IDENTIFICATION OF RESIDUES WITHIN THE 35-AMINO-ACID SPACING REGION OF ROUS SARCOMA VIRUS INTEGRASE THAT ARE IMPORTANT FOR INTERACTIONS WITH ITS VIRAL AND NONVIRAL DNA SUBSTRATES

Open Access
- Author:
- Raup-Konsavage, Wesley Merle
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 18, 2007
- Committee Members:
- Michael Katzman, Committee Chair/Co-Chair
David Joseph Spector, Committee Member
John Warren Wills, Committee Member
Leslie Joan Parent, Committee Member
Ira Joseph Ropson, Committee Member - Keywords:
- Rous Sarcoma Virus
retroviral integrase
integration - Abstract:
- Retrovirus replication requires insertion of the viral genomic DNA into the DNA of the host. This insertion is mediated by the viral integrase enzyme. A better understanding of how integrase interacts with these two DNA substrates is important for both the development of antiretroviral drugs and for improved retroviral vectors for gene therapy. Integrase specifically recognizes the viral DNA and removes the terminal nucleotides from the 3' end of each strand (this reaction is referred to as processing). The enzyme then inserts this trimmed DNA nonspecifically into the host DNA (this reaction is referred to as DNA joining). Interestingly, avian integrases are known to nick viral DNA at both the biologically relevant site and one nucleotide away from that site when reactions are done in the presence of manganese. However, mutations at residue 124 of Rous sarcoma virus (RSV) integrase yielded enzymes that are more specific for nicking at the biologically relevant site. One of these mutations, S124D, is markedly enhanced for processing activity, both in terms of specificity and effiency, but is dramatically impaired for DNA joining activity. Additional mutations were constructed to determine what properties of the aspartate substitution result in this activity profile. It was found that both the negative charge, the position of the negative charge, and the structure of the substituted amino acid all contributed to the S124D’s activity profile. Moreover, the S124D protein was found to be impaired for binding to nonviral DNA. All retroviral integrases contain a conserved D,D-35-E motif within the core domain. The three acidic residues are known to be required for enzymatic activity, but the importance of the conserved 35-amino-acid spacing is unknown. Mutations were constructed in a flexible loop within this 35-amino-acid spacing that altered the length of the spacing or the flexibility of the loop. It appears that the flexibility of the loop is more important for interactions with viral DNA than nonviral DNA. Additionally, enzymes with alterations to the spacing maintained enzymatic activity. However, virions containing these mutations were defective for replication, suggesting that the 35-amino-acid spacing is important for virus replication. Together, these data show that the D,D-35-E motif participates in distinct and separable functions for integrase acitivity.