Human-APOBEC3G-dependent restriction of porcine endogenous retrovirus replication is mediated by cytidine deamination and inhibition of DNA strand transfer during reverse transcription
Although human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hA3G)-mediated deamination is the major mechanism used to restrict the infectivity of a broad range of retroviruses, it is unclear whether porcine endogenous retrovirus (PERV) is affected by hA3G or porcine A3F (poA3F). To determine whether DNA deamination is required for hA3G- and poA3F-dependent inhibition of PERV transmission, we developed VSV-pseudotype PERV-B expressing hA3G, mutant hA3G-E67Q (encapsidation and RNA binding activity-deficient), mutant hA3G-E259Q (deaminase-deficient), or poA3F. hA3G-E67Q decreased virus infectivity by ~ 93% compared to the ~ 99% decrease of viral infectivity by wild-type hA3G, while hA3G-E259Q decreased the infectivity of PERV-B by ~ 35%. These data suggest that cytidine deamination activity is crucial for efficient restriction of PERV by hA3G, but cytidine deamination cannot fully explain the inactivation of PERV by hA3G. Furthermore, differential DNA denaturation PCR (3D-PCR) products from 293T cells infected with PERV-B expressing hA3G mutants were sequenced. G-to-A hypermutation was detected at a frequency of 4.1% for hA3G, 3.4% for hA3G-E67Q, and 4.7% for poA3F. These results also suggest that hA3G and poA3F inhibit PERV by a deamination-dependent mechanism. To examine the effect of hA3G on the production of PERV DNA, genomic DNA was extracted from 293T cells 12 h after infection with PERV expressing hA3G, and this DNA was used as template for real-time PCR. A 50% decrease in minus strand strong stop (-sss) DNA synthesis/transfer was observed in the presence of hA3G. Based on these results, we conclude that hA3G may restrict PERV by both deamination-dependent mechanisms and inhibition of DNA strand transfer during PERV reverse transcription.
This work was supported in part by DANKOOK ChemBio Specialization for Creative Korea-II.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2017R1D1A1B03032753).
Compliance with ethical standards
Conflict of interest
The author of this study has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 5.Kao S, Khan MA, Miyagi E, Plishka R, Buckler-White A, Strebel K (2003) The human immunodeficiency virus type 1 Vif protein reduces intracellular expression and inhibits packaging of APOBEC3G (CEM15), a cellular inhibitor of virus infectivity. J Virol 77:11398–11407CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Harris RS, Liddament MT (2004) Retroviral restriction by APOBEC proteins. Nature 4:868–877Google Scholar
- 22.Mbisa JL, Barr R, Thomas JA, Vandegraaff N, Dorweiler IJ, Svarovskaia ES, Brown WL, Mansky LM, Gorelick RJ, Harris RS, Engelman A, Pathak VK (2007) Human immunodeficiency virus type 1 cDNAs produced in the presence of APOBEC3G exhibit defects in plus-strand DNA transfer and integration. J Virol 81:7099–7110CrossRefPubMedPubMedCentralGoogle Scholar
- 28.Suspène R, Aynaud MM, Guétard D, Henry M, Eckhoff G, Marchio A, Pineau P, Dejean A, Vartanian JP, Wain-Hobson S (2011) Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism. Proc Natl Acad Sci USA 108:4858–4863CrossRefPubMedPubMedCentralGoogle Scholar
- 34.Kobayashi T, Koizumi Y, Takeuchi JS, Misawa N, Kimura Y, Morita S, Aihara K, Koyanagi Y, Iwami S, Sato K (2014) Quantification of deaminase activity-dependent and -independent restriction of HIV-1 replication mediated by APOBEC3F and APOBEC3G through experimental-mathematical investigation. J Virol 88:5881–5887CrossRefPubMedPubMedCentralGoogle Scholar