Analysis of amino acids in the beta7-beta8 loop of human immunodeficiency virus type 1 reverse transcriptase for their role in virus replication.
The HIV-1 p51/p66 reverse transcriptase (RT) heterodimer interface comprises, in part, intermolecular interaction of the loop region between beta-strands 7 and 8 (beta7-beta8 loop) in the p51 fingers subdomain with the p66 palm subdomain. In this study, for the first time in the context of infectious HIV-1 particles, we analyzed the contribution of amino acid residues (S134, I135, N136, N137, T139 and P140) in the beta7-beta8 loop for RT heterodimerization, enzymatic activity, and virus infectivity. Mutating asparagine 136 to alanine (N136A) reduced viral infectivity and enzyme activity dramatically. The N136A mutation appeared to destabilize the RT heterodimer and render both the p66 and p51 subunits susceptible to aberrant cleavage by the viral protease. Subunit-specific mutagenesis demonstrated that the presence of the N136A mutation in the p51 subunit alone was sufficient to cause degradation of RT within the virus particle. Alanine mutation at other residues of the beta7-beta8 loop did not affect either RT stability or virus infectivity significantly. None of the beta7-beta8 loop alanine mutations affected the sensitivity of virus to inhibition by NNRTIs. In the context of infectious virions, our results indicate a critical role of the p51 N136 residue within the beta7-beta8 loop for RT heterodimer stability and function. These findings suggest the interface comprising N136 in p51 and interacting residues in p66 as a possible target for rational drug design.
Mulky
Alok
A
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Vu
B Christie
BC
Conway
Joan A
JA
Hughes
Stephen H
SH
Kappes
John C
JC
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism*; D001216:Asparagine / Q000235:genetics; D019281:Dimerization; D015658:HIV Infections / Q000201:enzymology; D016333:HIV Protease / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology* / Q000472:pathogenicity / Q000502:physiology; D006801:Humans; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014771:Virion / Q000187:drug effects / Q000201:enzymology; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*
D000596:Amino Acids; D021122:Protein Subunits; D018894:Reverse Transcriptase Inhibitors; D001216:Asparagine; D054303:HIV Reverse Transcriptase; D016333:HIV Protease; D000409:Alanine