Successful Application of Hyperbranched Multidisplacement Genomic Amplification to Detect HIV-1 Sequences in Single Neurons Removed from Autopsy Brain Sections by Laser Capture Microdissection

摘要

To confirm studies suggesting that HIV-1 infects neurons and to determine whether CD8⁺ T lymphocytes traffic to HIV-1-infected neurons, we used laser capture microdissection to remove hippocampal neurons with and without perineuronal CD8⁺ T cells from AIDS patients with HIV-1 encephalitis (HIVE) or without HIVE and from normal controls. We used hyperbranched multidisplacement amplification for whole gene amplification (MDA-WGA) plus two rounds of PCR to amplify housekeeping sequences (HK⁺) and, in HK⁺ samples, to amplify HIV-1 gag, nef, and pol sequences. Sample size and, in single neurons, MDA-WGA correlated with housekeeping gene amplification (P < 0.05), whereas patient group and postmortem interval did not (P > 0.05). Neuronal viral sequences correlated with HIVE (43% vs. 13% and 0 in non-HIVE and controls, respectively) and, in HIVE cases, with perineuronal CD8⁺ T lymphocytes (70% in CD8⁺ samples vs. 37% of CD8⁻ samples). Our results suggest that MDA-WGA is a useful technique when analyzing DNA from single cells from autopsy brains, supporting prior studies that show that neurons may contain HIV-1 neuronal sequences in vivo. The association between neuronal infection and perineuronal CD8⁺ T cells supports our hypothesis that these cells specifically traffic to infected neurons but raises the possibility that CD8⁺ T cells, if infected, could transmit virus to neurons.