Alanine scanning mutagenesis of HIV-1 gp41 heptad repeat 1: insight into the gp120-gp41 interaction.
On the basis of mutagenesis, biochemical, and structural studies, heptad repeat 1 of HIV gp41 (HR1) has been shown to play numerous critical roles in HIV entry, including interacting with gp120 in prefusion states and interacting with gp41 heptad repeat 2 (HR2) in the fusion state. Moreover, HR1 is the site of therapeutic intervention by enfuviritide, a peptide analogue of HR2. In this study, the functional importance of each amino acid residue in gp41 HR1 has been systematically examined by alanine scanning mutagenesis, with subsequent characterization of the mutagenic effects on folding (as measured by incorporation into virions), association with gp120, and membrane fusion. The mutational effects on entry can be grouped into three classes: (1) wild type (defined as >40% of wild-type entry), (2) impaired (defined as 5-40% of wild-type entry), and (3) nonfunctional (defined as <5% of wild-type entry). Interestingly, the majority of HR1 mutations (77%) exhibit impaired or nonfunctional entry. Surprisingly, effects of mutations on folding, association, or fusion are not correlated to heptad position; however, folding defects are most often found in the N-terminal region of HR1. Moreover, disruption of the gp41-gp120 interaction is correlated to the C-terminal region of HR1, suggesting that this region interacts most closely with gp120. In summary, the sensitivity of gp41 HR1 to alanine substitutions suggests that even subtle changes in the local environment may severely affect envelope function, thereby strengthening the notion that HR1 is an attractive site for therapeutic intervention.
Sen
Jayita
J
Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607, USA.
Yan
Tianran
T
Wang
Jizhen
J
Rong
Lijun
L
Tao
Lin
L
Caffrey
Michael
M
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D015699:HIV Envelope Protein gp120 / Q000502:physiology*; D015700:HIV Envelope Protein gp41 / Q000235:genetics* / Q000502:physiology; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008561:Membrane Fusion; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D020449:Repetitive Sequences, Amino Acid; D053586:Virus Internalization
D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D000409:Alanine