Nucleic Acids Research, 2007, Vol. 35, No. 1 249 Figure 1. U1 snRNA variants targeted to HIV-1 are able to suppress HIV-1 gene expression. (A) Outline of HIV-1 genome structure. Sites targeted by U1 snRNA variants are indicated by numbered lines above and below reading frames. (B) Sequence logos showing the conservation of sequences targeted by five U1 snRNA variants. Selected 10 nt sequences identified by MSA are boxed, the height of the individual base reflecting the degree of conservation at that position among HIV-1 strains. (C) Effect of anti-HIV-1 U1 snRNAs on viral protein expression. Cells were transfected with a plasmid containing HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-), CMV PLAP and one of the indicated U1 expression constructs. Forty-eight hours post-transfection, cells were harvested and level of p24 production assessed by western blot. Effect on secreted alkaline phosphatase (SEAP) expression (D) was performed in parallel as outlined in Materials and Methods and data are shown. polyvinylidene diﬂuoride (PVDF) membranes. p24 blots were blocked in 1· phosphate-buffered saline (PBS), 5% milk, 0.05% Tween-20 then probed with mouse anti-p24 hybridoma supernatant overnight at 4 C. Donkey anti-mouse horseradish peroxidase (HRP)-conjugated antibody was appl- ied to the blots after washing at a 1:1000 dilution for 1 h, then washed four times prior to development with western lightning (Perkin–Elmer). To normalize for protein loading,