Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors

Ira B Dicker,Himadri K Samanta,Zhufang Li,Yang Hong,Yuan Tian,Jacques Banville,Roger R Remillard,Michael A Walker,David R Langley,Mark Krystal

doi:10.1074/jbc.m704935200

Ira B Dicker, Himadri K Samanta + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m704935200

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Abstract

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G(2)T(1)C(-1)A(-2)) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G(2):C(2) base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln(148) interacts with G(2), T(1), and C(-1) at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.

Highlights

WT [56], and the virus was defective for replication [57]
Defects in the N155H/abasic G2 combination (Table 2, row 5) were significantly magnified compared with either N155H complexes prepared with a WT LTR (Table 2, row 1) or WT IN complexes prepared with an abasic G2 (Table 2, row 5)
When G2 was replaced by inosine (Table 2, row 7), specific strand transfer activity and [3H]compound 1 affinity were restored to at least that of N155H assembled with the WT LTR, but assembly, improved, remained defective (17%)

Summary

Introduction

WT [56], and the virus was defective for replication [57]. Molecular modeling and crystallographic data have suggested that this loop is flexible (52, 58 – 61), implying it has a role in catalysis beyond that in IN/DNA assembly. Replacement of Gly140 [62], or a combination of Gly140 and Gly149 by alanines [58], or Gln148 by alanine [55], was reported to reduce or eliminate 3Ј-processing, strand transfer, and disintegration activities. The 5Ј CϪ1 overhang is reported to be required for efficient use of a pre-processed substrate in the strand transfer reaction. Changing CϪ1 to a T barely produced any end joining products [34]. Non-3Ј processed substrates, with substitutions of CϪ1

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