Figure 4

Unique molecular features identified in D17 and D24. (A) κB-site polymorphism in the viral enhancer. The nucleotide sequences within the enhancer region of D17 and D24 have been aligned with the three available infectious subtype C molecular clones using Clustal W program. Dashes represent gaps in the sequence and dots sequence homology. The sequence coordinates are as per the D24 molecular clone (accession number EF469243). The three canonical NF-κB sites and the upstream additional κB-element are highlighted. Note that at position 398, the molecular clone MJ4 (AF321523) contains a T to G variation in the C-NF-κB site that is expected to make this element non-functional. (B) Schematic representation of Rev expression variation in diverse subtypes of HIV-1. The C-terminus of D24 Rev is compared with that of diverse viral subtypes. The amino acid residues are represented in the single letter code. The coordinates of the amino acid residues are as per the HXB2 molecular clone (at the top). Subtypes A, A/E, G and C strains contain a 7 amino acid insertion at the residue 95 of HXB2 equivalent as shown. At residue 101, only subtype C strains typically contain a stop codon (highlighted by shading) that is non-functional in D24. A second stop codon (highlighted) at position 117 that terminates Rev in all non-subtype C strains is also non-functional in D24. Rev translation in D24 is terminated by a third stop codon (highlighted) located after a tyrosine residue at position 126. The expected size of the Rev proteins from different subtypes is shown on the right hand side. Stop codons are indicated by an asterisk; aa, amino acid. (C) Presence of two different variant forms of Rev in the molecular clones. Amino acid sequences at the C-terminus of all the 8 HIV-1 molecular clones reported in the present work (shaded) and the three subtype C infectious clones are aligned. Rev sequence of HXB2 is also included for comparison. Dots represent sequence homology and dashes gaps. The sequence coordinates are as per HXB2. Asterisks indicate putative stop codons (see 'B' above). Coexistence of two different variant forms of Rev in the donor provirus (D) and plasma virus (E). Using a pair of primers, Rev exon-2 was amplified in a DNA PCR using genomic DNA extracted from the PBMC of the donor BL94. Alternatively, viral RNA was extracted from the plasma sample, reverse transcribed and the exon-2 of Rev was amplified using template-specific primers. The sequence information of the anti-sense strand was obtained directly from the PCR or RT-PCR amplicons. The codon of the amino acid residue 101 is boxed. Presence of both 'C' and 'A' at position 8695 (amino acid residue 101 of Rev) on the anti-sense strand confirmed the simultaneous existence of two different forms of Rev in the provirus as well as in the plasma virus.