Journal: bioRxiv

Article Title: CD34 serves as an intrinsic innate immune guardrail protecting stem cells from replicating retroviruses

doi: 10.1101/2025.03.15.643450

Figure Lengend Snippet: ( A to C ) Virion incorporation of CD34. HEK293T cells were co-transfected with 10 μg HIV-1 DNA plus 2 μg of CD34 DNA (HIV + CD34) or with CD34 DNA only (CD34 only). Supernatants were harvested at 48 h and purified through 6%–18% OptiPrep gradient ultra-centrifugation. CD34 and viral p24 proteins were analyzed by western blot using antibodies against HIV-1 p24 or CD34. Shown is the representative of three repeats. ( D and E ) Schematic of the immunomagnetic capture assay for detecting CD34 on HIV-1 particles ( D ). HEK293T cells were co-transfected with HIV-1 DNA (1 μg) plus a CD34 expression vector (0.5–10 ng) or an empty vector. Supernatants were harvested at 48 h and incubated with magnetic beads coated with anti-CD34 antibody or an isotype control antibody. Captured particles were washed, eluted, and quantified for p24. p -values were based on three independent assays ( E ). ( F ) Effects of CD34 on HIV-1 Env incorporation. HEK293T cells were co-transfected with HIV-1 DNA plus a CD34 expression vector (10 and 50 ng) or an empty vector. Particles were harvested at 48 h and purified through a sucrose gradient. Virions were lysed and analyzed with western blot using anti-HIV-1 gp41 or p24 antibodies. Representative blots from three independent repeats are shown. ( G ) CD34 blocks HIV-1 virion attachment to target cells. Viral particles were produced by co-transfection of HEK293T cells with HIV-1 DNA (1 μg) and a CD34 expression vector or an empty vector (10 ng). HIV-1 p24-normalized viral particles were then assayed for attachment to target Rev-A3R5-GFP cells by western blot for cell-bound p24. Representative blots from three independent assays are shown. The protein band intensity was quantified and the relative ratios of vector/GAPDH and CD34/GAPDH were calculated. Vector/GAPDH ratios were assigned as “1”. ( H ) CD34 blocks HIV-1 virion attachment independent of Env interaction with cellular receptors. Viral particles were produced similarly by co-transfection of HEK293T cells with HIV-1 DNA or HIV-1(NL4-3/KFS) and a CD34 expression vector or an empty vector. HIV-1 p24-normalized viral particles were then assayed for attachment to target HeLa or HeLa JC.53 cells by western blot for cell-bound p24. Representative blots from three independent assays are shown. The protein band intensity was similarly quantified. All p -values were calculated using a two-tailed T-test.

Article Snippet: For HIV-1 p24 release assays, cells were co-transfected with 1 μg of HIV-1(NL4-3) DNA and the indicated amounts of pCMV3-CD34 or pCMV3-Empty vector using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Purification, Centrifugation, Western Blot, Expressing, Plasmid Preparation, Incubation, Magnetic Beads, Control, Produced, Cotransfection, Two Tailed Test

Journal: bioRxiv

Article Title: CD34 serves as an intrinsic innate immune guardrail protecting stem cells from replicating retroviruses

doi: 10.1101/2025.03.15.643450

Figure Lengend Snippet: (A) Cells were infected with HIV-1(89.6) for 3.5 h, washed, and then cultured for 10 days. Viral p24 release was quantified by ELISA using cell culture supernatant. ( B ) Kasumi-3 cells were electroporated with HIV-1(NL4-3) DNA, HIV-1(89.6) DNA, or a control empty vector (4 μg). Viral replication was quantified by p24 release using ELISA of the cell culture supernatant.

Article Snippet: For HIV-1 p24 release assays, cells were co-transfected with 1 μg of HIV-1(NL4-3) DNA and the indicated amounts of pCMV3-CD34 or pCMV3-Empty vector using Lipofectamine 2000 (Invitrogen).

Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: CD34 serves as an intrinsic innate immune guardrail protecting stem cells from replicating retroviruses

doi: 10.1101/2025.03.15.643450

Figure Lengend Snippet: (A) Kasumi-3 cells were electroporated with HIV-1(NL4-3) DNA, HIV-1(89.6) DNA, or a control empty vector (4 μg). Downregulation of surface CD34 was quantified at 4 and 7 days post electroporation. ( B ) HEK293T cells were transfected with a control empty vector or a CD34 expression vector (100 ng). Cells were also co-transfected with a CD34 expression vector plus HIV-1(NL4-3) DNA, a Gag expression vector, a Vpu expression vector, or a Nef expression vector (2 μg of each vector). Surface CD34 expression was quantified with flow cytometry at 48 h post co-transfection.

Article Snippet: For HIV-1 p24 release assays, cells were co-transfected with 1 μg of HIV-1(NL4-3) DNA and the indicated amounts of pCMV3-CD34 or pCMV3-Empty vector using Lipofectamine 2000 (Invitrogen).

Techniques: Control, Plasmid Preparation, Electroporation, Transfection, Expressing, Flow Cytometry, Cotransfection