Date of Award

2016-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Manuel Llano

Abstract

The lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin-bound protein essential for efficient lentiviral integration. Knockdown of this protein severely affects HIV-1 integration levels and thus itâ??s replication. Genome-wide studies have located LEDGF/p75 inside actively transcribed genes where it mediates lentiviral integration. Although this is clearly established, the molecular mechanisms of LEDGF/p75 in HIV-1 infection remain in part elusive.

Using protein-protein interaction assays, we demonstrated that LEDGF/p75 complexes with a chromatin remodeling complex, Facilitates Chromatin Transcription (FACT), a heterodimer of the structure specific recognition protein 1 (SSRP1) and the human homolog of suppressor of Ty 16 (hSpt16). The FACT complex is a histone chaperone that remodels nucleosomes in an ATP-independent fashion, providing Pol II access to the DNA during transcriptional elongation. Functional characterizations demonstrate a role for this novel complex
in the regulation of HIV-1 replication. shRNA-mediated partial knockdown of SSRP1 reduces HIV-1, but not Murine Leukemia Virus (MLV), infection in human CD4+ T cells. Similarly, SSRP1 knockdown affects infection by HIV-1-derived viruses that express genes from the viral LTR but not from an internal immediate-early CMV promoter, suggesting a role of SSRP1 in LTR-driven gene expression but not viral DNA integration. Detailed analysis of the interaction of LEDGF/p75 with the FACT complex indicates that LEDGF/p75 interacts with SSRP1 in an independent and direct manner as is shown by immunoprecipitation results of recombinant LEDGF and SSRP1 proteins. This interaction requires the PWWP
domain of LEDGF/p75 and the HMG domain of SSRP1. Together, our data demonstrate for the first time the association of LEDGF/p75 with the FACT complex and give further support to a role of SSRP1 in HIV-1 infection.

Language

en

Provenance

Received from ProQuest

File Size

119 pages

File Format

application/pdf

Rights Holder

Angelica Patricia Lopez

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Virology Commons

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