Background: The role of viral envelope (env) glycoproteins in HIV-induced neurological disease is unclear. Envelopes that confer more efficient infection and cell:cell fusion of brain macrophages and microglia may result in increased release of factors toxic for neurons. Such factors could include soluble forms of the envelope glycoprotein which may directly cause neuronal apoptosis. Most HIV-1 brain envelopes studied to date have been derived from viruses isolated by culture in primary blood cells. This process may select for and preferentially isolate HIV-1 strains best able to infect T-lymphocytes and against variants adapted for replication in cells resident in the brain. We hypothesized that envs derived by PCR from uncultured tissue samples would more closely represent envs present in vivo compared to envs derived by culture. Here, our objectives were 1) to amplify complete env genes from uncultured patient autopsy brain and lymph node tissue samples, 2) to prepare high titer reporter viruses carrying each env, and 3) to analyze the receptor and coreceptor-usage and cell tropism conferred by tissue-derived envs.

Methods: Nested PCR was used to amplify complete env genes from brain and lymph node samples of 5 patients (pts) who suffered from neurological disease including dementia. The V1V2 loops of these envs were sequenced to identify unique genotypes and to remove replicate envs from further study. Each env was cloned into the pSVIII env expression vector. Single-round reporter viruses were prepared by co-transfection into 293T cells together with an env-NL43 plasmid that provided all other HIV components required for virion production. Such reporter viruses thus carry the core of NL43 but carry the pt envelope of choice. Reporter viruses carrying pt envs were used in infectivity assays of cells to test co-receptor use, CD4-dependence, and cell tropism. Cell tropisms were tested on astrocytes, brain microvascular endothelial cells (BMVEC), and macrophages.

Results: The majority of envelopes whether from brain or lymph node tissues were tightly CCR5-specific, highly CD4-dependent and macrophage-tropic.

Conclusions: Our results indicate the presence of CCR5-specific, CD4-dependent viruses in the brain of pts with neurological disease.