Background:  Approximately 20% of the HIV-1-infected individuals have symptoms of viral meningitis. HIV-1 brain infections occur at an early stage in the virus's development, whereas HIV-associated dementia (HAD) occurs only at a later stage. Little is known about the nature of HIV-1 expansion within the brain of infected individuals. Even less is known about the effect this expansion has on viral evolution and the development of HAD. Importantly, the NEF protein in HIV-1 is known to down-regulate the expression of CD4⁺ cells and HAD has been associated with the over-activation of infected macrophages in the brain.

Methods:  The NEF protein was sequenced from multiple brain (meninges, frontal lobe, basal ganglia, occipital lobe, and cerebellum) and body (lymph node, spleen, and liver) compartments. In total, 757 complete NEF sequences were isolated from 5 HAD individuals. Sequences were codon-aligned manually using the SL89 protocol. Maximum likelihood (ML) and Bayesian trees were estimated by PhyML and MrBayes software, positive selection by PAML and HyPhy, and recombination tested using Lamarc program. Mutation analysis used both statistical and datamining methods in R and WEKA software.

Results:  The ML and Bayesian trees show compartmentalization for the majority (>80%) of brain sequences. The meninges is the brain region that harbors the most diverse population of the virus (p <0.05). The population of virus infecting the meninges is related to both brain (in 5 of 5 patients) and lymph node and spleen (in 3 of the 5) sequences. Codon selection analysis showed that 2 to 4% of the sites in NEF are under positive selection (w >2). Of 39 positive selected sites, 18 were related to amino acid site variation (>50% difference) between brain and body sequences. Coalescent results determine low level of intra-host viral recombination (average rate 0.002) and large population size (average Q 0.30).

Conclusions: Most of HIV-1 virus sequences are compartmentalized in the brain of 5 HAD individuals. The entry of HIV-1 virus into the brain was probably via the meninges, which harbors the most diverse viral population in the brain. Positive selection on the nef gene is involved in shaping the brain's viral population. Fixation of specific viral mutations is a process of selection followed by expansion. Our results support the scenario that HIV-1 expansion in the brain is caused by over-expression of infected macrophages, which may be responsible for the development of dementia.