Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase.
Integrase (IN) from human immunodeficiency virus, type 1 (HIV-1) exerts pleiotropic effects in the viral replication cycle. Besides integration, IN mutations can impact nuclear import, viral maturation, and reverse transcription. IN and reverse transcriptase (RT) interact in vitro, and the IN C-terminal domain (CTD) is both necessary and sufficient for binding RT. We used nuclear magnetic resonance spectroscopy to identify a putative RT-binding surface on the IN CTD, and surface plasmon resonance to obtain kinetic parameters and the binding affinity for the IN-RT interaction. An IN K258A substitution that disrupts reverse transcription in infected cells is located at the putative RT-binding surface, and we found that this substitution substantially weakens IN CTD-RT interactions. We also identified two additional IN amino acid substitutions located at the putative RT-binding surface (W243E and V250E) that significantly impair viral replication in tissue culture. These results strengthen the notion that IN-RT interactions are biologically relevant during HIV-1 replication and also provide insights into this interaction at the molecular level.
Wilkinson
Thomas A
TA
Department of Molecular & Medical Pharmacology, University of California, Los Angeles, California 90095, USA.
Januszyk
Kurt
K
Phillips
Martin L
ML
Tekeste
Shewit S
SS
Zhang
Min
M
Miller
Jennifer T
JT
Le Grice
Stuart F J
SF
Clubb
Robert T
RT
Chow
Samson A
SA
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D010449:Peptide Mapping; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D048348:Reverse Transcription / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology*
D019427:HIV Integrase; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase