Abstract

TUAA0201 - Oral Abstract Session

Unique regulatory mechanisms of CNS-derived HIV-1 LTRs associated with latency

Presented by Lachlan Gray (Australia).

L. Gray^1,2, D. Cowley^1,3, E. Crespan¹, C. Welsh^1,4, C. Mackenzie^1,5, P. Gorry^1,3,6, S. Wesselingh^1,7, M. Churchill<sup>1,3,4

1</sup>Burnet Institute, Centre for Virology, Melbourne, Australia, ²Monash University, Department of Biochemistry and Molecular Biology, Clayton, Australia, ³Monash University, Department of Medicine, Clayton, Australia, ⁴Monash University, Department of Microbiology, Clayton, Australia, ⁵Monash University, Department of Immunology, Melbourne, Australia, ⁶The University of Melbourne, Department of Microbiology and Immunology, Melbourne, Australia, ⁷South Australian Health and Medical Research Institute, Adelaide, Australia

Background: HIV-1 penetrates the central nervous system (CNS) during early infection, establishing a viral reservoir. While macrophages and microglia represent the sites of productive HIV-1 infection, astrocytes undergo a restricted/latent infection. We recently demonstrated that astrocytes are extensively infected and may therefore constitute a significant potential reservoir of HIV-1 within the CNS. Here, we analyzed HIV-1 promoters (LTR) from matched CNS and non-CNS compartments to determine their role in virus restriction within CNS-derived cells. Determining the regulatory mechanisms of CNS-derived LTRs is essential to understanding the CNS as a HIV-1 viral reservoir, and in developing strategies aimed at HIV-1 eradication.
Methods: HIV-1 LTR sequences from a cohort of HIV-1 autopsy subjects consisting of matched CNS- and non-CNS-derived isolates were examined and their activity was determined in T-cells and SVG astrocyte cells. Electrophoretic mobility shift assays (EMSA) were used to analyze transcription factor binding activity within the core and basal promoter regions of the LTR.
Results: CNS-derived LTRs demonstrated restricted basal transcriptional activity in both T-cells and SVG cells, and non-CNS-derived LTRs showed decreased activity in SVG cells. Restricted basal activity mapped to the three Sp binding motifs, previously shown to be essential for both Tat-independent and Tat-dependent activation of the LTR in T-cells. Further repression in astrocytes was observed due to elevated levels of the repressor Sp3 in SVG cells.
Conclusions: The reduced transcriptional activity observed for CNS-derived HIV-1 promoters was found to correlate with a reduction in Sp1 binding, which mapped to mutations within the core Sp binding motif. Transcriptional activity in SVG cells was further regulated by Sp3, which outcompeted Sp1 for Sp-motif binding. These data suggest that CNS-derived viruses have a reduced capacity to initiate viral transcription in astrocytes and highlights that unique transcriptional mechanisms exist within the CNS, ultimately affecting the fate of viral infection and the development of latency.