Figure 1

Cloning and expression of functional Vpu. (A) An alignment of the NL-Vpu (subtype B) and R5-Vpu (subtype C) protein sequences is shown. Boxes show sequence conservation; dark shading indicates sequence identity and light shading indicates conservative changes. The transmembrane domain, cytoplasmic helices and phosphoserine residues (black circles) are indicated. Residues are numbered according to the NL-Vpu sequence. (B) Phylogenetic comparison of the NL and R5 Vpu protein sequences to the consensus Vpu sequences for different HIV-1 subtypes available in the Los Alamos HIV sequence database http://www.hiv.lanl.gov/content/hiv-db/ALIGN_CURRENT/ALIGN-INDEX.html. The tree was drawn using the ClustalW (V1.4) algorithm in MacVector v7.2.2 (Accelrys). A Blosum similarity matrix was used with the following settings: Open Gap Penalty = 10; Extend Gap Penalty = 0.1; Delay Divergent = 40%; Gap Distance = 8. Numbers indicate the relative distance. (C) HeLa cells were transfected with wild type (Vpu+) or vpu-deficient (Vpu-) HIV-1 proviral DNA, the latter with empty vector (lanes 2 and 5) or an R5-Vpu expression vector (lanes 3 and 6), as described in Methods. Virions released in the culture medium (lanes 1–3) and intracellular virions (lanes 4–6) were estimated by western blotting with anti-p24 antibodies. The bands corresponding to the HIV-1 capsid precursors p55 and p41, and the mature p24 are indicated.