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Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
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Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Published on: December 4, 2007

Infectivity and structure of molecular clones obtained from two genetically transmitted Moloney leukemia proviral

K Harbers, A Schnieke, H Stuhlmann

    Nucleic Acids Research
    |April 24, 1982
    PubMed

    Insights

    Moloney leukemia virus (M-MuLV) integrated in mouse germ lines can activate infectious virus, but mutations at Mov-2 and Mov-10 loci prevent infectious virus synthesis, allowing only partial genome expression or non-infectious particle production.

    Area of Science:

    • Virology
    • Genetics
    • Molecular Biology

    Background:

    • The Mov-2 and Mov-10 mouse substrains harbor Moloney leukemia virus (M-MuLV) in their germ line.
    • These substrains exhibit variable activation of infectious M-MuLV at later ages (Mov-2) or not at all (Mov-10).

    Purpose of the Study:

    • To investigate the molecular basis for the differential activation of infectious M-MuLV in Mov-2 and Mov-10 mice.
    • To identify mutations within the M-MuLV provirus or flanking sequences that affect viral infectivity.

    Main Methods:

    • Molecular cloning of M-MuLV proviruses with flanking mouse sequences from Mov-2 and Mov-10 loci.
    • Restriction enzyme analysis to detect alterations in proviral genomes.
    • Transfection assays using cloned DNAs to assess XC plaque induction and viral infectivity in 3T3 cells.

    Main Results:

    • Restriction analysis showed no major deletions or insertions in the M-MuLV proviruses at Mov-2 and Mov-10 loci.
    • Both cloned DNAs induced XC plaques, indicating partial viral gene expression.
    • Transfected 3T3 cells did not produce infectious virus, and modifying 5' cellular sequences did not restore infectivity.

    Conclusions:

    • M-MuLV integrated at the Mov-2 and Mov-10 loci likely harbors a mutation preventing infectious virus synthesis.
    • The mutation allows for XC plaque induction, suggesting partial genome expression or the production of non-infectious viral particles.

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