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Journal of Virology, December 2008, p. 11669-11681, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01559-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Biochemical and Biophysical Characterization of a Chimeric TRIM21-TRIM5{alpha} Protein{triangledown}

Alak Kanti Kar,1 Felipe Diaz-Griffero,1 Yuan Li,1 Xing Li,1 and Joseph Sodroski1,2*

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 021152

Received 23 July 2008/ Accepted 5 September 2008

The tripartite motif (TRIM) protein, TRIM5{alpha}, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5{alpha} promotes premature uncoating of the capsid, thus blocking virus infection. Low levels of expression and tendencies to aggregate have hindered the biochemical, biophysical, and structural characterization of TRIM proteins. Here, a chimeric TRIM5{alpha} protein (TRIM5Rh-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5Rh-21R protein formed large aggregates, soluble fractions of the protein formed oligomers (mainly dimers), exhibited a protease-resistant core, and contained a high percentage of helical secondary structure. Cross-linking followed by negative staining and electron microscopy suggested a globular structure. The purified TRIM5Rh-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5Rh-21R protein specifically associated with human immunodeficiency virus type 1 capsid-like complexes; a deletion within the V1 variable region of the B30.2(SPRY) domain decreased capsid binding. Thus, the TRIM5Rh-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins.

* Corresponding author. Mailing address: Dana-Farber Cancer Institute, 44 Binney St.-JFB 824, Boston, MA 02115. Phone: (617) 632-3371. Fax: (671) 632-4338. E-mail: joseph_sodroski{at}dfci.harvard.edu

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

Journal of Virology, December 2008, p. 11669-11681, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01559-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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