An effective method for the quantitative detection of porcine endogenous retrovirus in pig tissues

Xenotransplantation shows great promise for providing a virtually limitless supply of cells, tissues, and organs for a variety of therapeutical procedures. However, the potential of porcine endogenous retrovirus (PERV) as a human-tropic pathogen, particularly as a public health risk, is a major concern for xenotransplantation. This study focus on the detection of copy number in various tissues and organs in Banna Minipig Inbreed (BMI) from 2006 to 2007 in West China Hospital, Sichuan University. Real-time quantitative polymerase chain reaction (SYBR Green I) was performed in this study. The results showed that the pol gene had the most copy number in tissues compared with gag, envA, and envB. Our experiment will offer a rapid and accurate method for the detection of the copy number in various tissues and was especially suitable for the selection of tissues or organs in future clinical xenotransplantation.     

Evidence and consequence of porcine endogenous retrovirus recombination

The genetic nature and biological effects of recombination between porcine endogenous retroviruses (PERV) were studied. An infectious molecular clone was generated from a high-titer, human-tropic PERV isolate, PERV-A 14/220 (B. A. Oldmixon, et al. J. Virol. 76:3045-3048, 2002; T. A. Ericsson et al. Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). To analyze this sequence and 15 available full-length PERV nucleotide sequences, we developed a sequence comparison program, LOHA(TM) to calculate local sequence homology between two sequences. This analysis determined that PERV-A 14/220 arose by homologous recombination of a PERV-C genome replacing an 850-bp region around the pol-env junction with that of a PERV-A sequence. This 850-bp PERV-A sequence encompasses the env receptor binding domain, thereby conferring a wide host range including human cells. In addition, we determined that multiple regions derived from PERV-C are responsible for the increased infectious titer of PERV-A 14/220. Thus, a single recombination event may be a fast and effective way to generate high-titer, potentially harmful PERV. Further, local homology and phylogenetic analyses between 16 full-length sequences revealed evidence for other recombination events in the past that give rise to other PERV genomes that possess the PERV-A, but not the PERV-B, env gene. These results indicate that PERV-A env is more prone to recombination with heterogeneous backbone genomes than PERV-B env. Such recombination events that generate more active PERV-A appear to occur in pigs rather frequently, which increases the potential risk of zoonotic PERV transmission. In this context, pigs lacking non-human-tropic PERV-C would be more suitable as donor animals for clinical xenotransplantation.     

Immunological relationships of an endogenous guinea pig retrovirus with prototype mammalian type B and type D retroviruses.

A retrovirus endogenous to guinea pig cells was earlier shown to be morphologically similar to type B and type D prototype retroviruses. Molecular hybridization techniques were used to show that guinea pig virus nucleotide sequences are endogenous to both domestic (Cavia porcellus) and indigenous (Cavia aperea) guinea pigs, but cannot be detected in the DNA of either other hystricomorph rodents or other mammals tested. Using radioimmunological techniques designed to detect interspecies relationships, the major internal polypeptide of guinea pig virus (p26) was shown to share three different sets of interspecies antigenic determinants with squirrel monkey retrovirus, viper retrovirus, and mouse mammary tumor virus. Thus, guinea pig virus appears to provide an evolutionary link between type B and D retroviruses.     

Molecular and enzymatic characterization of the porcine endogenous retrovirus protease

The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M(r) of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.     

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.A better understanding of the cellular and molecular basis of transplant rejection and the generation of transgenic donor animals bearing genes that mediate protection towards rejection (3, 24, 25) have stimulated approaches to use xenotransplantation, i.e., the therapeutic use of animal cells, tissues, and organs, to overcome the shortage of allogeneic transplants (7). Pigs are preferred as donors for xenotransplants (10).Major concerns have been raised about the possibility of introducing new microbial agents from the animal into the recipient, leading to xenozoonosis (2, 11, 18, 27). Viruses that are germ line transmitted, i.e., porcine endogenous retroviruses (PERV) (21), and DNA viruses that can persist without symptoms in their natural host and are transmitted via intrauterine or transplacentar pathways, e.g., herpesviruses (8), are of particular interest.Approximately 50 integration sites of PERV exist in the genomes of different pig breeds (1, 14, 21), and at least three classes are known (14, 28). Those classes, named PERV-A, -B, and -C (PERV-C is also known as PERV-MSL), display high sequence homology in the genes for group-specific antigens (gag) and polymerase (pol) but differ in the envelope (env) genes which determine the host range. In addition, the existence of multiple other PERV sequences in domestic pigs and their phylogenetic relatives has been described. However, only classes A, B, and C appear to be infectious (22).PERV that are released from different pig cell lines are able to infect human cells in vitro (15, 32, 33). PERV-C (1) is ecotropic compared to PERV-A and PERV-B, which are polytropic as deduced from pseudotype experiments utilizing the corresponding env genes (28).A retrospective investigation of 160 patients who had been treated with porcine cells and tissues showed no evidence for transmission of PERV (20); however, no long-term transplantation of a whole vascularized organ has been attempted so far. In contrast, a recent study utilizing NOD/SCID mice revealed PERV infection in several tissue compartments after transplantation of pig pancreatic islets, indicating the xenozoonotic potential of those retroviruses (31).Recently, we have reported the isolation of replication-competent PERV-B molecular clones derived from human embryonic kidney cells infected with PERV (293 PERV-PK) (5). In this communication, we describe the cloning and characterization of PERV-A and PERV-B proviral sequences derived from the porcine kidney cell line PK15 as well as the characterization of the molecular clone PERV-A(42); isolated from 293 PERV-PK cells (5). [Hereafter, clones derived from cell line 293 PERV-PK will be designated 293-PERV-B(33), 293-PERV-B(43), and 293-PERV-A(42); clones derived from cell line PK15 will be designated PK15-PERV-A(58), and so on.] Three proviruses, one PERV-B and two PERV-A clones, produce infectious and replication-competent particles upon transfection of susceptible cells and subsequent infection of different human cell lines. Thus, this study provides the first functional PERV-A and PERV-B clones isolated directly from the pig genome and allows the comparison of proviral PERV sequences from different origins at the molecular and cellular level.     

Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells

Galactose alpha1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.     

Characterization of two porcine endogenous retrovirus integration loci and variability in pigs

The pig (Sus scrofa) is a potential organ donor for man but porcine endogenous retroviruses (PERVs) represent an important concern for patients, and identification or engineering of PERV-free pigs suitable for xenotransplantation is a major undertaking. Consequently, studies of variability in pigs for the presence of PERVs at specific loci are a prerequisite. We identified genomic flanking sequences of two PERVs cloned in bacterial artificial chromosomes, a replication-competent PERV-A at locus 1q2.4 and a defective PERV-B at locus 7p1.1–2. PERV-A is embedded in the second repeat of a tandem of eight 190 bp repeats. A short duplicated 4 bp cellular motif, AGAC, was found at each flank of PERV-A and a degenerate 4 bp motif was found for PERV-B. At each locus, the PERV flanks matched expressed sequence tags available in public databases. Primer pairs were designed to amplify either genomic flanks or PERV-genomic junctions. Polymerase chain reaction screening was performed on pigs from 11 distinct Chinese breeds and from the European Large White breed. PERV-B at locus 7p1.1–2 was detected in all animals whereas the presence of PERV-A at locus 1q2.4 was variable. Our results suggest that a genetic selection can be designed to identify animals lacking a potentially active PERV at a specific locus and that Chinese and European pig breeds represent large biodiversity reservoirs to explore. Our results point also to the existence of PERVs that might be fixed in the pig genome, and that might not be eliminated by classical genetic selection.Accession numbers: Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under Accession numbers AY160111–AY160114     

The trans-activator gene of the human T cell lymphotropic virus type III is required for replication

The trans-activator gene (tat-III) of the human T lymphotropic virus type III (HTLV-III/LAV) is shown to regulate positively the expression of viral proteins. Viruses in which the tat-III gene is deleted are incapable of prolific replication and do not demonstrate cytopathic effects in T4+ cell lines. These defects can be fully complemented in cell lines that constitutively express the tat-III gene product. We conclude that the tat-III gene product is required for efficient replication of HTLV-III in T4+ cells, and for that reason is important for the cytopathic effects of virus infection. These observations predict that inhibitors of the tat-III gene product may constitute effective therapeutic agents.     

Complete nucleotide sequence of simian endogenous type D retrovirus with intact genome organization: evidence for ancestry to simian retrovirus and baboon endogenous virus.

A complete endogenous type D viral genome has been isolated from a baboon genomic library. The provirus, simian endogenous retrovirus (SERV), is 8,393 nucleotides long and contains two long terminal repeats and complete genes for gag, pro, pol, and env. The primer binding site is complementary to tRNA(Lys)3, like in lentiviruses. The env GP70 protein is highly homologous to that of baboon endogenous virus (BaEV). PCR analysis of primate DNA showed that related proviral sequences are present in Old World monkeys of the subfamily Cercopithecinae but not in apes and humans. Analysis of virus and host sequences indicated that the proviral genomes were inherited from a common ancestor. Comparison of the evolution of BaEV, exogenous simian retrovirus types 1 to 3 (SRV1 to SRV3), and SERV suggests that SERV is ancestral to both BaEV and the SRVs.     

Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1

The replication proteins Rep and Rep' of porcine circovirus type 1 (PCV1) are both capable of introducing and resealing strand discontinuities at the viral origin of DNA replication in vitro underlying genome amplification by rolling-circle replication. The PCV1 origin of replication encompasses the minimal binding site (MBS) of the Rep and Rep' proteins and an inverted repeat with the potential to form a stem-loop. In this study, both elements of the PCV1 origin were demonstrated to be essential for viral replication in transfected cells. Furthermore, investigation of conserved amino acid motifs within Rep and Rep' proteins revealed that the mutation of motifs I, II, and III and of the GKS box interfered with viral replication. In vitro studies demonstrated that motifs I to III were essential for origin cleavage, while the GKS box was dispensable for the initiation of viral replication. A covalent link between Rep/Rep' and the DNA after origin cleavage was demonstrated, providing a mechanism for energy conservation for the termination of replication.     

The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag

The sheep genome harbors approximately 20 endogenous retroviruses (enJSRVs) highly related to the exogenous Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, acts as a unique restriction factor by blocking JSRV in a transdominant fashion at a late stage of the retroviral cycle. To better understand the molecular basis of this restriction (termed JLR, for JSRV late restriction), we functionally characterized JSRV and enJS56A1 Gag proteins. We identified the putative JSRV Gag membrane binding and late domains and determined their lack of involvement in JLR. In addition, by using enJS56A1 truncation mutants, we established that the entire Gag protein is necessary to restrict JSRV exit. By using differentially tagged viruses, we observed, by confocal microscopy, colocalization between JSRV and enJS56A1 Gag proteins. By coimmunoprecipitation and molecular complementation analyses, we also revealed intracellular association and likely coassembly between JSRV and enJS56A1 Gag proteins. Interestingly, JSRV and enJS56A1 Gag proteins showed distinct intracellular targeting: JSRV exhibited pericentrosomal accumulation of Gag staining, while enJS56A1 Gag did not accumulate in this region. Furthermore, the number of cells displaying pericentrosomal JSRV Gag was drastically reduced in the presence of enJS56A1. We identified amino acid residue R21 in JSRV Gag as the primary determinant of centrosome targeting. We concluded that JLR is dependent on a Gag-Gag interaction between enJS56A1 and JSRV leading to altered cellular localization of the latter.     

Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C

Xenotransplantation of pig organs is complicated by the existence of polytropic replication-competent porcine endogenous retroviruses (PERV) capable of infecting human cells. The potential for recombination between ecotropic PERV-C and human-tropic PERV-A and PERV-B adds another level of infectious risk. Proviral PERV-C were characterized in MAX-T cells derived from d/d haplotype miniature swine. Three proviruses were cloned from a genomic library. Clone PERV-C(1312) generated infectious particles after transfection into porcine ST-IOWA cells. Electron microscopy revealed the same morphologies of virions in MAX-T cells and in ST-IOWA cells infected with cell-free PERV-C(1312) particles, indicating that MAX-T cells harbor one functional PERV-C provirus.     

Survival of early preimplantation porcine embryos after co-culture with cells producing an avian retrovirus

To determine the relative survival of porcine embryos after co-culture with cells producing an avian retrovirus, four-cell stage embryos were obtained from sows following synchronization with altrenogest and superovulation with gonadotropins. These embryos were randomly assigned to the following treatments: no manipulation (zona-intact); zona removed with acidified Tyrode's solution (zona-free); and zona removed followed by co-culture with D-17 canine cells producing an avian retrovirus vector derived from spleen necrosis virus (zona-free + co-culture). The survival rates of four-cell stage embryos to morulae or early blastocysts during a 48-h culture period were 93.3, 80.0 and 57.7% in zona-intact, zona-free and zona-free + co-culture groups, respectively. Following embryo transfer, the development of embryos to fetuses at six weeks of gestation was 37.5, 30.0 and 11.7% in zona-intact, zona-free and zona-free + co-culture groups. These results indicate that early preimplantation porcine embryos can develop to apparently normal fetuses following co-culture with cells producing a retrovirus, and the feasibility of this method for retrovirus-mediated gene transfer in pigs was demonstrated.