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Transcriptional regulation of human immunodeficiency virus type-1 in the central nervous system
thesisposted on 09.02.2017, 05:30 by Cowley, Daniel John
Infection with human immunodeficiency virus type-1 (HIV-1) is a significant global health challenge. Since first identified as the aetiological agent of acquired immunodeficiency syndrome (AIDS), approximately 60 million people have been infected and 25 million people have died of HIV-related disease. HIV-1 infection of the central nervous system (CNS) can lead to a number of neurological complications, presenting clinically as a spectrum of disease from asympotomatic neurological impairment to HIV-1 associated dementia (HAD). Despite advances in antiretroviral therapies the neurological complications of HIV-1 infection persist. Therefore, understanding the mechanisms of HIV-1 neuropathogenesis will help guide strategies designed to control HIV-1 within the CNS. HIV-1 transcription is regulated by the interaction of host and viral factors with cis-acting sequences within the HIV-1 long terminal repeat (LTR). The mechanisms regulating HIV-1 transcription in the CNS and their role in neuropathogenesis remain unclear. In this study, the transcriptional regulatory mechanisms in a panel of matched CNS- and lymphoid-tissue derived HIV-1 from subjects with HAD were examined. Compartmentalisation of HIV-1 LTR sequences was identified between viruses derived from CNS and lymphoid tissues. This compartmentalisation was associated with CNS specific sequence variations in conserved transcription factor binding motifs and tissue specific transcriptional activities, particularly in CNS derived astrocyte cells. By further analysing the HIV-1 encoded transactivator (Tat), CNS specific Tat sequences were identified in a subset of cohort subjects associated with heterogeneity in Tat stimulated LTR transcriptional activity. Multiple amino acid changes with functional significance were observed in both CNS- and lymphoid-tissue derived Tat. Notably, sequence changes were identified in Tat neurotoxic and chemotactic domains that segregated CNS- and lymphoid-tissue derived Tat, suggesting tissue specific differences in these properties. Sequence differences that segregated matched CNS- and lymphoidtissue derived LTR sequences were observed in the region spanning the three Sp transcription factor-binding motifs of the basal/core promoter. This LTR region has previously been reported to be essential for both basal and activated transcription in lymphoid tissues. Electrophoretic mobility shift assays performed using DNA probes with all three Sp sites in combination identified differences between CNS and lymphoid-derived LTRs in overall Sp1 binding affinity. When Sp sites were analysed in isolation the Sp1 binding activity of CNS-derived promoter distal Sp sites (Sp-III) was markedly reduced when compared to lymphoid-derived sites. Heterogeneity was observed in the Sp1 affinity of CNS and lymphoid-derived promoter medial (Sp-II) and promoter proximal (Sp-I) Sp binding sites. Furthermore, sequence changes both within and flanking the Sp sites were responsible for reduced Sp1 binding. Changes at the promoter distal (Sp-III) site largely dictated overall LTR affinity. Reduced Sp1 binding is predicted to influence LTR mediated transcriptional regulation. Taken together, this study emphasises CNS specific HIV-1 transcriptional regulatory mechanisms with potential implications to neuropathogenesis. The reduced Sp1 binding affinities and resulting decreased transcriptional activity of CNS-derived LTRs indicate a reduced capacity to initiate viral transcription and highlight mechanisms that control the development of HIV-1 latency.