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Updated: Sep 30, 2026
Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
Published on: December 4, 2007
Inserting a nuclear targeting signal into a replication-competent Moloney murine leukemia virus affects viral export
Jennifer A Seamon1, Kathryn S Jones, Christina Miller
1Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854, USA.
Abstract:
The effects of inserting reported nuclear localization signals (NLSs) into the Moloney murine leukemia virus (Mo-MuLV) integrase (IN) protein, within a replication-competent viral construct, were studied. In contrast to the virus harboring IN fused to the simian virus 40 (SV40) large T antigen NLS (SV40 NLS) (J. A. Seamon, M. Adams, S. Sengupta, and M. J. Roth, Virology 274:412-419, 2000), a codon-modified SV40 NLS was stably expressed during viral propagation. Incorporation of the codon-modified SV40 NLS into IN, however, altered the packaging of the Gag-Pol precursor in the virus; viral particles contained decreased levels of reverse transcriptase (RT) and IN. In addition, the virus showed delayed kinetics of viral DNA synthesis upon infection. A panel of infectious MuLVs containing alternative IN-NLS fusions was generated and assayed for cell cycle-independent infection. Viral infection with the NLS-tagged proteins, however, remained dependent on passage of the cells through mitosis. This finding has direct implications for engineering murine-based retroviral vectors for gene therapy.
Insights
Adding nuclear localization signals (NLSs) to Moloney murine leukemia virus (Mo-MuLV) integrase (IN) impacted viral particle composition and DNA synthesis. This Mo-MuLV integrase modification affects retroviral vector design for gene therapy.
Area of Science:
- Molecular Biology
- Virology
- Gene Therapy
Background:
- Nuclear localization signals (NLSs) are crucial for directing proteins into the cell nucleus.
- Moloney murine leukemia virus (Mo-MuLV) integrase (IN) is essential for viral integration into the host genome.
- Modifying viral components can alter replication and infectivity.
Purpose of the Study:
- To investigate the effects of incorporating nuclear localization signals (NLSs) into the Moloney murine leukemia virus (Mo-MuLV) integrase (IN) protein.
- To assess the impact of these modifications on viral replication, particle composition, and infection kinetics.
- To evaluate the potential of NLS-tagged IN for developing improved retroviral vectors for gene therapy.
Main Methods:
- Construction of replication-competent Mo-MuLV with codon-modified simian virus 40 (SV40) NLS fused to IN.
- Analysis of viral particle composition, including reverse transcriptase (RT) and IN levels.
- Assay of viral DNA synthesis kinetics post-infection.
- Generation and testing of infectious MuLVs with alternative IN-NLS fusions for cell cycle-independent infection.
Main Results:
- A codon-modified SV40 NLS was stably expressed during viral propagation.
- Incorporation of the NLS-tagged IN altered Gag-Pol precursor packaging, reducing RT and IN levels in viral particles.
- The modified virus exhibited delayed viral DNA synthesis kinetics.
- Infection remained dependent on cellular passage through mitosis, even with NLS-tagged proteins.
Conclusions:
- Fusion of NLSs to Mo-MuLV IN affects viral particle content and replication efficiency.
- Retroviral vector infection mediated by NLS-tagged IN remains dependent on the cell cycle.
- Findings have implications for the design of murine-based retroviral vectors for gene therapy applications.
