Research On Stable Expression Of Porcine Apobec3F In PK15Cells And Its Mutational Inhibition On Perv

The apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) protein is an innate anti-viral factor. Besides lipid metabolism and antibody diversification, its major biological function also involves in the inhibition of many retrotransposons, retroviruses, and some DNA viruses which is even more important. It has been shown that APOBEC protein can efficiently inhibit the replication of HIV-1, HBV, MLV and PERV viruses by its cytidine deminases mechanism. Being packaged into virion and realeased in new target cells, APOBEC triggers the deamination of cytosine in the first cDNA strand during viral reverse transcription, leading to high levels of dCâ†'dU hypermutation of viral minus-strand cDNAs (plus-strand is Gâ†'A), and finally results in the inability of virus to normally replicate or assembled. As an important member of the innate immune defences, APOBEC is becoming a hot research topic due to its novel mechanism of viral restriction. Xenotransplantation of porcine cells, tissues and organs are considered to be one of the feasible approaches to surmount the shortage of human donor materials. However, the fact that pigs naturally contain PERV becomes the major obstacle to pig-to-human xenotransplantation. It was shown that PERV could infect human cells and it make recipients suffer from immunodeficiency diseases. A number of strategies have been devised to reduce or eliminate the risk of PERV infection in xenograft recipients, but all of them are not completely effective and prohibitively expensive. Pigs naturally contain a kind of APOBEC, APOBEC3F, which has been confirmed to restrict HIV-1and MLV. However, whether it can inhibit PERV replication is still inconclusive. If APOBEC3F can effectively restrict PERV transmission, the goal of clearing PERV or controlling its titer in a relatively low level by porcine own immune mechanisms may be achieved, and the potential risk of PERV in xenotransplantation can be effectively controlled. This study here wants to find out whether porcine APOBEC3F can inhibit replication of PERV and the possible mechanisms. The major work includes the following aspects:Firstly, we constructed a recombinant expression plasmid pMSCV-FLAG-A3F-GFP with porcine APOBEC3F gene by retroviral vector system. Then the recombinant plasmids together with pVSV-G and pGag-Pol, were co-transfected into HEK293T packaging cells to prepare the pseudotype virus, which was able to infect PK15cells. The PK15cell line which stably expressed porcine APOBEC3F was obtained by G418selection and integration and expression of porcine APOBEC3F gene can be detected in those cells. These results provided a model for the further research of inhibition of porcine endogenous retrovius by porcine APOBEC3F.Secondly, quantitative PCR was adopted to detect PERV mRNA in PK15cells which stably expressed porcine APOBEC3F. The results showed that the copy of PERV mRNA was only31%of the normal level; By contrast, siRNA expression vector was constructed for porcine APOBEC3F knockdown. The quantitative PCR analysis results showed that the copy of PERV was6-fold Compared with the control. These results indicate that porcine APOBEC3F contributes to the inhibiting of PERV replication in swine cells.Thirdly, the existence of interaction between porcine APOBEC3F and PERV-Gag was verified using the yeast two-hybrid assay, and Western blot analysis confirmed that porcine APOBEC3F was packaged into PERV particles. Meanwhile, Gâ†'A hypermutation occurred in the presence of porcine APOBEC3F by analyzing genome sequence of PK15cells which stably expressed pig APOBEC3F. The above results suggest that it is highly possible that porcine APOBEC3F is packaged into viron requiring PERV-Gag, and finally inhibit PERV by cytidine deamination. To sum up, here we confirmed that porcine APOBEC3F could inhibit replication of PERV, and it is highly possible that porcine APOBEC3F retrict PERV attributed to cytidine deamination. This study laid a foundation for further study on mechanism of APOBEC3F’s inhibition to PERV, and provided the support for possible existing resistent effect of PERV to APOBEC3F.