Date of Award

2010-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Manuel Llano

Abstract

LEDGF/p75 is an important cellular co-factor for lentiviral integration. LEDGF/p75-deficient cells are markedly resistant to HIV-1 infection and re-expression of the wild type protein rescues infectivity. Although the molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet known, this co-factor activity requires the interaction of LEDGF/p75 with both the host chromatin and viral integrase. In order to evaluate the involvement of other LEDGF/p75 regions in HIV-1 infection we constructed a panel of deletion mutants targeting clusters of charged residues that are evolutionarily conserved and predicted to be post-translationally modified. These mutants were evaluated for their ability to rescue HIV-1 infection in a LEDGF/p75-deficient human T cell line (TL3 cells) and to interact with chromatin and integrase. Our results indicate that serine residues 271, 273, and 275 are involved in the HIV-1 cofactor role of LEDGF/p75. HIV-1 infection is impaired in LEDGF/p75-deficient CD4+ T cells expressing the LEDGF/p75 mutant S271A/S273A/S275A to levels observed with LEDGF/p75 mutants where chromatin binding is deficient. However, this mutation did not affect chromatin or integrase binding. More importantly, this mutant tethers EGFP-tagged integrase to the host chromatin in cells. According to five different global phosphoproteomic studies done by other laboratories, these residues are targeted for phosphorylation in both human and mouse cells. In correlation with this, in silico analysis predicted that these serine residues are a substrate for Protein Kinase Casein Kinase II. This data strongly suggests that the role of LEDGF/p75 in HIV-1 integration may also involve other protein functions in addition to integrase and chromatin binding.

Language

en

Provenance

Received from ProQuest

File Size

117 pages

File Format

application/pdf

Rights Holder

Jose Adrian Garcia

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