Preliminary Study On Interaction Between Porcine Transferrin Receptor And PPV-VP2

Transferrin receptor(TfR), which locates on the cell membrane, is a transmembrane protein andcan transport the iron ions with transferrin in biological fluids into cells. Porcine parvovirus(PPV),which belongs to parvovirus genus, consists by a linear single-stranded 5000 nucleotides negative DNAgenome. PPV encodes three structural proteins(VP1, VP2 and VP3) and three non-structural proteins(NS1, NS2 and NS3). Porcine parvovirus VP2 accounts for about 80% of the virus capsid and canself-assemble virus-like particles.The initiation process of virus replication starts with the combinationwith the cell surface receptors, which is also one of the crucial factors that affect virus host specificity,pathogenicity and tissue tropism. Researches have confirmed that canine parvovirus virus(CPV) andfeline panleukopenia virus(FPV), which belong to the same family with PPV, could infect cells throughthe combination with cell surface TfR by VP2. Although the capsid protein amino acid sequences arenot identical of CPV and PPV, there are high homology, about 50~60%. Consequently, it is possibly thatPPV VP2 could combine with porcine TfR. However, there is little clear evidence that the transferrinreceptor is a receptor for porcine parvovirus, and indeed some evidence suggests that the transferrinreceptor is not a receptor for PPV. In order to clarify whether TfR is the receptor of PPV. Thisexperiment prepared some elementary work, including the establishment of quantitative real-time PCRassays, preliminary identification of protein interactions and HEK293 cell line stably expressing TfRand so on.The real-time PCR assays based on SYBR Green for detection PPV VP2, TfR and β-actin wereestablished in this study. The results indicated that the established assays were highly specific andsensitive with a detection limit of less than 10 copies, and the coefficient of variation were less than 3%.The assays with high sensitivity, specificity and reproducibility could be used to detect and quantifyPPV and TfR. The established assays were used to detect the proliferation of PPV and mRNA of TfRtranscript level in ST cells infected with PPV. The results showed that a certain amount of TfR wereused for PPV to self-proliferate in ST cells and the consumption characteristics of TfR was consistentwith the characteristics of potential cell surface receptors of PPV.Co-immunoprecipitation is an effective method to study protein-protein interactions. Recombinantplasmids targeting for VP2 and TfR were constructed in this study. And confocal microscopy confirmedthat TfR could colocalize with the PPV VP2 protein in the cell membrane. Co-immunoprecipitation wasused to verify whether there exist the interaction between the two protein. Results indicated that: there isno direct interaction between the two protein, or do not have the interaction.To further explore the interaction between VP2 and TfR, HEK293 cell line stably expressing TfRwas constructed and the results showed that integration efficiency of TfR gene in this cell line was morethan 90% and the expression efficiency of TfR protein was also high. However, HEK293-TfR cell linecouldn’t be infected with PPV.The above results show that TfR couldn’t serve as a cell surface receptor of PPV. The cell surfacereceptor of PPV remains to be further studied.