SUMMARY Xenotransplantation may be a solution to overcome the shortage of organs for the treatment of patients with organ failure, but it may be associated with the transmission of porcine microorganisms and the development of xenozoonoses. Whereas most microorganisms may be eliminated by pathogen-free breeding of the donor animals, porcine endogenous retroviruses (PERVs) cannot be eliminated, since these are integrated into the genomes of all pigs. Human-tropic PERV-A and -B are present in all pigs and are able to infect human cells. Infection of ecotropic PERV-C is limited to pig cells. PERVs may adapt to host cells by varying the number of LTR-binding transcription factor binding sites. Like all retroviruses, they may induce tumors and/or immunodeficiencies. To date, all experimental, preclinical, and clinical xenotransplantations using pig cells, tissues, and organs have not shown transmission of PERV. Highly sensitive and specific methods have been developed to analyze the PERV status of donor pigs and to monitor recipients for PERV infection. Strategies have been developed to prevent PERV transmission, including selection of PERV-C-negative, low-producer pigs, generation of an effective vaccine, selection of effective antiretrovirals, and generation of animals transgenic for a PERV-specific short hairpin RNA inhibiting PERV expression by RNA interference.

The human endogenous retrovirus family HTDV/HERV-K codes for the viral particles observed in teratocarcinoma cell lines. Two types of proviral genomes exist; these differ in the presence or absence of a stretch of 292 nucleotides. This sequence comprises the amino-terminal part of the env gene, the putative signal peptide, which overlaps in part with the carboxy terminus of the pol gene. Type 2 genomes containing this sequence presumably more closely reflect the structure of the infectious, replication-competent retrovirus ancestors of the HERV-K family than do type 1 genomes that lack the sequence. In human teratocarcinoma cell lines, both variants are expressed. Type 1 genomes, in which pol and env genes are fused, are deficient in splicing. Type 2 transcripts are spliced to subgenomic env mRNA and smaller messages. A doubly spliced transcript encodes a short open reading frame, preliminarily designated cORF (R. Löwer, K. Boller, B. Hasenmeier, C. Korbmacher, N. Mueller-Lantzsch, J. Löwer, and R. Kurth, Proc. Natl. Acad. Sci. USA 90:4480-4484). The genomic organization of cORF resembles that of nonprimate lentivirus rev genes: the first exon comprises nearly the entire signal peptide of env, and the second exon is derived from a different reading frame in the 3 part of the genome. A nucleolar localization signal, which is also a putative RNA binding domain, as well as a sequence with similarities to the Rev effector domain consensus sequence is present in the first exon. Secondary structure analysis reveals similarities to basic helix-loop-helix proteins. cORF is a small protein with an apparent molecular mass of 14 kDa which accumulates in the nucleolus as has been described for Rev proteins.

The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.

Of all human endogenous retroviruses known today, HERV-K is the only one that has been shown to produce viral particles. While the first of the approximately 30 HERV-K sequences integrated into the human genome more than 40 million years ago, evidence is accumulating that HERV-K was active more recently, provirus HERV-K113 being the youngest sequence found. However, it is unclear which HERV-K sequences code for the viral particles that are produced by human germ-cell tumours or melanomas. Here, we show that the provirus HERV-K113, cloned into a baculovirus expression vector, is capable of producing intact particles of retroviral morphology, exhibiting the typical structure of those particles that were characterized in cell lines derived from human germ-cell tumours. Thus, the HERV-K113 sequence is a candidate for particle production in vivo and for an active human endogenous retrovirus of today.

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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.

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