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

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.

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Correspondence to RLG Schrijvers.

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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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Schrijvers, R., De Rijck, J., Gijsbers, R. et al. LEDGF/p75 is critical but not essential for multiple-round HIV 1 replication. JIAS 13, O10 (2010). https://doi.org/10.1186/1758-2652-13-S3-O10

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

Keywords

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