Identification of amino acid residues in the human immunodeficiency virus type-1 reverse transcriptase tryptophan-repeat motif that are required for subunit interaction using infectious virions.
The human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) functions as a heterodimer (p51/p66), which makes disruption of subunit interactions a possible target for antiviral drug design. Our understanding of subunit interface interactions has been limited by the lack of virus-based approaches for studying the heterodimer. Therefore, we developed a novel subunit-specific mutagenesis approach that enables precise molecular analysis of the heterodimer in the context of infectious HIV-1 particles. Here, we analyzed the contributions of amino acid residues comprising the Trp-motif to RT subunit interaction and function. Our results reveal important inter- and intra-subunit interactions of residues in the Trp-motif. A tryptophan cluster in p51 (W398, W402, W406, W414), proximal to the interface, was found to be important for p51/p66 interaction and stability. At the dimer interface, residues W401, Y405 and N363 in p51 and W410 in p66 mediate inter-subunit interactions. The W401 residue is critical for RT dimerization, exerting distinct effects in p51 and p66. Our analysis of the RT heterodimerization enhancing non-nucleoside RT inhibitor (NNRTI), efavirenz, indicates that the effects of drugs on RT dimer stability can be examined in human cells. Thus, we provide the first description of subunit-specific molecular interactions that affect RT heterodimer function and virus infection in vivo. Moreover, with heightened interest in novel RT inhibitors that affect dimerization, we demonstrate the ability to assess the effects of RT inhibitors on subunit interactions in a physiologically relevant context.
Mulky
Alok
A
Department of Microbiology, University of Alabama at Birmingham, AL 35294, USA.
Sarafianos
Stefan G
SG
Jia
Yujiang
Y
Arnold
Eddy
E
Kappes
John C
JC
D000480:Alkynes; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D048588:Benzoxazines; D001665:Binding Sites; D002460:Cell Line; D003521:Cyclopropanes; D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010078:Oxazines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011533:Proviruses / Q000737:chemistry; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000737:chemistry / Q000201:enzymology / Q000502:physiology
D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D010078:Oxazines; D021122:Protein Subunits; D014364:Tryptophan; D054303:HIV Reverse Transcriptase; C098320:efavirenz