Figure 4. SL2mut variants of anti-HIV U1 snRNAs retain anti-viral activity. (A) Cells were transfected with HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-) and vector expressing anti-HIV #1/#5 SL2mut snRNA either alone or in combination. Forty-eight hours post-transfection, cells were harvested and lysates analyzed for p24 expression by western blot. (B) Cells were transfected with HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-) and vector expressing anti-HIV #1/#5 wild-type, SL2mut, or SL1mut snRNAs either alone or in combination. Total RNA was subsequently extracted and levels of viral RNA, and GAPDH RNA assayed by northern blot. Shown is representative gel of the results obtained. Identity of viral RNAs, GAPDH RNA and U1 snRNA variants (lower) are as indicated. the gene expression machinery engaged in mRNA synthesis, processing, export and surveillance (34–36) suggesting that disruption of RNA processing will compromise proper gene expression. Binding of U1 snRNP upstream of the AAUAAA signal in the 30-terminal exon inhibits the polyadenylation step in 30 end formation by disrupting the protein–protein interac- tions in the polyadenylation machinery (20). In contrast, interaction between U1 snRNP and the major splice donor site downstream of the HIV-1 50 LTR poly(A) site inactivates the cleavage step in a U1 70 K dependent manner and is thought to be important for production of full-length genomic RNA (29). Here we demonstrated, for the ﬁrst time, that the same component of the splicing machinery recruited by HIV for its survival can be re-programmed to direct its demise. Nucleic Acids Research, 2007, Vol. 35, No. 1 253 Figure 5. Cells stably expressing anti-HIV #5 U1 snRNA show reduced capacity to support HIV-1 replication. HeLa CD4+ cells lines were isolated following transfection with pBABE vectors expressing without insert (puro), U1wt, or anti-HIV #5. Following selection for stable integrants, the pooled clones were challenged with HIV-1 and p24 production assayed five days post-infection by ELISA. Shown are the averages of two independent experiments and a total of four independent assays. Asterisk indicates values that are different from control cells (Mock) at a P-value <0.05. The target site selection algorithm employed in this study does not account for possible local secondary structure formation which might explain the failure of several of the aHIV constructs to elicit a signiﬁcant reduction in viral protein production. However, dramatic reduction of viral structural protein expression by ﬁve of the constructs (#1, #3, #5, #7 and #12) was observed in this study with little or no effect on expression of the co-transfected SEAP vector. Synergistic inhibition of HIV gene expression by U1 target- ing was likely achieved at two levels. The sites targeted are common to all viral transcripts, so expression of all viral pro- teins is suppressed by U1 targeting. Hence, reduction of viral RNA is not only achieved by direct destabilization of the RNA but also by loss of Tat and the transactivation of the HIV-1 LTR (37). It should be noted that maximum possible inhibition of viral gene expression was likely not observed in this study. The assembly of functional U1 snRNP particles takes at least 16 h (22,38). Therefore, a delay exists between the initiation of gene expression from the co-transfected pro- virus and when signiﬁcant nuclear accumulation of anti-HIV U1 snRNPs has occurred. In addition to establishing the anti-viral activity of the modiﬁed U1 snRNAs, we also demonstrate that activity of the aHIV U1 snRNAs is dependent upon proper assembly of the U1 snRNP. Mutations which affected any component of this RNP abolished function, the sole exception being point mutants within stem–loop 2 that affect U1A binding. Assembly of the Sm core is a requisite for import of U1 snRNA (38) into the nucleus while U1 70K has been shown to be required for inhibiting the cleavage/polyadenylation reaction (18,29). These observations suggest that it is unlikely that these constructs are functioning solely in an anti-sense manner. The partial activity of the SL2mut variant of aHIV #1 and #5 raises the possibility that they could be used in gene therapy protocols should the intact form of the snRNA prove to have deleterious side effects. The high activity of U1 snRNA variants targeted to HIV-1 and their distinct mechanism of action raises the possibility that they could be used in a complementary fashion with