252 Nucleic Acids Research, 2007, Vol. 35, No. 1 Figure 3. HIV-1 Suppression is dependent upon the integrity of the U1 snRNP. (A) Schematic of U1 snRNA structure with the location and nature of U1 mutants indicated. Shown are the regions of a-HIV U1 #1 and #5 that were mutated to identify the protein components of the snRNP required to suppress viral gene expression. Mutations were as follows: deletion of stem– loop 1 (DSL1), deletion of stem–loop 2 (DSL2), substitutions in stem–loop 1 (SL1mut), substitutions in the loop region of stem–loop 2 (SL2mut) and mutations in the Sm binding region that abolishes assembly of the Sm core complex (DSm). (B and C) Effect of anti-HIV-1 #5/#1 mutation on Suppression of HIV-1 Gene Expression Cells were transfected with a plasmid containing HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-) and mutants of anti-HIV #5 (B) or #1 (C). Forty-eight hours post-transfection, cells were harvested and level of p24 production assessed by western blot. whether the inhibition seen in this study was operating through the same or another mechanism, we examined whether the response was dependent upon proper assembly of the U1 snRNP particle. If mutants in the various protein assembly domains of U1 snRNA do not alter the response, then only base-pairing potential would be deemed necessary for the loss of HIV-1 expression (suggestive of an anti-sense mechanism). Five variants of aHIV #5 U1 snRNA were gen- erated to probe the requirement of the various regions of the U1 snRNA for function: DSL1 or substitutions in stem–loop 1 that results in loss of U1 70 K binding (SL1mut), deletion of stem–loop 2 (DSL2) or substitutions in stem–loop 2 that results in loss of U1A binding (SL2 mut), and mutations within the Sm binding region (DSm) that abolish assembly of the Sm core (Figure 3A) (29). Cotransfection of these mutant U1 snRNAs with proviral DNA revealed four of the ﬁve mutants had no inhibitory activity; expression of HIV-1 p24 was similar to that seen with wt U1 snRNA (Figure 3B). Only aHIV #5 SL2mut retained some inhibitory activity although signiﬁcantly less than that of the unmodiﬁed variant. To assess whether this phenomenon was limited to only one construct or was valid for all U1 variants, both SL1mut and SL2mut mutations were introduced in the context of anti- HIV #1. As shown in Figure 3C, the same spectrum of effects was observed, mutation of SL1 leading to abolition of anti- HIV activity while SL2 mutants retained low but signiﬁcant anti-viral activity. The residual anti-viral activity of the SL2 mutants raised the possibility that these constructs may serve as alternatives should the unmodiﬁed anti-viral U1 RNAs have deleterious effects on the cell. To probe this issue further, we assessed the anti-viral activities of the SL2mut constructs alone or in combination (Figure 4). Consistent with their reduced activity, higher amounts of each plasmid were required to repress HIV-1 gene expression (data not shown). However, when used in combination, signiﬁcant inhibition of viral p24 expression (to background) was achievable, albeit using higher amounts of plasmid. To test whether this effect was attributable to a similar effect on viral RNA expression, Northern blots were also carried out. As shown in Figure 4B, when used in combination SL2 mutants of anti-HIV #1 and #5 led to a signiﬁcant reduction in HIV-1 RNA levels. A similar response is not evident upon expression of the SL1 variants of the same constructs (Figure 4B). Cells stably expressing anti-HIV U1 snRNAs show reduced capacity to support HIV replication While the experiments to date have clearly established the anti-HIV properties of the U1 constructs generated, we were interested in examining their properties under conditions that more accurately mimicked the in vivo situation. Toward this end, stable cells expressing one of the anti-viral U1 constructs was generated and challenged with virus. As shown in Figure 5, monitoring of p24 production in the media follow- ing viral challenge revealed a reduction in HIV-1 replication in only the cell line expressing the anti-HIV U1 snRNA vari- ant. Little to no change in p24 levels was seen with cells expressing either of the control vectors (U1 wt, U16A). Although the inhibition of HIV-1 replication seen in the context of the stable cell lines is less than that observed in transient assays (see Figure 1), subsequent analysis has revealed that expression of the U1 constructs is considerably below that obtained in the transient assays (data not shown). Current efforts are directed at improving the expression of the modiﬁed U1 snRNAs. DISCUSSION Maturation of pre-mRNA in eukaryotes involves a set of factors that modify the primary transcript into an export competent mRNP (messenger ribonucleoprotein) complex, the formation of which signals its release from the site of transcription and export into the cytoplasm for translation (30). These processing events include capping, splicing, 30 end cleavage and polyadenylation. Proper 30 end formation promotes transcription termination (15) and nuclear export of the mRNA (31) and enhances the stability and translation of the mature transcript in the cytoplasm (32,33). Recent evi- dence implicates the existence of extensive coupling between