Introduction: Being on the verge of clinical application xenotransplantation (XTx) using pigs as organ donors experienced a tremendous progress. However, XTx still bears a zoonotic risk of which the potential transmission of porcine endogenous retroviruses (PERV) needs to be surveyed. Despite the great efforts to eliminate this risk e.g. by the selection of PERV-C free pigs with low expression of possibly functional PERV-A/-B and by the genome-wide inactivation of PERV using CRISPR/Cas9, the effect of superinfection resistance (SIR) was not investigated. SIR features a viral characteristic to prevent reinfection (superinfection). In the case of PERV, the underlying mechanism is unclear. It has not been determined whether cells harboring functional PERV are protected and how it works e.g. by downregulation of its host receptor or by expression of proviral genes and assembly of viral particles. In this study, we investigated SIR for PERV-C in a newly developed in vitro model to pursue the mechanism and to show its protective effect.

Method: We have designed three PERV-C constructs based on the native PERV-C(5683) virus carrying a hemagglutinin (HA) tag at different positions of the env gene (SP-HA, HA-VRA, and RPep-HA). The recombinant PERV-C viruses were functionally characterized by analyses of RNA and Env protein expression, reverse transcriptase activity, and infectivity.

HA-tagged viruses that were found to be functional were used in superinfection experiments. Here, ST-IOWA cells positive for PERV-C(5683) were challenged with the HA-tagged PERVs. Subsequently, the challenged cells were analyzed for eight weeks for signs of SI using the HA-tag as a marker.

Results: It was shown that two of three viral constructs, SP-HA and RPep-HA, were comparable to the PERV-C(5683) wild-type virus, whereas HA-VRA did not show replication competence. The challenge experiment revealed that PERV-C-HA viruses are not able to superinfect PERV-C(5683) producing ST-IOWA cells. They neither integrate into the genome nor show any signs of PERV-C expression. PERV-C(5683) positive cells are resistant against novel PERV‑C infection.

Conclusions: The mechanism of SIR applies to PERV. Hence, targeted viral expression or production of defective PERV may serve as a protection mechanism to prevent reinfection of cells and possibly donor animals with exogenous PERV. In addition, PERV-C-HA may be used as a novel tool for further in vitro and in vivo infection studies gaining knowledge into viral invasion and pathogenesis e.g. host cell reservoir that will contribute to the virological safety regarding PERV in the course of XTx.