This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chiu, R.
Right arrow Articles by Grandgenett, D. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiu, R.
Right arrow Articles by Grandgenett, D. P.
Right arrowPubmed/NCBI databases
*Substance via MeSH

 Previous Article  |  Next Article 

Journal of Virology, June 2003, p. 6482-6492, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6482-6492.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular and Genetic Determinants of Rous Sarcoma Virus Integrase for Concerted DNA Integration

Roger Chiu and Duane P. Grandgenett*

St. Louis University Health Sciences Center, Institute for Molecular Virology, St. Louis, Missouri 63110

Received 4 December 2002/ Accepted 3 March 2003

Site-directed mutagenesis of recombinant Rous sarcoma virus (RSV) integrase (IN) allowed us to gain insights into the protein-protein and protein-DNA interactions involved in reconstituted IN-viral DNA complexes capable of efficient concerted DNA integration (termed full-site). At 4 nM IN, wild-type (wt) RSV IN incorporates ~30% of the input donor into full-site integration products after 10 min of incubation at 37°C, which is equivalent to isolated retrovirus preintegration complexes for full-site integration activity. DNase I protection analysis demonstrated that wt IN was able to protect the viral DNA ends, mapping ~20 bp from the end. We had previously mapped the replication capabilities of several RSV IN mutants (A48P and P115S) which appeared to affect viral DNA integration in vivo. Surprisingly, recombinant RSV A48P IN retained wt IN properties even though the virus carrying this mutation had significantly reduced integrated viral DNA in comparison to wt viral DNA in virus-infected cells. Recombinant RSV P115S IN also displayed all of the properties of wt RSV IN. Upon heating of dimeric P115S IN in solution at 57°C, it became apparent that the mutation in the catalytic core of RSV IN exhibited the same thermolabile properties for 3' OH processing and strand transfer (half-site and full-site integration) activities consistent with the observed temperature-sensitive defect for integration in vivo. The average half-life for inactivation of the three activities were similar, ranging from 1.6 to 1.9 min independent of the IN concentrations in the assay mixtures. Wt IN was stable under the same heat treatment. DNase I protection analysis of several conservative and nonconservative substitutions at W233 (a highly conserved residue of the retrovirus C-terminal domain) suggests that this region is involved in protein-DNA interactions at the viral DNA attachment site. Our data suggest that the use of recombinant RSV IN to investigate efficient full-site integration in vitro with reference to integration in vivo is promising.

* Corresponding author. Mailing address: St. Louis University Health Sciences Center, Institute for Molecular Virology, 3681 Park Ave., St. Louis, MO 63110. Phone: (314) 577-8411. Fax: (314) 577-8406. E-mail: Grandgdp{at}slu.edu.

Journal of Virology, June 2003, p. 6482-6492, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6482-6492.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Oh, J., Chang, K. W., Hughes, S. H. (2008). Integration of Rous Sarcoma Virus DNA: a CA Dinucleotide Is Not Required for Integration of the U3 End of Viral DNA. J. Virol. 82: 11480-11483 [Abstract] [Full Text]  
  • Ebina, H., Chatterjee, A. G., Judson, R. L., Levin, H. L. (2008). The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein. J. Biol. Chem. 283: 15965-15974 [Abstract] [Full Text]  
  • Pandey, K. K., Bera, S., Zahm, J., Vora, A., Stillmock, K., Hazuda, D., Grandgenett, D. P. (2007). Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate. J. Virol. 81: 12189-12199 [Abstract] [Full Text]  
  • Pandey, K. K., Sinha, S., Grandgenett, D. P. (2007). Transcriptional Coactivator LEDGF/p75 Modulates Human Immunodeficiency Virus Type 1 Integrase-Mediated Concerted Integration. J. Virol. 81: 3969-3979 [Abstract] [Full Text]  
  • Li, M., Craigie, R. (2005). Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration. J. Biol. Chem. 280: 29334-29339 [Abstract] [Full Text]  
  • Sinha, S., Grandgenett, D. P. (2005). Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells. J. Virol. 79: 8208-8216 [Abstract] [Full Text]  
  • Busschots, K., Vercammen, J., Emiliani, S., Benarous, R., Engelborghs, Y., Christ, F., Debyser, Z. (2005). The Interaction of LEDGF/p75 with Integrase Is Lentivirus-specific and Promotes DNA Binding. J. Biol. Chem. 280: 17841-17847 [Abstract] [Full Text]  
  • Vora, A., Bera, S., Grandgenett, D. (2004). Structural Organization of Avian Retrovirus Integrase in Assembled Intasomes Mediating Full-site Integration. J. Biol. Chem. 279: 18670-18678 [Abstract] [Full Text]  
  • Auerbach, M. R., Shu, C., Kaplan, A., Singh, I. R. (2003). Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting. Proc. Natl. Acad. Sci. USA 100: 11678-11683 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»