Journal: Cell chemical biology

Article Title: Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.

doi: 10.1016/j.chembiol.2023.12.017

Figure Lengend Snippet: Figure 2. Construction and validation of an amber-free primary HIV-1 system (A) Alternations of four amber TAG termination codons of pol, vif, vpu, rev open reading frames (ORFs) in full-length wild-type HIV-1Q23 BG505 to ochre TAA termination codons, generating an amber-free HIV-1 genome. (B–D) Amber-free HIV-1Q23 BG505 yields an approximate 10-fold increase in released infectivity (B) and an enhanced Env expression (C) and retains similar susceptibility to potent dodecameric CD4 molecules (sCD4D1D2–Igatp, D) compared to the amber-abundant wild type counterpart. (B) Release of virus infectivity (mean ± SD) of the mutants carrying a termination codon change at Pol from amber to ochre (TAG to TAA) (left), and amber-free viral particles (right) in comparison to that of the wild type counterpart. The red arrow points to a 5- to 10-fold increase in infectivity caused by a single termination codon change at Pol. For comparisons, infectivity for the wild-type HIV-1 was measured separately and thus shown twice in graph (B). Where indicated as ‘‘Amber-Suppression’’ or ‘‘S,’’ a plasmid encoding tRNAPyl/NESPylRSAF was co-transfected with the HIV-1 genome, and the TCO* at 250 mM was added to the media. In contrast, only HIV-1 genome was transfected under the ‘‘Non-Suppression’’ or ‘‘NS’’ condition (gray bar in B). WT or wild type, the original amber-abundant HIV-1Q23 BG505. (C) Env processing and incorporation (Figure S2) of viral particles were assessed by SDS-PAGE of virus supernatants, followed by western blotting using the polyclonal anti-gp120 and anti-HIV-IG. Repeated twice. (D) Neutralization curves (mean ± SD) of amber-free and amber-abundant viral particles by SCD4D1D2-Igatp molecules.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Streptavidin Invitrogen Cat #S888 60% (w/v) Opti-prep Sigma Cat #D1556 Nitrobenzyl alcohol Sigma Cat #N12821 Protocatechuic acid Sigma Cat # 37580 Protocatechuic-3,4-dioxygenase Sigma Cat #P8279 Acetone, EM-Grade, Glass-Distilled Electron Microscopy Sciences Cat # 10015 SPY555-Actin Cytoskeleton CAT # CY-SC202 Critical commercial assays KAPA SYBR FAST qPCR Master Mix (2X) Kit KAPA Biosystems Cat # KK4600 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat # 16158 Experimental models: cell lines HEK293T ATCC Cat # CRL-3216 TZM-bl NIH HIV Reagent Program Cat # ARP-8129 Experimental models: organisms/strains HIV-1 lentiviral particles carrying envelope This paper N/A Oligonucleotides Primers for constructing plasmids, see Table S4 This paper N/A Recombinant DNA Wildtype HIV-1Q23 BG505 Julie Overbaugh Lab, Fred Hutch Cancer Center N/A Amber-free HIV-1Q23 BG505 This paper N/A tRNAPyl/NESPylRSAF Edward Lemke Lab, Johannes Gutenberg – University Mainz N/A Amber-free HIV-1Q23 BG505 N136TAG V4A1 This paper N/A Amber-free HIV-1Q23 BG505 V1Q3 S401TAG This paper N/A Amber-free HIV-1Q23 BG505 N136TAG S401TAG This paper N/A Amber-free HIV-1Q23 BG505 N136TAG S413TAG This paper N/A HIV-1-InGluc Walther Mothes Lab, Yale University N/A Deposited data Replication-incompetent amber-free HIV1Q23 BG505 deltaRT Addgene Cat # 213007 Replication-competent amber-free HIV1Q23 BG505 This paper Mendeley Data (https://doi.org/10.17632/ d9ww5mh46d.1) Software and algorithms GraphPad Prism v8.4.3 GraphPad https://www.graphpad.com/ MATLAB Mathworks https://www.mathworks.com/ PyMOL Schrödinger https://pymol.org/2/ Chimera University of California, San Francisco https://bio3d.colorado.edu/SerialEM/ SerialEM software package David N. Mastronarde, University of Colorado Boulder https://bio3d.colorado.edu/SerialEM/ IMOD software package David N. Mastronarde, University of Colorado Boulder https://bio3d.colorado.edu/imod/ Other Trans-Blot Turbo system Bio-Rad N/A ProFlex PCR System Applied biosystems N/A NanoSight instrument Malvern Panalytical N/A (Continued on next page) Cell Chemical Biology 31, 1–15.e1–e7, March 21, 2024 e2

Techniques: Biomarker Discovery, Infection, Expressing, Virus, Comparison, Plasmid Preparation, Transfection, SDS Page, Western Blot, Neutralization

Journal: Cell chemical biology

Article Title: Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.

doi: 10.1016/j.chembiol.2023.12.017

Figure Lengend Snippet: Figure 3. Efficient ncAA incorporation into dually amber-tagged Env on amber-free HIV-1 virus that retains sensitivities to trimer-specific neutralizing antibodies (A) Scheme showing tag insertion sites and denotations of labeling tags in BG505 gp120 subunit. (B) Release of infectivity (mean ± SD) on TZM-bl cells from HEK293T cells transfected with plasmids encoding singly amber-tagged, hybrid peptide/amber- tagged, dually amber-tagged EnvBG505 in the context of an amber-free Q23 backbone (HIV-1Q23 BG505). (C) Env processing and incorporation of amber-free, dually amber-tagged Env (N136TAG S401TAG and N136TAG S413TAG), or hybrid peptide/amber-tagged Env (N136TA V4A1 and V1Q3 S401TAG) viral particles. Proteins were analyzed by immunoblotting using a polyclonal antiserum against HIV-1 gp120. (D) A representative cryo-EM image of amber-free and two dually amber-tagged HIV-1 viral particles, respectively. Scale bar: 50 nm. (E) Neutralization curves (mean ± SD) of amber-free and dually amber-tagged HIV-1 viral particles by trimer-specific antibodies, including PG9, PG16, and PGT151. Dose-dependent relative infectivity was normalized to the mean value determined in the absence of the corresponding ligand. All viral particles generated in this study were prepared using 100% of indicated HIV-1 constructs during transfection.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Streptavidin Invitrogen Cat #S888 60% (w/v) Opti-prep Sigma Cat #D1556 Nitrobenzyl alcohol Sigma Cat #N12821 Protocatechuic acid Sigma Cat # 37580 Protocatechuic-3,4-dioxygenase Sigma Cat #P8279 Acetone, EM-Grade, Glass-Distilled Electron Microscopy Sciences Cat # 10015 SPY555-Actin Cytoskeleton CAT # CY-SC202 Critical commercial assays KAPA SYBR FAST qPCR Master Mix (2X) Kit KAPA Biosystems Cat # KK4600 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat # 16158 Experimental models: cell lines HEK293T ATCC Cat # CRL-3216 TZM-bl NIH HIV Reagent Program Cat # ARP-8129 Experimental models: organisms/strains HIV-1 lentiviral particles carrying envelope This paper N/A Oligonucleotides Primers for constructing plasmids, see Table S4 This paper N/A Recombinant DNA Wildtype HIV-1Q23 BG505 Julie Overbaugh Lab, Fred Hutch Cancer Center N/A Amber-free HIV-1Q23 BG505 This paper N/A tRNAPyl/NESPylRSAF Edward Lemke Lab, Johannes Gutenberg – University Mainz N/A Amber-free HIV-1Q23 BG505 N136TAG V4A1 This paper N/A Amber-free HIV-1Q23 BG505 V1Q3 S401TAG This paper N/A Amber-free HIV-1Q23 BG505 N136TAG S401TAG This paper N/A Amber-free HIV-1Q23 BG505 N136TAG S413TAG This paper N/A HIV-1-InGluc Walther Mothes Lab, Yale University N/A Deposited data Replication-incompetent amber-free HIV1Q23 BG505 deltaRT Addgene Cat # 213007 Replication-competent amber-free HIV1Q23 BG505 This paper Mendeley Data (https://doi.org/10.17632/ d9ww5mh46d.1) Software and algorithms GraphPad Prism v8.4.3 GraphPad https://www.graphpad.com/ MATLAB Mathworks https://www.mathworks.com/ PyMOL Schrödinger https://pymol.org/2/ Chimera University of California, San Francisco https://bio3d.colorado.edu/SerialEM/ SerialEM software package David N. Mastronarde, University of Colorado Boulder https://bio3d.colorado.edu/SerialEM/ IMOD software package David N. Mastronarde, University of Colorado Boulder https://bio3d.colorado.edu/imod/ Other Trans-Blot Turbo system Bio-Rad N/A ProFlex PCR System Applied biosystems N/A NanoSight instrument Malvern Panalytical N/A (Continued on next page) Cell Chemical Biology 31, 1–15.e1–e7, March 21, 2024 e2

Techniques: Virus, Labeling, Infection, Transfection, Western Blot, Cryo-EM Sample Prep, Neutralization, Generated, Construct

Journal: Cell chemical biology

Article Title: Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.

doi: 10.1016/j.chembiol.2023.12.017

Figure Lengend Snippet: Figure 6. Trafficking of amber-free HIV-1Q23 BG505 virions carrying 100% click-labeled Env N136TAG-Cy5 in TZM-bl HeLa cells (A) Live cell imaging of SPY555-Actin (magenta) labeled TZM-bl cells with an internalization event of a single virion with click-labeled Env N136TAG-Cy5 (green). The HIV-1 particle of interest initially associated with the cell periphery is marked with a dashed yellow circle. (B) Representative single particle trajectory of the click-labeled HIV-1 internalization event from panel A. The single HIV-1 trajectory is overlayed on the source image to demonstrate the depth of virus internalization into the cell. (C) Representative single particle intensity trace for the particle of interest from panel A. The internalization event is indicated with the black arrowhead on the panel. (D) Instantaneous particle speed demonstrating an increase in virus trafficking speed during internalization into the cell, consistent with virus transport in endosomes. The internalization event is signified with an arrow. (E) Mean square displacement (MSD) plot of the particle from panel A demonstrating that the particle of interest undergoes directional motion for a fraction of its trajectory coinciding with the internalization even.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Streptavidin Invitrogen Cat #S888 60% (w/v) Opti-prep Sigma Cat #D1556 Nitrobenzyl alcohol Sigma Cat #N12821 Protocatechuic acid Sigma Cat # 37580 Protocatechuic-3,4-dioxygenase Sigma Cat #P8279 Acetone, EM-Grade, Glass-Distilled Electron Microscopy Sciences Cat # 10015 SPY555-Actin Cytoskeleton CAT # CY-SC202 Critical commercial assays KAPA SYBR FAST qPCR Master Mix (2X) Kit KAPA Biosystems Cat # KK4600 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat # 16158 Experimental models: cell lines HEK293T ATCC Cat # CRL-3216 TZM-bl NIH HIV Reagent Program Cat # ARP-8129 Experimental models: organisms/strains HIV-1 lentiviral particles carrying envelope This paper N/A Oligonucleotides Primers for constructing plasmids, see Table S4 This paper N/A Recombinant DNA Wildtype HIV-1Q23 BG505 Julie Overbaugh Lab, Fred Hutch Cancer Center N/A Amber-free HIV-1Q23 BG505 This paper N/A tRNAPyl/NESPylRSAF Edward Lemke Lab, Johannes Gutenberg – University Mainz N/A Amber-free HIV-1Q23 BG505 N136TAG V4A1 This paper N/A Amber-free HIV-1Q23 BG505 V1Q3 S401TAG This paper N/A Amber-free HIV-1Q23 BG505 N136TAG S401TAG This paper N/A Amber-free HIV-1Q23 BG505 N136TAG S413TAG This paper N/A HIV-1-InGluc Walther Mothes Lab, Yale University N/A Deposited data Replication-incompetent amber-free HIV1Q23 BG505 deltaRT Addgene Cat # 213007 Replication-competent amber-free HIV1Q23 BG505 This paper Mendeley Data (https://doi.org/10.17632/ d9ww5mh46d.1) Software and algorithms GraphPad Prism v8.4.3 GraphPad https://www.graphpad.com/ MATLAB Mathworks https://www.mathworks.com/ PyMOL Schrödinger https://pymol.org/2/ Chimera University of California, San Francisco https://bio3d.colorado.edu/SerialEM/ SerialEM software package David N. Mastronarde, University of Colorado Boulder https://bio3d.colorado.edu/SerialEM/ IMOD software package David N. Mastronarde, University of Colorado Boulder https://bio3d.colorado.edu/imod/ Other Trans-Blot Turbo system Bio-Rad N/A ProFlex PCR System Applied biosystems N/A NanoSight instrument Malvern Panalytical N/A (Continued on next page) Cell Chemical Biology 31, 1–15.e1–e7, March 21, 2024 e2

Techniques: Labeling, Live Cell Imaging, Single Particle, Virus