APOBEC3G-UBA2 fusion as a potential strategy for stable expression of APOBEC3G and inhibition of HIV-1 replication.
BACKGROUND
Although APOBEC3G protein is a potent and innate anti-HIV-1 cellular factor, HIV-1 Vif counteracts the effect of APOBEC3G by promoting its degradation through proteasome-mediated proteolysis. Thus, any means that could prevent APOBEC3G degradation could potentially enhance its anti-viral effect. The UBA2 domain has been identified as an intrinsic stabilization signal that protects protein from proteasomal degradation. In this pilot study, we tested whether APOBEC3G, when it is fused with UBA2, can resist Vif-mediated proteasomal degradation and further inhibit HIV-1 infection.


RESULTS
APOBEC3G-UBA2 fusion protein is indeed more resistant to Vif-mediated degradation than APOBEC3G. The ability of UBA2 domain to stabilize APOBEC3G was diminished when polyubiquitin was over-expressed and the APOBEC3G-UBA2 fusion protein was found to bind less polyubiquitin than APOBEC3G, suggesting that UBA2 stabilizes APOBEC3G by preventing ubiquitin chain elongation and proteasome-mediated proteolysis. Consistently, treatment of cells with a proteasome inhibitor MG132 alleviated protein degradation of APOBEC3G and APOBEC3G-UBA2 fusion proteins. Analysis of the effect of APOBEC3G-UBA2 fusion protein on viral infectivity indicated that infection of virus packaged from HEK293 cells expressing APOBEC3G-UBA2 fusion protein is significantly lower than those packaged from HEK293 cells over-producing APOBEC3G or APOBEC3G-UBA2 mutant fusion proteins.


CONCLUSIONS
Fusion of UBA2 to APOBEC3G can make it more difficult to be degraded by proteasome. Thus, UBA2 could potentially be used to antagonize Vif-mediated APOBEC3G degradation by preventing polyubiquitination. The stabilized APOBEC3G-UBA2 fusion protein gives stronger inhibitory effect on viral infectivity than APOBEC3G without UBA2.
Li
Lin
L
Department of Pathology, University of Maryland, 10 South Pine Street, MSTF700A, Baltimore, MD 21201, USA. dearwood@sina.com
Liang
Dong
D
Li
Jing-yun
JY
Zhao
Richard Y
RY
D000071480:APOBEC-3G Deaminase; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D015870:Gene Expression; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014779:Virus Replication / Q000187:drug effects*; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D011993:Recombinant Fusion Proteins; C470301:UBA2 protein, human; D054320:vif Gene Products, Human Immunodeficiency Virus; D025821:Polyubiquitin; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase; D044764:Ubiquitin-Activating Enzymes; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde