T cells contain an RNase-insensitive inhibitor of APOBEC3G deaminase activity.
The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected. However, to date, the specific deaminase activity of endogenous A3G in human CD4+ T cells has not been examined directly. Here, we compared deaminase activity of endogenous and exogenous A3G in various human cell lines using a standard assay and a novel, quantitative, high-throughput assay. Exogenous A3G in epithelial cell lysates displayed deaminase activity only following RNase treatment, as expected given that A3G is known to form an enzymatically inactive RNA-containing complex. Surprisingly, comparable amounts of endogenous A3G from T cell lines or from resting or activated primary CD4+ T cells exhibited minimal deaminase activity, despite RNase treatment. Specific deaminase activity of endogenous A3G in H9, CEM, and other T cell lines was up to 36-fold lower than specific activity of exogenous A3G in epithelial-derived cell lines. Furthermore, RNase-treated T cell lysates conferred a dose-dependent inhibition to epithelial cell lysates expressing enzymatically active A3G. These studies suggest that T cells, unlike epithelial-derived cell lines, express an unidentified RNase-resistant factor that inhibits A3G deaminase activity. This factor could be responsible for reduced levels of hypermutation in T cells, and its identification and blockade could offer a means for increasing antiretroviral intrinsic immunity of T cells.
Thielen
Beth K
BK
Department of Pathobiology, University of Washington, Seattle, Washington, USA.
Klein
Kevin C
KC
Walker
Lorne W
LW
Rieck
Mary
M
Buckner
Jane H
JH
Tomblingson
Garrett W
GW
Lingappa
Jaisri R
JR
D000071480:APOBEC-3G Deaminase; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology*; D002460:Cell Line; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D012260:Ribonucleases / Q000378:metabolism; D014162:Transfection
D012260:Ribonucleases; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase