TRIM5α/Cyclophilin A-Modified MDBK Cells for Lentiviral-Based Gene Editing

Area of Science:

• Virology
• Molecular Biology
• Gene Editing

Background:

• Lentiviral vectors are crucial for genetic modification but show limited efficiency in Madin-Darby bovine kidney (MDBK) cells.
• HIV-1 restriction factors, such as TRIM5α, impede lentiviral infection in certain cell types.

Purpose of the Study:

• To enhance lentiviral gene modification efficiency in MDBK cells.
• To investigate the roles of TRIM5α and cyclophilin A (CypA) in restricting lentiviral infection in bovine cells.

Main Methods:

• Depletion of TRIM5α using siRNA to assess its impact on lentiviral infection.
• Generation of a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9).
• CRISPR/Cas9-mediated knockout of TRIM5α in MDBK-iCas9 cells (MDBK-iCas9^TRIM5α-/-) via sgRNA transfection and single-cell cloning.
• Treatment with cyclosporine A to evaluate its effect on lentiviral infectivity.

Main Results:

• TRIM5α depletion significantly enhanced HIV-1 infection in MDBK cells.
• The MDBK-iCas9^TRIM5α-/- cell line exhibited increased permissiveness to lentivirus compared to wild-type (MDBK-WT) cells.
• Cyclosporine A treatment markedly increased lentiviral infectivity in both TRIM5α knockout and wild-type MDBK cells, indicating CypA acts independently of TRIM5α as an inhibitor.
• Combined targeting of TRIM5α and CypA shows potential for significantly boosting lentiviral infection.

Conclusions:

• Bovine TRIM5α and CypA are key inhibitors of lentiviral infection in MDBK cells.
• Targeting both TRIM5α and CypA offers a potent strategy to enhance lentiviral transduction efficiency in bovine cells.
• This approach provides a valuable tool for studying bovine virus-host interactions and advancing gene engineering in bovine cell systems.