Abstract

The HIV-1 Envelope (Env) glycoprotein trimers undergo structural changes upon interacting with host receptor CD4 and coreceptors (CCR5/CXCR4) for virus entry. As the only surface-exposed viral protein, Env is a key antibody target but evades recognition through structural changes. Studying Env dynamics with respect to the coreceptor binding region (V3 loop) provides insights into virus entry and immune evasion. Fluorescence labeling via genetic code expansion (amber–TAG suppression) offers a minimally invasive approach for fluorescence microscope-based Env dynamics studies. Here, two V3-related dual-amber Env constructs (A135TAG-P308TAG, P308TAG-E395TAG) were generated by incorporating two non-canonical amino acids (ncAAs) at consecutive amber codons in Env on virions. Infectivity and immunoblotting showed 10-15% amber suppression efficiency, successful Env expression, and incorporation on virions that exhibit wildtype-like virion particle size. Our results pave the way for attaching two fluorophores to dual ncAAs incorporated in viral Env via click-chemistry, enabling structural dynamics from the V3 perspective.

Date of publication

Summer 5-19-2025

Document Type

Thesis

Language

english

Persistent identifier

http://hdl.handle.net/10950/4854

Committee members

Dr. Maolin Lu, Dr. Vijaya Mohan Rao Lella, Dr. Guohua Yi, Dr. Pierre Neuenschwander, Dr. Osamu Sato

Degree

M.S Biotechnology

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