Background:  HIV-1 infection of the central nervous system (CNS) is a significant cause of morbidity and cognitive impairment. During infection, limited viral trafficking between tissues allows HIV-1 to evolve independently and become genetically compartmentalized. Earlier attempts at viral genetic characterization have focused on the study of cerebrospinal fluid (CSF), blood, and some HIV DNA from CNS tissues, but few studies have systematically compared brain-derived HIV-1 RNA and DNA sequences. Here we sampled both HIV-1 DNA and RNA directly from post mortem brain frontal cortex and splenic tissue from 9 HIV-1-infected subjects to better assess viral population genetics in CNS.

Methods:  To minimize resampling and polymerase chain reaction (PCR) error, each DNA/c-DNA product underwent 3 to 4 individual PCR amplifications of C2-V3 env using high-fidelity polymerases and were then pooled to generate 15 to 20 clonal sequences (45 to 80 clonal sequences per patient). Phylogenies were constructed using neighbor joining and maximum likelihood methods executed in PHYLIP and HYPHY. Compartmentalization was assessed with the Slatkin-Maddison test performed with MacClade. Diversity was estimated by measurement of pair-wise distances and predicted glycosylation patterns with N-glycosite.

Results:  All 9 subjects exhibited partitioning of C2-V3 sequences between tissues, according to the Slatkin-Maddison test (p <0.001). Furthermore, partitioning between Brain DNA and RNA sequences was also evident in 8 of 9 cases (p <0.01). HIV diversity was greater for DNA sequences than RNA sequences in brain. These findings are compatible with selective expression of a subset of viral variants from HIV provirus in brain that may be most fit for replication in CNS. Genetic distances from brain-derived sequences to the inferred most recent common ancestor (MRCA) were, on average, shorter than from splenic sequences to the MRCA. No differences in extent of glycosylation was appreciated.

Conclusions:  HIV-1 infecting the CNS shows evidence of compartmentalization. Only a subset of pro-viruses populating the CNS appears to be expressed, possibly reflecting CNS-specific constraints on viral fitness. In cases demonstrating the greatest degree of compartmentalization, brain-derived sequences were more closely related to the MRCA compatible with the hypothesis that brain infection is established early during primary infection and evolution of viral variants in the CNS may not proceed at the same pace as in lymphoid tissues.