Biochemical and biophysical characterization of a chimeric TRIM21-TRIM5alpha protein.
The tripartite motif (TRIM) protein, TRIM5alpha, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5alpha 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 TRIM5alpha protein (TRIM5(Rh)-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5(Rh)-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 TRIM5(Rh)-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5(Rh)-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 TRIM5(Rh)-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins.
Kar
Alak Kanti
AK
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115, USA.
Diaz-Griffero
Felipe
F
Li
Yuan
Y
Li
Xing
X
Sodroski
Joseph
J
D000818:Animals; D002213:Capsid / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D019281:Dimerization; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology; D011506:Proteins / Q000737:chemistry* / Q000502:physiology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012261:Ribonucleoproteins; D018411:Spodoptera; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D000099207:SS-A Antigen
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D044767:Ubiquitin-Protein Ligases; D000099207:SS-A Antigen; C482587:TRIM5(alpha) protein, rhesus monkey
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