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Journal of Virology, December 2008, p. 11682-11694, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01562-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of a Recombinant TRIM5{alpha} Protein That Restricts Human Immunodeficiency Virus Type 1 Replication{triangledown} ,{dagger}

Charles R. Langelier,1 Virginie Sandrin,1 Debra M. Eckert,1 Devin E. Christensen,1 Viswanathan Chandrasekaran,1 Steven L. Alam,1 Christopher Aiken,2 John C. Olsen,3 Alak Kanti Kar,4 Joseph G. Sodroski,4 and Wesley I. Sundquist1*

Department of Biochemistry, University of Utah, Salt Lake City, Utah 84112-5650,1 Department of Microbiology and Immunology, Vanderbilt University School of Medicine, A-5301 Medical Center North, Nashville, Tennessee 37232-2363,2 Department of Medicine, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,3 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, Massachusetts 021154

Received 23 July 2008/ Accepted 5 September 2008

The rhesus monkey intrinsic immunity factor TRIM5{alpha}rh recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5{alpha} proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5{alpha}rh protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5{alpha} proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5{alpha} proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5{alpha} proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.

* Corresponding author. Mailing address: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112-5650. Phone: (801) 585-5402. Fax: (801) 581-7959. E-mail: wes{at}biochem.utah.edu

{triangledown} Published ahead of print on 17 September 2008.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, December 2008, p. 11682-11694, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01562-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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