Ubiquitin-fusion as a strategy to modulate protein half-life: A3G antiviral activity revisited.
The human APOBEC3G (A3G) is a potent inhibitor of HIV-1 replication and its activity is suppressed by HIV-1 virion infectivity factor (Vif). Vif neutralizes A3G mainly by inducing its degradation in the proteasome and blocking its incorporation into HIV-1 virions. Assessing the time needed for A3G incorporation into virions is, therefore, important to determine how quickly Vif must act to induce its degradation. We show that modelling the intracellular half-life of A3G can induce its Vif-independent targeting to the ubiquitin-proteasome system. By using various amino acids (X) in a cleavable ubiquitin-X-A3G fusion, we demonstrate that the half-life (t1/2) of X-A3G can be manipulated. We show that A3G molecules with a half-life of 13 min are incorporated into virions, whereas those with a half-life shorter than 5 min were not. The amount of X-A3G incorporated into virions increases from 13 min (Phe-A3G) to 85 min (Asn-A3G) and remains constant after this time period. Interestingly, despite the presence of similar levels of Arg-A3G (t1/2=28 min) and Asp-A3G (t1/2=65 min) into HIV-1 Deltavif virions, inhibition of viral infectivity was only evident in the presence of A3G proteins with a longer half-life (t1/2 > or = 65 min).
Cadima-Couto
Iris
I
URIA-Centro Patogénese Molecular, Faculdade de Farmácia da Universidade Lisboa, Lisboa 1649-059, Portugal.
Freitas-Vieira
Acilino
A
Nowarski
Roni
R
Britan-Rosich
Elena
E
Kotler
Moshe
M
Goncalves
Joao
J
D000071480:APOBEC-3G Deaminase; D002460:Cell Line; D003564:Cytidine Deaminase / Q000378:metabolism*; D015658:HIV Infections / Q000378:metabolism / Q000821:virology; D015497:HIV-1 / Q000502:physiology; D006207:Half-Life; D006801:Humans; D025801:Ubiquitin / Q000378:metabolism*; D014779:Virus Replication; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D025801:Ubiquitin; D054320:vif Gene Products, Human Immunodeficiency Virus; C514847:vif protein, Human immunodeficiency virus 1; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase