Journal: bioRxiv

Article Title: Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes

doi: 10.1101/2021.09.14.460218

Figure Lengend Snippet: (a) Experimental scheme for GCE and click-labeling. The system used here requires the introduction of an amber stop codon (UAG) at a specific site into the coding sequence of the protein of interest. A genetically engineered bio-orthogonal tRNA / aminoacyl-tRNA synthetase pair mediates incorporation of a non-canonical amino acid (ncAA) at the chosen position. In a second step, a highly reactive group of the ncAA is covalently linked to a fluorophore carrying a cognate reactive group (e.g., a tetrazine group reacting with a cyclopropane group at the ncAA via strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC)). Image created with BioRender.com (b) Morphology of HIV-1*CA14 ncAA assembly sites and particles. HEK293T cells were co-transfected with pNLC4-3*CA14 TAG and pNESPlyRS-eRF1dn-tRNA and grown in the presence of 550 µM CpK. At 44 h p.t., cells were fixed, embedded, and analyzed by thin-section EM as described in materials and methods. (c,d) Virus production. Click labeled particles were prepared from the supernatant of HEK293T cells co-transfected with either pNLC4-3* or pNLC4-3*CA14 TAG and pNESPlyRS-eRF1dn-tRNA. Cells were grown in the presence of 500 µM CpK as described in materials and methods. Particle yield in the final preparations was determined via quantitation of RT activity (SG-PERT assay;) (c) and by determination of CA amounts using quantitative immunoblot as described in materials and methods (d) . The graphs show mean values and SD from four independent experiments. (e) Immunoblot analysis of virus preparations. Particle lysates were separated by SDS-PAGE, and proteins were transferred to nitrocellulose membranes by semi-dry blotting. Viral proteins were detected using polyclonal antisera raised against recombinant HIV-1 RT or CA. Bound antibodies were detected by quantitative immunofluorescence with a Li-COR CLx infrared scanner, using secondary antibodies and protocols according to the manufacturer’s instructions. An asterisk indicates non-specific reactivity with bovine serum albumin carried over from the medium (f) In-gel fluorescence. Particle lysates prepared as in (e) were separated by SDS-PAGE, and the acrylamide gel was scanned using a Li-COR CLx infrared scanner set at an emission wavelength of 700 nm.

Article Snippet: CD4+ T cells were isolated using EasySep TM Direct Human T Cell Isolation Kit (Stemcell technologies, GER) according to the manufacturer’s instructions and activated by incubation in the presence of 100 U/ml IL-2 (Sigma Aldrich) and T Cell TransActTM human (Miltenyi Biotec, GER) for 72 h.

Techniques: Labeling, Sequencing, Transfection, Virus, Quantitation Assay, Activity Assay, Western Blot, SDS Page, Semi Dry Blot, Recombinant, Immunofluorescence, Fluorescence, Acrylamide Gel Assay

Journal: bioRxiv

Article Title: Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes

doi: 10.1101/2021.09.14.460218

Figure Lengend Snippet: (a) Activated CD4 + T cells were infected with HIV-1*CA14 SiR (MOI∼0.8) for 24 h before DMSO/PF74 treatment for 1 h, fixation, and methanol extraction. Samples were immunostained against CA (green) and laminA (blue). Images show a single z slice through the cell. Enlargements show the particle marked by the arrowhead. Scale bars: 10 µm (overview) and 1 µm (enlargement). (b) Data analyzed from the experiment outlined in (a). The graph shows the number of CA positive foci per nucleus in cells infected with HIV-1* (n=35 cells, mean=0.85) or HIV-1*CA14 SiR (n= 73 cells, mean=0.51). Pooled data from 6 different blood donors are shown. Grey lines show median and interquartile lines. (c) CA(SiR) intensities of nuclear objects in infected and activated CD4 + T cells at an MOI∼0.8 (n=13; mean=12,485 ± 7,445 a.u.) and an MOI ∼8 (n=7; mean=39,502 ± 18,025 a.u.). MOI was determined in TZM-bl cells. Grey lines show median and interquartile lines. (d-f) Nuclear cone-shaped capsids detected by CLEM-ET. SupT1 cells were treated with 1 µM aphidicolin (APC) for 16 h to prevent cell division, before infection with HIV-1*CA14 SiR virions (2.3 µU RT/cell, corresponds to an MOI∼0.4 determined in TZM-bl cells). At 24 h p.i., cells were cryo-immobilized by high-pressure freezing, freeze substituted, and further processed for CLEM and ET as described in materials and methods. (d) SDCM image of a 250-nm thick resin section of the cell infected with HIV-1*CA14 SiR virions (magenta), post-stained with Hoechst (blue) and decorated with multi-fluorescent fiducials (Fd) for correlation. The arrowhead in the enlargement of the boxed region indicates a CA(SiR) signal within the Hoechst-stained nuclear region. Scale bars: 1 µm (overview) and 200 nm (enlargement). (e) Computational slices through tomographic reconstructions at the correlated region boxed in (d) with views highlighting the presence of clustered capsid-reminiscent structures (black arrowheads) in the nuclear region. Nu, nucleus; NPC, nuclear pore complex; NE, nuclear envelope. Scale bar: 100 nm. (f) Segmented and isosurface rendered structure of the cones detected in (e). Magenta: capsid, yellow: NE, cyan: NPC. See also supplementary movie 1.

Article Snippet: CD4+ T cells were isolated using EasySep TM Direct Human T Cell Isolation Kit (Stemcell technologies, GER) according to the manufacturer’s instructions and activated by incubation in the presence of 100 U/ml IL-2 (Sigma Aldrich) and T Cell TransActTM human (Miltenyi Biotec, GER) for 72 h.

Techniques: Infection, Extraction, Staining

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements.

doi: 10.1096/fj.13-236273

Figure Lengend Snippet: Figure 1. Detection of transcription factors HNF1/ and Cdx1/2 in human colon cancers and cell lines expressing various amounts of B3GALT5 LTR transcript. A) RNA extracted from cancers and cell lines was reverse transcribed, and normalized amounts of the resultant first-strand cDNAs were mixed with the indicated amounts of competitor (truncated) cDNAs and subjected to PCR (25 and 35 cycles for -actin and B3GALT5 LTR, respectively), using primers specific to -actin and B3GALT5 LTR transcript, respectively. Note the different amounts of B3GALT5 LTR competitor cDNA used in the case of colon cancers, cell lines (present figure), and matched pairs of colon biopsies (Supplemental Fig. S1A). Twenty percent of each amplification reaction was electrophoresed in a 1% agarose gel and visualized by ethidium bromide staining. The target doublet corresponded to the alternative splicing that had been reported (10). Representative gels are shown. Samples were not all run on the same gel, as indicated by the vertical white spaces between the gels. B) Densitometric scanning of gel images was performed to quantitate the amounts of B3GALT5 LTR transcript, which were calculated from a standard curve and normalized to the amounts calculated for -actin. Results are means sd of 3 determinations. Note the different scales used for colon cancer and the cell lines. C) Nuclear extracts (5–10 g of protein) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane that was blotted with anti-HNF1 antibody (recognizing both HNF1 and HNF1), or anti-Cdx1, anti-Cdx2, or anti-histone H3 antibodies, followed by an HRP-labeled secondary antibody and chemiluminescence detection. Longer exposures were necessary to detect a visible spot for Cdx1 and Cdx2. For comparative quantitation, see Supplemental Fig. S1B.

Article Snippet: Aliquots of nuclear extracts (5–10 g of protein) were separated by 10% SDS-PAGE; transferred to a nitrocellulose membrane (Trans-Blot SD Semi-Dry Transfer Cell; Bio-Rad Laboratories S.r.l., Milan, Italy); and blotted with rabbit polyclonal anti-HNF1 / (sc-8986, 1:200; Santa Cruz Biotechnology Italia, Milan, Italy), rabbit polyclonal anti-H3 (D2B12 4620, 1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-Cdx1 (PAB4713, 1:200; Abnova, Taipei, Taiwan), or mouse monoclonal antiCdx2 (M01, 1:200; Abnova) antibodies, according to our published protocol (18).

Techniques: Expressing, Reverse Transcription, Agarose Gel Electrophoresis, Staining, Alternative Splicing, SDS Page, Membrane, Labeling, Quantitation Assay