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The palindromic LTR-LTR junction of Moloney murine leukemia virus is not an efficient substrate for proviral
L I Lobel1, J E Murphy, S P Goff
1Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Abstract:
We generated viral constructs to test the hypothesis that the major substrate on retroviral DNA that is utilized for proviral DNA integration is the palindromic sequence, termed the LTR-LTR junction, normally present in circular molecules formed by joining the two termini of linear proviral DNA. Recombinant viral genomes were built which carried a selectable marker and an extra copy of the LTR-LTR junction from a cloned circular provirus. The junction sequence in each case was positioned such that its use during integration would lead to an easily detected, aberrantly integrated proviral DNA. Analysis of DNA from cells infected with the virus constructs showed that the introduced junction sequence is used at least 1,000-fold less efficiently than the natural sequences at the ends of the genome. This suggests that a linear or more exotic DNA intermediate is most likely the true precursor for the integration reaction.
Insights
Researchers tested if the LTR-LTR junction in circular retroviral DNA is key for integration. Findings show this junction is rarely used, suggesting linear DNA intermediates are more likely for proviral DNA integration.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Retroviral DNA integration is crucial for viral replication and pathogenesis.
- The precise DNA substrate utilized during integration remains incompletely understood.
- Circular molecules containing an LTR-LTR junction are formed from linear proviral DNA termini.
Purpose of the Study:
- To investigate the hypothesis that the LTR-LTR junction is the major substrate for proviral DNA integration.
- To determine the efficiency of the LTR-LTR junction in retroviral integration compared to natural integration sites.
Main Methods:
- Generation of recombinant viral constructs with an additional LTR-LTR junction sequence.
- Introduction of a selectable marker for easy detection of integrated proviral DNA.
- Analysis of DNA from infected cells to quantify integration events at the introduced junction.
Main Results:
- The introduced LTR-LTR junction sequence was utilized for integration at least 1,000-fold less efficiently than natural end sequences.
- Aberrantly integrated proviral DNA resulting from the use of the introduced junction was rarely detected.
- Natural sequences at the ends of the viral genome are highly preferred for integration.
Conclusions:
- The LTR-LTR junction is not the major substrate for proviral DNA integration.
- A linear or other non-circular DNA intermediate is the likely precursor for retroviral integration.
- This finding refines our understanding of the retroviral replication cycle and integration mechanisms.