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Molecular and enzymatic characterization of the porcine endogenous retrovirus protease.

ABSTRACT: 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.

SUBMITTER: Blusch JH

PROVIDER: S-EPMC136392 | biostudies-literature | 2002 Aug

REPOSITORIES: biostudies-literature

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Molecular and enzymatic characterization of the porcine endogenous retrovirus protease.

Blusch Jürgen H JH Seelmeir Sigrid S von der Helm Klaus K

Journal of virology 20020801 15

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 ...[more]

PMID: 12097607

Similar Datasets

Susceptibility of the porcine endogenous retrovirus to reverse transcriptase and protease inhibitors.

Project description:Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC(50)s and IC(90)s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC(50)s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culture-based, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC(50)s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine.

| S-EPMC114002 | biostudies-literature

Detection and characterization of porcine endogenous retrovirus in porcine plasma and porcine factor VIII.

Project description:The pig genome contains porcine endogenous retroviruses (PERVs) capable of infecting human cells. Detection of infectious retrovirus in porcine peripheral blood mononuclear cells and endothelial cells suggested to us that pig plasma is likely to contain PERV. Both PERV env sequences and viral reverse transcriptase (RT) activity were detected in all plasma samples isolated from four NIH minipigs. To detect infectious virus from plasma, we performed a culture assay using three cell lines of feline, swine, and human origin that had previously been shown to be permissive for PERV. Infectious virus was successfully cultured from all four NIH minipig plasmas on the swine cell line ST-IOWA. Using RT-PCR with env-specific primers, we could detect expression of PERV class C envelope in the supernatant of ST-IOWA cells that had been exposed to each pig plasma. We next examined a pig plasma derivative, Hyate:C (porcine factor VIII), and found evidence of PERV particles, since all six lots examined were positive for PERV RNA and RT activity. However, infectious virus could not be detected in clinical lots of Hyate:C, suggesting that the manufacturing process might reduce the load of infectious virus to levels below detectable limits of the assay. Detection of infectious virus in porcine plasma confirms and extends the previous findings that certain porcine cells express PERV when manipulated in vitro and clearly demonstrates that there are porcine cells that express infectious PERV constitutively in vivo.

| S-EPMC114208 | biostudies-literature

Characterization of porcine endogenous retrovirus gamma pro-pol nucleotide sequences.

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| S-EPMC136799 | biostudies-literature

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

Project description: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.

| S-EPMC1617276 | biostudies-literature

Characterization of porcine endogenous retrovirus clones from the NIH miniature pig BAC library.

Project description:Pigs have been considered as donors for xenotransplantation in the replacement of human organs and tissues. However, porcine endogenous retroviruses (PERVs) might transmit new infectious disease to humans during xenotransplantation. To investigate PERV integration sites, 45 PERV-positive BAC clones, including 12 PERV-A, 16 PERV-B, and 17 PERV-C clones, were identified from the NIH miniature pig BAC library. The analysis of 12 selected full-length sequences of PERVs, including the long terminal repeat (LTR) region, identified the expected of open reading frame length, an indicative of active PERV, in all five PERV-C clones and one of the four PERV-B clones. Premature stop codons were observed in only three PERV-A clones. Also, eleven PERV integration sites were mapped using a 5000-rad IMpRH panel. The map locations of PERV-C clones have not been reported before, thus they are novel PERV clones identified in this study. The results could provide basic information for the elimination of site-specific PERVs in selection of pigs for xenotransplantation.

| S-EPMC3168785 | biostudies-literature

Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases.

Project description:Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro, suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3. The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5' TGC for A3Z2 and A3Z2-Z3 and 5' CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo, thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation.

| S-EPMC3126140 | biostudies-literature

Evidence and consequence of porcine endogenous retrovirus recombination.

Project description: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.

| S-EPMC533951 | biostudies-literature

Regulation of porcine endogenous retrovirus release by porcine and human tetherins.

Project description:The risk of transmission of porcine endogenous retrovirus (PERV) is one of the major safety issues in xenotransplantation. Human tetherin, recently described as an antiviral protein able to inhibit the release of enveloped viruses, and its porcine homologue were shown to inhibit PERV release from producer cells, establishing themselves as candidate molecules to suppress PERV production in porcine xenografts by animal engineering.

| S-EPMC2820912 | biostudies-literature

Mouse retrovirus mediates porcine endogenous retrovirus transmission into human cells in long-term human-porcine chimeric mice.

Project description:Porcine endogenous retrovirus (PERV) is a potential pathogen in clinical xenotransplantation; transmission of PERV in vivo has been suggested in murine xenotransplantation models. We analyzed the transmission of PERV to human cells in vivo using a model in which immunodeficient NOD/SCID transgenic mice were transplanted with porcine and human lymphohematopoietic tissues. Our results demonstrate, we believe for the first time, that human and pig cells can coexist long-term (up to 25 weeks) without direct PERV infection of human cells. Despite the transplantation of porcine cells that did not produce human-tropic PERV, human cells from the chimeric mice were frequently found to contain PERV sequences. However, this transmission was due to the pseudotyping of PERV-C (a virus without human tropism) by xenotropic murine leukemia virus, rather than to de novo generation of human-tropic PERV. Thus, pseudotyping might account for the PERV transmission previously observed in mice. The absence of direct human cell infection following long-term in vivo coexistence with large numbers of porcine cells provides encouragement regarding the potential safety of using pigs that do not produce human-tropic PERV as source animals for transplantation to humans.

| S-EPMC514590 | biostudies-literature

Susceptibility of porcine endogenous retrovirus to anti-retroviral inhibitors.

Project description:BACKGROUND:Porcine endogenous retrovirus (PERV) is an endogenous retrovirus that poses a risk of iatrogenic transmission in the context of pig-to-human xenotransplantation. The lack of a means to control PERV infection in the context of pig-to-human xenotransplantation is a major concern in the field. In this study, we set out to evaluate the ability of currently licensed anti-HIV drugs, and other types of anti-retroviral compounds, to inhibit PERV infection in vitro. METHODS:We used target cells stably expressing one of the known PERV viral receptors, an infectious molecular clone, PERV-A 14/220, and at least one drug from each class of anti-retroviral inhibitors as well as off-label drugs shown to have anti-viral activities. The susceptibility of PERV-A 14/220 LacZ to the anti-retroviral drugs was determined from infected cells by histochemical staining. RESULTS:We extend the results of previous studies by showing that, in addition to raltegravir, dolutegravir is found to have a potent inhibitory activity against PERV replication (IC50 8.634 ±0.336 and IC50 3.06 ± 0.844 nM, respectively). The anti-HIV drug zidovudine (AZT) showed considerable anti-PERV activity with IC50 of 1.923 ±0.691 ?M as well. CONCLUSIONS:The study results indicate that some of the licensed anti-retroviral drugs may be useful for controlling PERV infection. However, the efficacy at nanomolar concentrations put forward integrase inhibitors as a drug that has the potential to be useful in the event that xenotransplantation recipients have evidence of PERV transmission and replication.

| S-EPMC4873160 | biostudies-literature

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