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Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and
M J Roth1, P Schwartzberg, N Tanese
1Department of Biochemistry, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, Piscataway 08854.
Abstract:
The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms. Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration.
Insights
Moloney murine leukemia virus integration (IN) protein mutations disrupted viral DNA integration. Seven lethal mutations blocked 3' recessed termini formation, essential for provirus integration.
Area of Science:
- Retroviral molecular biology
- Protein structure-function relationships
- Viral integration mechanisms
Background:
- The pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, crucial for proviral DNA integration.
- Understanding the IN protein's function is vital for comprehending retroviral replication cycles.
Purpose of the Study:
- To investigate the role of specific regions within the Moloney murine leukemia virus IN protein in DNA binding and viral integration.
- To elucidate the functional consequences of mutations affecting conserved cysteine residues in the IN protein.
Main Methods:
- Generated linker insertion and single-base substitution mutations within the IN protein coding region.
- Assayed mutant IN proteins for DNA-binding activity in vitro.
- Reconstructed mutant viruses and analyzed viral DNA processing and integration intermediates.
Main Results:
- Mutations altering conserved cysteine residues impacted IN protein DNA binding, likely due to altered protein folding.
- Seven of eight mutations were lethal when reconstituted into full-length virus.
- Lethal mutations blocked the formation of 3' recessed termini in viral DNA, a prerequisite for integration.
Conclusions:
- The integrity of specific regions within the Moloney murine leukemia virus IN protein, particularly those involving conserved cysteines, is essential for its function in viral integration.
- Disruption of 3' recessed termini formation is a critical bottleneck caused by lethal IN mutations, preventing provirus establishment.