A Versatile Tool for Live-Cell Imaging and Super-Resolution Nanoscopy Studies of HIV-1 Env Distribution and Mobility

Abstract

The envelope glycoproteins (Env) of HIV-1 mediate cell entry through fusion of the viral envelope with a target cell membrane. Intramembrane mobility and clustering of Env trimers at the viral budding site are essential for its function. Previous live-cell and super-resolution microscopy studies were limited by lack of a functional fluorescent Env derivative, requiring antibody labeling for detection. Introduction of a bio-orthogonal amino acid by genetic code expansion, combined with click chemistry, offers novel possibilities for site-specific, minimally invasive labeling. Using this approach, we established efficient incorporation of non-canonical amino acids within HIV-1 Env in mammalian cells. The engineered protein retained plasma membrane localization, glycosylation, virion incorporation, and fusogenic activity, and could be rapidly and specifically labeled with synthetic dyes. This strategy allowed us to revisit Env dynamics and nanoscale distribution at the plasma membrane close to its native state, applying fluorescence recovery after photo bleaching and STED nanoscopy, respectively.

Recent trends in click chemistry as a promising technology for virus-related research

“…Recently, metabolic incorporation of a clickable group into viral protein was reported as another strategy to label a virus or viral protein for investigating the behavior of viral proteins (Fernandez and Freed, 2017;Lin et al, 2013;Nikic et al, 2014;Plass et al, 2012;Sakin et al, 2017). One protocol is based on genetic code expansion.…”

“…As the ncAA bears a ring-strained alkyne or alkene, the ncAA is clicked to azide-or tetrazine-containing dyes via SPAAC or SPIEDAC reactions, and thus, the mobility of the target viral protein, such as HIV-1 Env, can be measured in a real-time manner ( Fig. 1b) (Fernandez and Freed, 2017;Nikic et al, 2014;Sakin et al, 2017). Moreover, HIV-1 particles can be generated by adding eGFP-tagged Gag to the system, and therefore, the Env accumulating sites of particle budding can be visualized by stimulated emission depletion (STED) nanoscopy (Fernandez and Freed, 2017;Sakin et al, 2017).…”

“…(a) Virions were labeled with quantum dots (QDs) via copper-free click chemistry by linking azide-clicked virions to the dibenzocyclooctyne-derived QDs, resulting in intact, fluorescence-labeled and infectious virions (Hao et al, 2012). (b) An amber (UAG) stop codon was inserted into the HIV Env protein by site-directed mutagenesis, followed by coexpression of the protein with an aminoacyl tRNA synthase/tRNA and noncanonical amino acid (ncAA) in the target cell (Fernandez and Freed, 2017;Sakin et al, 2017). Subsequently, the ncAA was incorporated at the amber stop codon, allowing the continuous translation through the amber stop codon (Fernandez and Freed, 2017;Sakin et al, 2017).…”

“…(b) An amber (UAG) stop codon was inserted into the HIV Env protein by site-directed mutagenesis, followed by coexpression of the protein with an aminoacyl tRNA synthase/tRNA and noncanonical amino acid (ncAA) in the target cell (Fernandez and Freed, 2017;Sakin et al, 2017). Subsequently, the ncAA was incorporated at the amber stop codon, allowing the continuous translation through the amber stop codon (Fernandez and Freed, 2017;Sakin et al, 2017). When H-Tet-Cy5 was added, the Env was labeled between ncAA and tetrazine via a click reaction (Fernandez and Freed, 2017;Sakin et al, 2017).…”

“…Subsequently, the ncAA was incorporated at the amber stop codon, allowing the continuous translation through the amber stop codon (Fernandez and Freed, 2017;Sakin et al, 2017). When H-Tet-Cy5 was added, the Env was labeled between ncAA and tetrazine via a click reaction (Fernandez and Freed, 2017;Sakin et al, 2017). (c) Viral glycoprotein was labeled with a metabolically incorporated unnatural sugar (Ac4ManNAz), followed by a click reaction with organic fluorescent dyes, allowing virus-cell fusion to be visualized during the infection .…”