5 CCTGCCATC CGGAGT-30). Viral challenge assays To assess the ability of the anti-HIV U1 constructs to suppress HIV-1 replication, HeLa CD4+ cells lines were generated expressing vector alone, U1 6A, or anti-HIV #5. Following selection for stably transduced cells (using puromycin), pooled clones were subsequently used in the viral challenge assay. For HIV-1 infection, 0.5 · 106 cells were plated into tissue culture 6-well, ﬂat-bottom, dishes. After culture for 24 h, cells were infected with 0.3 moi of HIV-1IIIB virus for 1hr at 37oC as previously described (26). The cells were then washed three times with PBS lacking Ca2+/Mg2+ and left in 2 ml of media. On day three of culture, 0.5 ml aliquots were collected and assayed for production of virus by using HIV-1 p24 antigen ELISA kit (Beckman-Coulter Inc.). RESULTS Computation-based selection of anti-HIV target sites in the 30-terminal exon of viral transcripts U1 snRNP mediated inhibition of polyadenylation requires localization of the modiﬁed U1 snRNP particle to the 30- terminal exon of the target transcript (22). This is accom- plished by base pairing the 50 end of the U1 snRNA in its associated RNP complex to a complementary region in the target pre-mRNA. To achieve selective targeting of U1 snRNA to sequences in HIV-1, we not only identiﬁed con- served sequences in the virus but also eliminated the U1 sequences having a signiﬁcant number of interactions with host cell mRNAs. Due to the 10 nt length of the targeted sequence, binding sites are expected to occur approximately once in 106 bases, corresponding to 3000 occurrences in the human genome. However, since exons constitute only 2% of the human genome (27) and inhibition by U1 snRNP particles requires base-pairing to unstructured regions in the 30-terminal exon of transcripts containing AAUAAA type poly(A) signals (22), the speciﬁcity of inhibition can be sub- stantially improved with judicious selection of target sites. In light of the few published studies on U1 snRNA-mediated reduction of target gene expression (21,22), rational design of anti-HIV U1 constructs based on the characteristics of known effective target sites cannot be achieved at this point. To facilitate the discovery of HIV sequences suscepti- ble to U1-mediated inhibition that would provide broad cov- erage for all HIV-1 strains, a MSA of consensus sequences of the env and nef regions of all M and O group HIV-1 subtypes was generated and a number of highly conserved 10 bp sequences identiﬁed in the MSA. The number of BLAST hits returned for a given 10 bp sequence against HIV-1 sequences in GenBank was determined and deﬁned as the Raw Score (SR-value). The magnitude of the SR-value is pro- portional to the extent of conservation observed in the MSA. This value was then scaled according to the number of BLAST hits in the human genome returned for each sequence to derive the Final Score (SF-value). Sequences with the least hits upon scanning of the human genome were deemed suitable for targeting and placed in a list of candidate sites. Selection of target sites within highly conserved regions of the HIV-1 genome allows the identiﬁcation of universal tar- gets common to all subtypes. In addition, the conservation of these sites among HIV-1 subtypes suggests that mutations within these regions would have detrimental effects to the virus. Using this computational strategy, multiple anti-HIV targets were identiﬁed in the 30 LTR proximal region of HIV-1 of which ﬁve were chosen for initial evaluation. The sequence and position of a subset of the selected target sites are shown in Figure 1A and B. All selected sites reside in the env or nef region and are present in the 30-terminal exon of all viral transcripts. Selection of these target sites ensures that the expression of all viral proteins will be affected. Targeting of the HIV-1 30-terminal exons with anti-HIV U1 constructs inhibits viral gene expression at both the protein and RNA level Having selected a number of sites within the terminal exon of HIV-1 that met the desired criteria, ﬁve U1 expression vec- tors were generated in which the ﬁrst 10 nt of the transcript was able to base pair to one of the sites. To measure their anti-viral capacity, U1 snRNA expression plasmids were co-transfected with plasmid HxBruR /RI that contains a HIV-1 provirus in which the RT and IN genes have been deleted. In addition to the ﬁve anti-HIV U1 variants, parallel transfections were performed with vectors expressing unmodiﬁed U1 snRNA (U1 wt), a point mutant of U1 (U1 6A) that abolished activity in splicing, and a U1 variant targeted to chloramphenicol acetyl transferase (CAT) (22). Effect of U1 snRNA expression on HIV-1 gene expression was monitored by western blot for p24. As shown in Figure 1C, three of the ﬁve anti-HIV constructs tested (#1, #3 and #5) resulted in a signiﬁcant reduction in HIV-1 gene expression. In contrast, U1 6A and U1 a-CAT had little to no effect relative to U1 wt (see Figure 1C). Parallel examination of the effect of these vectors on a non-target RNA (secreted alkaline phosphatase, SEAP) failed to show signiﬁcant levels of inhibition (Figure 1D). The failure to see any large changes in SEAP expression suggests that the viral inhibition obser- ved cannot be attributed to gross toxicity to the cell. In light of this success, we continued to evaluate additional sites within the HIV-1 provirus for their susceptibility to this inhibitory strategy. As detailed in Table 1, 15 sites in total were targeted of which only ﬁve yielded signiﬁcant reduction on viral p24 expression.