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Background:  The nef gene of the primate lentiviruses has been studied extensively because of its multiple immunsuppressive and replicative effects. Patient studies have revealed important virulence capacities of the gene and the role of Nef protein is a vital aspect of HIV pathogenesis. It is a truly pluripotent protein with multiple functional domains contained within 206 amino acids. Due to the overlap of the nef-ORF with the 3’ long-term repeat (LTR), some restraints of the evolutionary plasticity might be expected. We set out to assess the polymorphism in the nef gene in Danish patients.

Methods:  We established a patient cohort of long-term nonprogressors (LTNP) consisting of 14 patients with well-controlled HIV-1 infections (median infected time 15.3 years) with a stable CD4⁺ cell count (median count in 2004: 571; median decline (1997-2004) 17.4 CD4⁺ cells/year). Peripheral blood mononuclear cells were isolated by Ficoll-paque gradient centrifugation and proviral DNA was extracted by the Qiagen whole blood purification kit. Nef was amplified by nested polymerase chain reaction, cloned in pGem vector, and sequenced. Three sequences were obtained from each patient and used to produce a patient consensus sequence. As a control group, 15 patients randomly chosen from the out-patient clinic were included and the nef gene was amplified in a similar manner.

Results:  The patients in the LTNP cohort were stratified according to the CD4 cell decline over a 7-year period from 1997-2004. The results revealed 4 patients with a T138C mutation previously reported to confer decreased replicative capacities in cell culture settings. None of the patients in the control group harbored the T138C mutation. Earlier studies of LTNP associated with Nef defects have not focused on this mutation. A search in GeneBank of nef sequences containing the cysteine mutation produced multiple hits. Selecting sequences that contained a description of patient disease status, the data revealed a high prevalence of T138C among AIDS patients with a long asymptotic infection history. A phylogenetic comparison of all sequences compiled from the LTNP cohort documented a heterogeneous viral origin, ruling out a common infectious source.

Results:  The functional nature of the Nef protein that is impeded by this mutation and its relationship to long term nonprogression remains unclear.

Keywords: Nef; Disease progression; Virulence