Nucleic Acids Research, 2007, Vol. 35, No. 1 251 Figure 2. Suppression of HIV-1 gene expression occurs at the level of both viral protein and RNA. Cells were transfected with proviral DNA (pHxb2 R-/RI-) and 4 mg of U1 wt/aCAT expression vector or increasing amounts of aHIV #1/#5 (0.25, 0.5, 1, 2 or 4 mg) alone or in combination. Forty-eight hours post-transfection, cells were harvested and either (A) used to examine HIV-1 p24 expression or (B) RNA extracted and levels of RNA determined by northern blot. Representative gels are shown indicating the position of the viral RNAs (upper), GAPDH (middle) and U1 snRNA variants (lower). Having established the potency of a subset of the U1 aHIV-1 constructs, we tested the relative efﬁcacy of the anti-HIV U1 targeting vectors alone or in combination. Two U1 aHIV-1 vectors were selected to assess their potency (U1 aHIV-1 #1 and U1 aHIV-1 #5). The two anti-HIV U1 targeting vectors were co-transfected individually or in com- bination with the proviral clone HxBruR /RI at increasing concentrations. The level of viral protein and RNA accumu- lation at 48 h post-transfection was assessed by western and northern blots, respectively. As shown in Figure 2A, the anti- HIV U1 constructs differed in the amount of plasmid required to suppress viral structural protein (p24 and p55) expression, anti-HIV #5 requiring 2 mg of transfected plasmid while anti- HIV #1 achieved the same degree of suppression at lower levels (0.5 mg of transfected plasmid). Combination of the two anti-HIV constructs (#1 and #5) was able to suppress viral gene expression at all concentrations tested. To explore the basis for the observed response, the effect of the U1 constructs on viral RNA levels was subsequently explored (Figure 2B). Cotransfection with vectors expressing anti- HIV #1 or #5 alone led to signiﬁcant reduction in viral RNA levels. Further reduction was achieved when the two con- structs were added in combination. In contrast, little or no change in viral RNA levels was detectable upon cotransfec- tion with the control vectors (U16A, U1 anti-CAT, data not shown). Inhibitory activity is dependent on proper assembly of a U1 snRNP particle Modiﬁed U1 snRNAs have been used in the past to suppress HIV-1 gene expression, but in that instance more extensive regions of base-pairing (61–68 bases) were used along with deletion of stem–loop 1 (DSL1) of the resultant RNA (28). Loss of HIV-1 expression in that study was attributed to RNA degradation due to an anti-sense mechanism. To test