The number of a U3 repeat box acting as an enhancer in long terminal repeats of polytropic replication-competent porcine endogenous retroviruses dynamically fluctuates during serial virus passages in human cells.

The organization and transcriptional regulation of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) are unknown. We have studied the activity of LTRs from replication-competent molecular clones by performing luciferase reporter assays. The LTRs differ in the presence and number of 39-bp repeats located in U3 that confer strong promoter activity in human, simian, canine, feline, and porcine cell lines, whereas for LTRs devoid of the repeats, the promoter strength was significantly reduced. As the activity of a heterologous simian virus 40 promoter and a homologous repeat-deficient LTR was elevated by four 39-bp repeats independently of its orientation and location, the repeat box complies with the definition of an enhancer. During serial virus passaging of molecular PERV clones on human 293 cells, proviral LTRs demonstrated adaptation of transcriptional activity by dynamic changes of the number of 39-bp repeats in the course of up to 12 passaging cycles.

Comparison of replication-competent molecular clones of porcine endogenous retrovirus class A and class B derived from pig and human cells.

Vertically transmitted endogenous retroviruses pose an infectious risk in the course of pig-to-human transplantation of cells, tissues, and organs. Two classes of polytropic type C porcine endogenous retroviruses (PERV) which are infectious for human cells in vitro are known. Recently, we described the cloning and characterization of replication-competent PERV-B sequences from productively infected human cells (F. Czauderna, N. Fischer, K. Boller, R. Kurth, and R. R. Tönjes, J. Virol. 74:4028-4038, 2000). Here, we report the isolation of infectious molecular PERV-A and PERV-B clones from pig cells and compare these proviruses with clones derived from infected human 293 cells. In addition to clone PERV-A(42) derived from 293 cells, four "native" full-length proviral PERV sequences derived from a genomic library of the porcine cell line PK15 were isolated. Three identical class A clones, designated PK15-PERV-A(42), PK15-PERV-A(45), and PK15-PERV-A(58), and one class B clone, PK15-PERV-B(213), were characterized. PK15-PERV-B(213) is highly homologous but distinct from the previously described clone PERV-B(43). PK15-PERV-A(58) demonstrates close homology to PERV-A(42) in env and to PERV-C in long terminal repeat, gag, and pro/pol sequences. All three PERV clones described here were replication competent upon infection of susceptible cell lines. The findings suggest that the pig genome harbors a limited number of infectious PERV-A and -B sequences.

Generation and testing of a highly specific anti-serum directed against porcine endogenous retrovirus nucleocapsid.

Advances in xenotransplantation offer chances to alleviate the shortage of human donor organs. The discovery that pig endogenous retroviruses (PERV) can infect human cells in vitro has stimulated the discussion on infectious risk in xenotransplantation. A molecular and immunologic monitoring of xenograft recipients and of donor animals for putative infection with PERV and other microorganisms is inevitable. In this report, we describe the generation and testing of a highly specific anti-serum directed against the PERV nucleocapsid protein. The Gag amino acid (aa) sequence of PERV class B was used to define immunogenic domains by computer analysis. A peptide corresponding to the C-terminal 19 aa of the 10 kDa (p10) nucleocapsid (NC) portion of the Gag polyprotein was used to immunize rabbits. The generated serum was tested using recombinant PERV Gag protein expressed in insect cells, purified PERV virus particles and human 293 cells transfected or infected with PERV, respectively. Test methods included Western blotting, indirect immunofluorescence, immunoperoxidase assay and ELISA. The PERV anti-serum provides a tool that is instrumental for detection of a potential agent of zoonosis. It can be used for screening of donor animals and xenograft recipients in the course of xenotransplantation procedures.