Genetic analysis of human immunodeficiency virus type 1 nef in Portugal: subtyping, identification of mosaic genes, and amino acid sequence variability

Extending our previous genetic characterization of human immunodeficiency virus type 1 (HIV‐1) strains circulating in Portugal, we here report the first phylogenetic and putative amino acid sequence variability analyses of nef accessory gene. Viral sequences (n = 53) were amplified by nested PCR from proviral DNA purified from peripheral blood mononuclear cells of HIV‐1 infected individuals (n = 49). Phylogenetic inference analysis demonstrated a distribution of the viral sequences between subtypes A (sub‐subtype A1), B, D, F (sub‐subtype F1), G, H, and J, with subtypes G and B accounting altogether for more than half of the genotypes found. A significant number of the proviral DNA sequences analyzed (18.4%) were shown to correspond to intragenic nef recombinants, with the majority having the typical CRF02_AG nef structure. In addition, three novel intragenic recombinant structures were found (B/G/B, CRF02_AG/H, and D/G). From phylogenetic analysis, it was concluded that part of the non‐recombinant nef genes might have actually been amplified from mosaic viruses: CRF06_cpx, CRF14_BG, and a new env A/nef J recombinant. While comparing all the putative Nef sequences, significant amino acid sequence variability was observed. However, most of the described nef functional motifs were relatively well conserved in the majority of the sequences analyzed and numerous amino acid changes fell outside these regions. The results presented unambiguously endorse the high level of complexity of HIV‐1 epidemics in Portugal.