Identification of cellular factors binding to acetylated HIV-1 integrase

Abstract

The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070–3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein–protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291–297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle.

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Abbreviations

HIV-1:

Human immunodeficiency virus-1

IN:

Integrase

PIC:

Pre-integration complex

GDBD:

Gal4 DNA binding domain

GAD:

Gal4 activation domain

HAT:

Histone acetyltransferase catalytic domain

wt:

Wild type

mut:

Mutated

His:

Histidine

Ade:

Adenine

Ac-Lys:

Acetylated lysines

TEV:

Tobacco etch virus protease site

HA:

Hemagglutinin epitope tag

WB:

Western blot analysis

α:

Antibodies

NLS:

Nuclear localization signal

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Acknowledgment

This work was supported by grants from the EU FP7 (THINC, HEALTH-F3-2008-201032) and by the ISS Italian AIDS Program (grant number 40G.17).

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Correspondence to Awatef Allouch.

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Allouch, A., Cereseto, A. Identification of cellular factors binding to acetylated HIV-1 integrase. Amino Acids 41, 1137–1145 (2011). https://doi.org/10.1007/s00726-009-0444-3

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Keywords

  • HIV-1 integrase
  • Histones acetyltransferase
  • Acetylation
  • Tethered catalysis system
  • Yeast two-hybrid screening
  • Integrase binding factors