Background:  In our investigation if co-receptor usage among subtype C HIV isolates from patients on ART, we have identified a dual-tropic (R5X4) virus from 1 patient that contains an unusual 5-amino acid insert within the V3 loop of envelope and appears to confer X4 tropism.

Methods:  Virus was isolated from peripheral blood mononuclear cells (PBMC) in 2001 and again in 2003 from a patient in Harare, Zimbabwe, who had been receiving lamivudine (3TC), stavudine (D4T), and saquinavir. Biological clones were derived from each isolate by limiting dilution culture in PBMC. TZM cells expressing luciferase in the presence of X4 (AMD3100) or R5 (TAK779) inhibitors were used to determine co-receptor tropism. Codon usage for human and HIV genomes was extracted from the codon usage database (www.kazusa.or.jp/codon).

Results:  The initial isolate from 2001 uses the co-receptor R5 and biological clones derived from this isolate also exclusively use R5. The isolate from 2003 switched co-receptor usage to both X4 and R5. Sequencing of env from both isolates revealed an unusual 15 nucleotide insert in the V3 loop just after the crown region. A BLAST search of the Los Alamos HIV database revealed that this insert was not found in any region of HIV among the sequences in the database, while a search of Genbank indicated that the sequence is found in the human genome. It is present in the transcription factor AP-2 in chromosome 6, as well as within DNA from chromosomes 10 and 16. The translated sequence of the insert, RSYTP, includes a serine at position 2 encoded by TCC, used in human genes at a frequency of 17.7/1000 codons compared with 4.7/1000 codons in HIV. Analysis of the sequence with the insert removed using a position-specific scoring matrix (PSSM—http://ubik.microbiol.washington.edu/computing/pssm/) indicates that the virus would be more likely to use R5 for entry. The insert increases the V3 loop length from 35 to 40 amino acids and increases the net charge, resulting in a virus with both X4 and R5 tropism in phenotypic assays of biologic clones.

Conclusions:  Immune escape, drug resistance, and co-receptor switching are usually the result of multiple point mutations. In this unusual virus, the V3 loop accommodated a15-base-pair insert, homologous to human transcription factor DNA. Recombination, incorporating DNA from the host, provides an additional mechanism for increasing HIV diversity.