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  • Oral presentation
  • Open Access

LEDGF/p75 is critical but not essential for multiple-round HIV 1 replication

  • 1Email author,
  • 1,
  • 1,
  • 2,
  • 2 and
  • 1
Journal of the International AIDS Society201013(Suppl 3):O10

https://doi.org/10.1186/1758-2652-13-S3-O10

Published: 04 November 2010

Keywords

  • Homologous Recombination
  • Escape Mutant
  • Knockout Cell
  • Nalm6 Cell
  • Vector Transduction

Background

Via its interaction with the cellular cofactor LEDGF/p75, HIV-1 integration is targeted towards active genes. Several strategies were used to show the important role of LEDGF/p75 in viral replication. After RNAi-mediated knockdown of LEDGF/p75, residual replication was observed, possibly supported by minute LEDGF/p75 protein levels. Mouse knockout fibroblasts were generated, enabling analysis of high-titer, single-round lentiviral vector transduction, but not multiple-round replication. To enable evaluation of multiple-round replication in the complete absence of LEDGF/p75, a human LEDGF/p75 knockout cell line was generated (-/-), leaving the p52 splice variant intact.

Methods

By homologous recombination in Nalm6 cells exons 10 to 13, coding for the LEDGF/p75 integrase binding domain (IBD) were deleted. As a result, a truncated protein was formed in which the C-terminal region of LEDGF/p75 (aa 325-530) was replaced by a new 9 aa tail.

Results

Correct homologous recombination was verified by southern blot analysis and DNA sequencing. Absence of LEDGF/p75 specific mRNA was verified by Q-PCR. Western blot analysis revealed the presence of the truncated protein. Using 454 sequencing, the HIV integration site profile was determined. In line with data published earlier, integration in the +/+ and +/- cells was favoured in transcription units. In the absence of LEDGF/p75, integration occurs away from genes and a preference for CpG islands emerges. Multiple-round HIV replication in LEDGF/p75 -/- and +/- cells revealed a delayed replication of two weeks.

Conclusions

Our results corroborate LEDGF/p75 as a critical but not essential cofactor for HIV replication in human cells. We are currently evaluating LEDGF/p75 knockout escape mutants to understand how HIV is capable of replicating in the absence of LEDGF/p75.

Authors’ Affiliations

(1)
Molecular Virology and Gene Therapy, KULeuven, Leuven, Belgium
(2)
School of Medicine Philadelphia, University of Pennsylvania, Philadelphia, USA

Copyright

© Schrijvers et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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