The Study On The Replication Inhibiton Effect Of Porcine Endogenous Retrovirus By Porcine APOBEC3F

Innate immunity is the first line of defence against pathogens infection, plays an important role in pathogen clearance. Recently, several novel cellular factors were discovered, such as apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) family, the tripartite motif protein (TRIM) family. These molecules were found to be able to inhibit virus replication and reduce virus load in infected cells, indicating their potential usese for antivirus theropy.APOBEC protein family consists of activation-induced deaminase (AID), APOBEC1, APOBEC2, APOBEC3 (A to H). All of these proteins have cytidine deaminase activity. The antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F) were well known. It was reported that A3G and A3F could effectively inhibit the replication of endogenous and exogenous retroviruses, as well as the replication of HBV. These two proteins both have two zinc-coordinating DNA cytosine deaminase motifs. The cytosine deaminase motifs deaminates dC to dU in nascent minus-strand viral DNA, resulting in G-to-A hypermutation in the plus strand DNA to inhibit the replication of viruses. The antiviral activity of these molecules depends on packaging into the virions , and play antiviral effect in the next round infected cells. APOBEC molecules inhibit virus repication directly and their antiviral mechanism is a unique which make them very promising theropy targets for virus infection.Pigs are considered to be suitable donors for xenotransplantation. However, PERV (Porcine Endogenous Retrovirus, PERV) in pigs could infect human cells in vitro, PERV TM protein is similar with HIV TM, presenting the function of inhibiting the activity of immune cells. Therefore, biosafety of porcine organs raised great safty concerns. The researchers have tried all kinds of defferent methods to remove the PERV from the donor pigs or its organ without desired results. If we can remove PERV or inhibit PERV replication using the pig`s own defence system, then the PERV concer in xenotransplantation biosafety risks can be effectively controlled. We know that there is a APOBEC- pA3F(Porcine APOBEC3F, pA3F) molecular in pigs body. In 2007, Jónsson SR reported that pA3F could not inhibit PERV, while D((?))rrschuck E in 2011 demonstrated human A3F and pA3F both could inhibit the replication of PERV. Here we would like study the function of pA3F in PERV replication and explore its possible mechanism:1. pA3F could inhibit the replication of PERV in vitro. Gain-of-function and loss-of-function analyses in PK-15cells were implemented. Experimental data showed that pA3F expression was in negative correlation with PERV pol mRNA and PERV reverse transcriptase activity. The most notable changes were observed in the 30μg transfection dose group of the pCDNA3-Blag/pA3F expression vector, the expression changes PERV mRNA and reverse transcriptase were down regulated by 0.252±0.032 fold and 6.85±0.69pg/well respectively when compared with the control group (p<0.05), and the results showed a significant dose dependent manner; By contrast, in pA3F knockdown cells PERV pol mRNA levels and PERV reverse transcriptase activity had been increased obviously compared with the control group (p<0.05) , and the most effectivly up regulation were observed in the 740-764bp(which is the location in the pA3F gene) siRNA sequence group, the changes were 4.172±0.251 fold and 88.58±1.46pg/well respectively. These results indicate that pA3F contributes to the inhibiting effect of PERV replication in pig cells.2. PERV-Gag is required for the antivirol activity of pA3FTo investigate the mechanism of the inhibiting effect of pA3F on PERV replication, we postulated that PERV-gag maybe required for pA3F viron packaging. Coimmunoprecipitation demonstrated the formation of pA3F/PERV-Gag complex. Confocal microscopy analysis indicated that these two proteins colocalize in PK-15 cells. More over, the virons harvested after pA3F transfection showed the presence of pA3F by immubloting method, take together, these results suggested that PERV-Gag is required for the virion package of pA3F and its antiviral activity.To sum up, here we found that pA3F could inhibit replication of PERV by viron pakege through PERV-gag. Modulation of pA3Fexpress may represent a novel target to remove PERV from pigs tissues and thus provide a resolution strafely to the sfety concern of pigs transplant to human.