Carolyn A. Wilson

Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus

ABSTRACT We previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082–3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.

Porcine Endogenous Retrovirus Transmission Characteristics of an Inbred Herd of Miniature Swine

ABSTRACT Here we report the identification of inbred miniature swine that failed to produce human-tropic replication-competent porcine endogenous retroviruses (HTRC PERVs), using in vitro coculture assays. When HTRC PERVs were isolated from transmitting animals, all were recombinant viruses, with the receptor-binding domain of PERV-A combining with PERV-C-related sequences.

Porcine endogenous retrovirus infects but does not replicate in nonhuman primate primary cells and cell lines.

ABSTRACT Porcine endogenous retroviruses (PERV) can infect human cell lines in vitro; hence, there is a presumed risk of viral exposure to a recipient when pig cells are transplanted into humans (xenotransplantation). Nonhuman primates (NHP) are considered a potential permissive animal model to study the risk of in vivo infection of PERV after xenotransplantation. We set out to determine whether PERV can infect and replicate in NHP primary cells or established cell lines from African green monkey, rhesus macaque, and baboon. We confirm that the NHP cell lines under investigation were infected with PERV as measured by detection of viral DNA and RNA by PCR and reverse transcription (RT)-PCR, respectively, indicating that a functional receptor must be present on the cell surface. However, the load of detectable viral DNA in infected NHP cells declined over time, and the cells never had detectable reverse transcriptase activity. Utilizing quantitative real-time TaqMan PCR we found detectable levels of unintegrated DNA intermediates, but the levels were approximately 100-fold lower compared to HEK 293 cells infected with PERV. Virions released from infected NHP cells could productively infect naïve human cell lines, HEK 293 and HeLa, as shown by RT-PCR and RT assay. However, naïve NHP cells remained negative in RT-PCR and RT assay after exposure to virions from infected NHP cells. Together our data demonstrate that NHP cells are not permissive to productive replication by PERV, presumably due to inefficient cell entry and replication. In light of these observations, the appropriateness of NHP as suitable animal models to study PERV infection in vivo needs to be reevaluated.

Mice Transgenic for a Human Porcine Endogenous Retrovirus Receptor Are Susceptible to Productive Viral Infection

ABSTRACT Porcine endogenous retrovirus (PERV) is considered one of the major risks in xenotransplantation. No valid animal model has been established to evaluate the risks associated with PERV transmission to human patients by pig tissue xenotransplantation or to study the potential pathogenesis associated with PERV infection. In previous work we isolated two genes encoding functional human PERV receptors and proved that introduction of these into mouse fibroblasts allowed the normally nonpermissive mouse cells to become productively infected (T. A. Ericsson, Y. Takeuchi, C. Templin, G. Quinn, S. F. Farhadian, J. C. Wood, B. A. Oldmixon, K. M. Suling, J. K. Ishii, Y. Kitagawa, T. Miyazawa, D. R. Salomon, R. A. Weiss, and C. Patience, Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). In the present study we created mice transgenic for human PERV-A receptor 2 (HuPAR-2). After inoculation of transgenic animals with infectious PERV supernatants, viral DNA and RNA were detected at multiple time points, indicating productive replication. This establishes the role of HuPAR-2 in PERV infection in vivo; in addition, these transgenic mice represent a new model for determining the risk of PERV transmission and potential pathogenesis. These mice also create a unique opportunity to study the immune response to PERV infection and test potential therapeutic or preventative modalities.

Detection and Characterization of Porcine Endogenous Retrovirus in Porcine Plasma and Porcine Factor VIII

ABSTRACT 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.

Human CD59 Incorporation into Porcine Endogenous Retrovirus Particles: Implications for the Use of Transgenic Pigs for Xenotransplantation

ABSTRACT Transgenic pigs have been engineered to express human CD59 (hCD59) in order to suppress hyperacute rejection of xenotransplants in human recipients. In this study, porcine endogenous retrovirus (PERV) was produced in a porcine cell line expressing hCD59 in order to examine the effect of this complement control protein on PERV neutralization by human sera. hCD59 was found to be incorporated into PERV particles produced from engineered ST-IOWA cells. PERV incorporation of hCD59 resulted in a dramatic inhibition of complement-mediated virolysis by human serum. However, incorporation of hCD59 had no effect on neutralization of PERV by human serum, as measured in infectivity assays. Our results suggest that the use of organs from hCD59 transgenic pigs will inhibit complement-mediated virolysis, but will not compromise the protective effects of human sera on the neutralization of PERV particles.

Pseudotyping of Porcine Endogenous Retrovirus by Xenotropic Murine Leukemia Virus in a Pig Islet Xenotransplantation Model

The potential of porcine endogenous retrovirus (PERV) as a human pathogen, particularly as a public health risk, is a major concern for xenotransplantation. In vitro PERV transmission to human cells is well established. Evidence from human/pig hematopoietic chimeras in immunodeficient mice suggests PERV transmission from pig to human cells in vivo. However, recently Yang et al. demonstrated in such a model that PERV‐C, a nonhuman‐tropic class, could be transmitted via pseudotyping by xenotropic murine leukemia virus (X‐MLV). We developed a mouse pig islet xenotransplant model, where pig and human cells are located in physically separate compartments, to directly assess PERV transmission from a functional pig xenograft. X‐MLV efficiently pseudotypes all three classes of PERV, including PERV‐A and ‐B that are known to productively infect human cell lines and PERV‐C that is normally not infectious for human cells. Pseudotyping also extends PERVs natural tropism to nonpermissive, nonhuman primate cells. X‐MLV is activated locally by the surgical procedure involved in the tissue transplants. Thus, the presence and activation of endogenous X‐MLV in immunodeficient mice limits the clinical significance of previous reports of in vivo PERV transmission from pig tissues to human cells.

Sequence Analysis of Porcine Endogenous Retrovirus Long Terminal Repeats and Identification of Transcriptional Regulatory Regions

ABSTRACT Porcine cells express endogenous retroviruses, some of which are infectious for human cells. To better understand the replication of these porcine endogenous retroviruses (PERVs) in cells of different types and animal species, we have performed studies of the long terminal repeat (LTR) region of known gammaretroviral isolates of PERV. Nucleotide sequence determination of the LTRs of PERV-NIH, PERV-C, PERV-A, and PERV-B revealed that the PERV-A and PERV-B LTRs are identical, whereas the PERV-NIH and PERV-C LTRs have significant sequence differences in the U3 region between each other and with the LTRs of PERV-A and PERV-B. Sequence analysis revealed a similar organization of basal promoter elements compared with other gammaretroviruses, including the presence of enhancer-like repeat elements. The sequences of the PERV-NIH and PERV-C repeat element are similar to that of the PERV-A and PERV-B element with some differences in the organization of these repeats. The sequence of the PERV enhancer-like repeat elements differs significantly from those of other known gammaretroviral enhancers. The transcriptional activities of the PERV-A, PERV-B, and PERV-C LTRs relative to each other were similar in different cell types of different animal species as determined by transient expression assays. On the other hand, the PERV-NIH LTR was considerably weaker in these cell types. The transcriptional activity of all PERV LTRs was considerably lower in porcine ST-IOWA cells than in cell lines from other species. Deletion mutant analysis of the LTR of a PERV-NIH isolate identified regions that transactivate or repress transcription depending on the cell type.

Comparison of cDNAs encoding the gibbon ape leukaemia virus receptor from susceptible and non-susceptible murine cells

The gibbon ape leukaemia virus (GaLV) family of type C retroviruses consists of five closely related viral isolates, GaLV SF, GaLV SEATO, GaLV Br, GaLV H and simian sarcoma-associated virus. The cDNA encoding the human receptor for GaLV SEATO had previously been isolated. We now demonstrate that all of the above GaLVs can use the human form of the GaLV receptor to infect cells. All murine cells analysed to date have been found to be resistant to infection by GaLVs owing to the absence of a functional GaLV receptor. We have now identified a murine cell line which is unique in its susceptibility to GaLV infection. This cell line was established from a Japanese feral mouse, Mus musculus molossinus. We cloned and sequenced the cDNA for the receptor expressed in these cells and compared it to the cDNA for the GaLV receptor expressed in resistant murine cells such as NIH 3T3 (derived from M. m. musculus) and MDTF (derived from M. dunni tail fibroblasts). The crucial region for GaLV infection (the fourth extracellular domain) from the functional M. m. molossinus GaLV receptor is quite divergent from the same region of the M. m. musculus and M. dunni proteins, but similar to that of the functional human GaLV receptor. These results confirm the importance of the amino acids of this region in GaLV receptor function.

Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus.

Real time PCR technology was applied to the development of assays for detection and quantitation of porcine endogenous retrovirus (PERV) RNA and DNA sequences in tissues and cells of human or animal origin. A plasmid construct encoding the PERV-pol gene or the in vitro transcribed RNA derived from the plasmid (cRNA) serves as a standard template for amplification of a 178 bp fragment. This study showed that the detection of this target sequence was linear over a range from 20 copies to 2 million copies of the plasmid and from 100 copies to 1 million copies of the cRNA. In addition, amplification of the target sequence was not inhibited by the presence of exogenous genomic DNA. These results demonstrate that a real time (TaqMan-based) PCR or RT-PCR assay can provide a sensitive, reproducible, and robust method for detecting and quantifying PERV DNA or RNA sequences in samples of human or guinea pig origin.