The human immunodeficiency virus (HIV-1) invades the central nervous system (CNS) early after infection, causing a slowly progressive dementia, the acquired immunodeficiency syndrome dementia complex (ADC) in a significant number of patients. Given the magnitude of HIV-1 infection, ADC is one of the most common causes of dementia in the US, especially in the young. Viral load in the brain correlates with ADC and the pathologic hallmarks of HIV-1 encephalitis, but those regions of the viral genome associated with ability to replicate within the CNS have not been completely defined. We have shown previously that transgenic mice constructed with the HIV-1 long terminal repeat (LTR) of strains JR-CSF and JR-FL, derived from the CNS of a patient with ADC, express a reporter gene in the CNS, while mice constructed with the blood-derived IIB LTR do not. This suggests that DNA sequences within the LTR may be responsible, in part, for tissue-specific gene expression and replication displayed by HIV-1. To help define these sequences, we have analyzed 56 LTR clones isolated from the brains of four HIV-1-infected patients. In these samples, we found at least five separate quasispecies of LTRs, with a significant amount of variation seen both within and between brain samples. The vast majority of variation occurred upstream of the NF-kappaB and Sp1 transcription factor binding sites in the LTR, within the NF-AT and LEF-1 binding sites, and at several locations outside of transcription factor binding sites. At many of these locations, our brain-derived LTR clones shared unique substitutions with the CNS-derived JR-CSF and JR-FL LTRs, in comparison to blood-derived IIIB LTR. Our hypothesis is that one or more substitutions in the upstream DNA sequences of the HIV-1 LTR contribute to tissue-specific, and, possibly, cell-specific, gene expression within the CNS. The goal of this proposal is to define specific LTR sequence variations associated with varying levels of gene expression and transcription factor binding in different cell types relevant to the CNS. We have constructed vectors expressing the reporter gene beta-galactosidase under the control of various HIV-1 LTRs, including IIIB, JR-FL and a subset of out brain-derived LTRs from each of the five quasispecies. We have begun transiently transfecting these vectors into cultured T lymphocytes, macrophages, astrocytes and neurons, looking for differences in gene expression between various clones in different cell types. Finally, we will perform DNase protection and electrophoretic mobility shift assays using nuclear extracts from the above cells to define the specific regions of the LTR which differentially bind to their nuclear proteins. These studies will define HIV-1 LTR regions associated with cell-specific gene expression in the CNS, and potentially provide novel strategies to attack HIV-1 infection in the CNS, and important cause of dementia in young Americans.
| Ross, H L; Gartner, S; McArthur, J C et al. (2001) HIV-1 LTR C/EBP binding site sequence configurations preferentially encountered in brain lead to enhanced C/EBP factor binding and increased LTR-specific activity. J Neurovirol 7:235-49 |
