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. 2008 Feb 13;82(8):3921–3931. doi: 10.1128/JVI.01558-07

FIG. 3.

FIG. 3.

Mutations decreasing the strength of 5′ss D2 decrease splicing to 3′ss A1 and expression of Vif. (A) Total RNA samples from 293T cells transfected with the indicated plasmids were analyzed by RT-PCR using primer pair BSS and SJ4.7A, specific for completely spliced ∼1.8-kb viral mRNA. HIV-1 mRNA species are indicated on the right side. (B) Total RNA samples from transfected cells were analyzed by qRT-PCR for vif and β-actin mRNAs, and the normalized levels of vif mRNA for the indicated mutants are expressed relative to that of the wild type. (C) Proteins isolated from transfected cells of the wild-type pNL4-3 and the indicated mutants were analyzed by Western blotting using antibodies to Vif and β-tubulin. (D) Northern blotting analysis of total RNA isolated from cells transfected with the wild-type pNL4-3 or the indicated mutants. The positions of the unspliced (∼9-kb), the incompletely spliced (∼4-kb), and the completely spliced (∼1.8-kb) mRNAs are indicated. (E) RT activity of cell-free supernatants from cells transfected with the wild-type pNL4-3 or the indicated mutants.