Hypermutation of ApoB mRNA by rat APOBEC-1 overexpression mimics APOBEC-3 hypermutation.
APOBEC-3 proteins induce C-to-U hypermutations in the viral genome of various viruses and have broad antiviral activity. Generally, only a small proportion of viral genomes (<10(-)(2)) are hypermutated by APOBEC-3s, but often many cytidines (up to 40%) are converted into uridine. The mechanism of this unique selective hypermutation remains unknown. We found that rat APOBEC-1 overexpression had a hypermutation pattern similar to that of APOBEC-3s on its substrate apolipoprotein B (apoB) mRNA. Transient plasmid transfection of rat APOBEC-1 resulted in 0.4% and 1.8% hypermutations with apoB mRNA in HepG2 and McA7777 cells, respectively. The low frequency of hypermutated apoB mRNA targets was enriched by differential DNA denaturation PCR at 72-76 °C, with hypermutation levels increasing up to 67%. Up to 69.6% of cytidines in HepG2 and up to 75.5% of cytidines in McA7777 cells were converted into uridines in the hypermutated apoB mRNA. When rat APOBEC-1 was overexpressed by adenovirus, the hypermutation frequency of apoB mRNA increased from 0.4% to ∼20% and was readily detected by regular PCR. However, this higher expression efficiency only increased the frequency of hypermutation, not the number of affected cytidines in hypermutated targets. Rat APOBEC-1 hypermutation was modulated by cofactors and eliminated by an E181Q mutation, indicating the role of cofactors in hypermutation. The finding of an APOBEC-3 hypermutation pattern with rat APOBEC-1 suggests that cofactors could also be involved in APOBEC-3 hypermutation. Using hepatitis B virus hypermutation, we found that KSRP increased APOBEC-3C and APOBEC-3B hypermutation. These data show that, like rat APOBEC-1 hypermutation, cellular factors may play a regulatory role in APOBEC-3 hypermutation.
Department of Laboratory Medicine, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Zhigang
Z
Chen
Thomas L
TL
Eggerman
Alexander V
AV
Bocharov
Irina N
IN
Baranova
Tatyana G
TG
Vishnyakova
Roger J
RJ
Kurlander
Gyorgy
G
Csako
Amy P
AP
Patterson
D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D003562:Cytidine / Q000378:metabolism; D003564:Cytidine Deaminase / Q000096:biosynthesis* / Q000235:genetics; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010957:Plasmids; D051381:Rats; D014162:Transfection; D014529:Uridine / Q000096:biosynthesis
D001055:Apolipoproteins B; D003562:Cytidine; D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase; D014529:Uridine