Figure 2.

Fragmentation-based ²H-edited NMR approach and observed Ψ^CES secondary structure. (A) The DIS loop of Ψ^CES served as the fragmentation site and was substituted by a stretch of intermolecular G-C base pairs. (B) Fragment annealing efficiency as measured by native polyacrylamide gel electrophoresis. (C) 2D NOESY spectra of uniformly labeled A^2rC^r-, A^2rU^r-, and A^2rC^rU^r-Ψ^CES and segmentally-labeled fr-A^2r:U^r- and fr-A^2rC^r:U^r-Ψ^CES samples used to make long-range NOE assignments. (D) Similarities in NOESY spectra obtained for A^2rC^rU^r-labeled [5′-L^ΔPBS]₂ and Ψ^CES confirm that the tandem three-way junction structure is present in both constructs. (E) NMR-derived secondary structure of Ψ^CES. Black and blue arrows denote A-H₂ NOEs observable in Ψ^CES and fr-Ψ^CES samples, respectively; red arrows highlight NOEs shown in panel (C and D); thin arrows denote very long-range NOEs. (F) Packaging of native HIV-1_NL4-3 5′-L and 5′-L^ΔPBS RNAs under competition conditions assayed by means of ribonuclease protection. P, undigested probe; M, RNA sizes marker. Lanes 1 and 2: native HIV-1_NL4-3 helper versus test vectors containing 5′-L^ΔPBS (1) or native HIV-1_NL4-3 (2). Lane 3: HIV-1_NL4-3 helper expressed without test RNA. Lane 4: mock transfected-cells. Samples obtained from transfected cells (Cells) or viral containing media (Virus) are indicated. Bands corresponding to host 7SL RNA, HIV-1_NL4-3 helper RNA (Ψ⁺) and co-packaged test RNAs (Test) are labeled.