Long-Term Effects on HEK-293 Cell Line After Co-Culture with Porcine Endogenous Retrovirus

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Long-Term Effects on HEK-293 Cell Line After Co-Culture With Porcine Endogenous Retrovirus

P. Yu, L. Zhang, S.F. Li, Y.P. Li, J.Q. Cheng, Y.R. Lu, and H. Bu

ABSTRACT Xenotransplantation of pig organs, tissues, and cells bears the risk of interspecies transmission of porcine endogenous retrovirus (PERV). To evaluate the long-term effect of PERV infection on human cells, human embryonic kidney cell line HEK-293 cells were co-cultured with PERV produced by the porcine kidney PK15 cell line for 24 hours and the infected HEK-293 cells were continually cultured for 6 months. PERV-gag, pol gene and gag protein were detected in infected HEK-293 cells by PCR and immunofluorescent staining. PERV from the supernatant of infected HEK-293 cells was same as that from PK15 in morphology. The concentration of reverse transcriptase in the supernatant of infected HEK-293 cells was almost 200 times lower than that of PK15 cells. Except that infected HEK-293 cells doubled a little earlier than the control and infected cells grew in serum-free medium poorly, further study for cell morphology and growth showed no significant difference between infected HEK-293 cells and uninfected control. These results suggested although PERV from PK15 could infect human cells in vitro, there was no significant acute effect attributable to PERV infection on the growth of HEK-293 cells by 6 months culture.

N RECENT YEARS the number of candidates waiting France) to remove the cell debris. Then the inoculum was used to I for organ transplantation has greatly outpaced the infect HEK-293 cells. After co-culture for 24 hours the infection supply of human organs available, which leads to a renewed supernatant in the culture medium of HEK-293, cells were re- interest in pig-to-human xenotransplantation as an alterna- moved and replaced with the complete medium. Then the infected tive.1 However, safety concerns over the risk of xenozoono- HEK-293 cells grew in the complete medium for 6 months and sis have raised because of the potential risk of zoonotic subcultured every 3 days. transmission of porcine endogenous retrovirus (PERV) from pig to human in xenotransplantation.2,3 Immunoelectron Microscopy PERV are proviruses, inherited in a stable Medelian fashion.4 Although a lot of experiments demonstrate the The supernatant of PK15 cells and infected HEK-293 cells was successful infection of human cells, as well as nonhuman harvested to examine the morphology of PERV by electron primate cells by PERV,5,6 the long-term effect of PERV microscopy (EM). Rabbit anti-PERV-gag antibody (kindly pro- infection on human cells is rarely reported. In this study, vided by Paul-Ehrlich-Institute, Germany) was used to concentrate PERV DNA, mRNA, and PERV-gag protein in the in- viral pellets. fected human cells were all detected. Some biological aspects of infected human cells after 6 months infection were further evaluated. From Key Laboratory of Transplant Engineering and Immunol- ogy, Ministry of Health, West China Hospital, Sichuan University, MATERIALS AND METHODS PR China. Cell Culture and PERV Infection Supported by the Natural Science Foundation of China (Grant Pig kidney cell line PK15 cells (ATCC, CCL-33) and human No. 30070725). embryonic kidney cell line HEK-293 cells (ATCC, CRL-1573) were Address reprint requests to Hong Bu, Key Laboratory of cultured in DH containing 10% new bovine serum (NBS). The Transplant Engineering and Immunology, Ministry of Health, culture supernatant of PK15 cells was harvested after 3 days culture West China Hospital, Sichuan University, 610041, PR. China. and passed through a 0.45-␮m low protein-binding ﬁlter (Milipore, E-mail: [email protected]

496 Transplantation Proceedings, 37, 496–499 (2005) 转载中国科技论文在线 http://www.paper.edu.cn

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RT Assay Detection of RT Activity Detection of PERV reverse transcriptase in the supernatant of The average concentrations of reverse transcriptase were PK15 cells, infected and uninfected HEK-293 cells employed a 221 pg/mL, 1.3 pg/mL, or 0 pg/mL in the supernatant of commercial reverse transcriptase assay kit (Roche, Germany). PK15, infected and uninfected HEK-293 cells, respectively. The level of RT activity in infected HEK-293 cells was PCR and RT-PCR nearly 200 times lower than that in PK15 cells. DNA and RNA of PK15 cells, the infected and the uninfected HEK-293 cells were extracted using a GFX Genomic Blood DNA Examination of PERV Infection on HEK-293 Purification Kit (Amerham, USA), and Promega RNA Extraction Kit (Promega, USA). Primers specific for PERV-gag, pol, env-A, PERV-gag, pol DNA presented in the genome of infected -B, -C gene and pig mitochondrion cytochrome B oxidase gene HEK-293 cells (Fig 1A) and PERV mRNA efficiently were cited in papers.7,8 GAPDH was used as an internal standard.9 expressed (Fig 1B). There was no pig mitochondrion cytochrome B oxidase gene in the infected HEK-293 cells (Fig 1C), Immunofluorescent Staining which implied there was no PK15 cells microchimerism. As same as that in PK15, the subtypes of PERV in infected Rabbit anti–PERV-gag antibody and FITC-goat–anti-rabbit IgG HEK-293 cells are PERV-A and -B (Fig 1D). (DAKO, Denmark) was used to detect PERV-gag protein, and the results were examined by Laser Scanning Confocal Microscopy (LSCM, MRC1024ES; Bio-Rad). Expression of PERV Protein LSCM showed PERV protein expressed in the cytoplast of Cell Morphology and Growth PK15 and infected HEK-293 cells, while uninfected control Infected HEK-293 cells and uninfected control grew in six-well showed negative signal (Fig 2). Quantity analysis by Laser- plates for light microscopy examination. Total DNA/cell was Pix Image Analysis Software (Bio-Rad, USA) demon- detected by fluorometric assay using the Hoechst dye 33258 strated the fluorescence intensity of PERV-gag protein in (Sigma, USA) and the fluorescence was measured at 350nm and PK15 and infected HEK-293 cells was 78.89 Ϯ 11.86 and 460nm in fluorophotometer (Shimadzu, Japan). To detect cell 66.22 Ϯ 15.38 respectively, and there was distinct difference doubling time equal numbers of cells were plated in 24-cell plates between them (Student t-test, P Ͻ .05). and cells were counted 24, 36, 48, 60, 72, and 84 hours after plating. Multiple experiments were performed to minimize the error. Equal numbers of cells were plated in 24-well plates, which culture Effect of PERV Infection on the Growth of HEK-293 Cells medium containing 0, 1%, 2%, 3%, 4%, 8%, or 10% NBS. Then Infected and uninfected HEK-293 cells showed no signifi- the alive cells were counted after 24 hours to evaluate the serum cant difference in morphology when grown in DH contain- dependence of infected cells. ing 10% NBS. Total DNA of uninfected control and infected cells were 7.45 Ϯ 0.15 ␮g and 8.02 Ϯ 0.10 ␮g, RESULTS respectively (Fig 3A), and there was no statistic difference PERV Morphology between them (Student t-test, P Ͼ .05). Otherwise, infected PERV particles in the harvested supernatant of PK15 and HEK-293 cells doubled a little earlier than the uninfected infected HEK-293 cells were same in electron microscopy, control in DH containing 10% NBS (Fig 3B). Infected HEK- which were round and about 120 nm in size. PERV particles 293 cells grew poorly in serum-free medium; the uninfected had dense virus core and capsid. control could survive in DH without serum (Fig 3C). The

Fig 1. (A, B) M, Marker; lane 1, 4, positive control PK15; lane 2, 5, uninfected control; lane 3, 6, infected HEK-293. (C) M, Marker; lane 1, positive control PK15; lane 2, uninfected control; lane 3, infected HEK-293. (D) M, Marker; lane 1, 3, 5, positive control PK15; lane 2, 4, 6, infected HEK-293. 中国科技论文在线 http://www.paper.edu.cn

498 YU, BU, ZHANG ET AL

Fig 2. The expression of PERV-gag protein (green ﬂuorescence) in LSCM examination (ϫ600). Blue ﬂuorescence labelled by DAPI represented cell nuclear. (A) Positive control PK15. (B) Uninfected control. (C) Infected HEK-293.

infected cells and uninfected control could grow well in DH detected were actually from infected HEK-293 cells ge- containing 8% NBS (Fig 3C). nome. The positive results of RT-PCR indicated PERV in infected HEK-293 cells were active and could efficiently DISCUSSION replicate. Otherwise, detection of envA, B, C genes in infected HEK-293 demonstrated the subtypes of PERV Xenotransplantation offers a potential solution to the short- were PERV-A and -B, which were same as that in PK15. age of donor organs for transplantation. But the use of These results clearly showed that PERV sequence in in- porcine organs, tissues, and cells is associated with the risk fected HEK-293 cells was complete and effective, which of PERV transmission.5,10 In recent years concerns over the produced infective PERV particles. changes of PERV after infection to other cells and the effect of PERV infection on host cells have been raised. In PERV-gag protein was also detected in infected HEK- this study, we showed that the ultrastructure of PERV did 293 cells, which indicated the functional expression of not alter after infection to human cells, and by morphologic PERV in infected human cells. Compared with PK15, the criteria PERV belongs to typical mammalian C-type vi- expression of PERV-gag protein in infected HEK-293 ruses. It is reported these viruses released by infected decreased slightly; however, the presence of virus protein HEK-293 cells can infect fresh human cells in vitro.10 offered a greater possibility of causing disease after infec- Therefore, the complete structure of virus is of great tion. In addition, RT assay demonstrated the reverse tran- importance for its infectivity. scriptase of PERV in infected HEK-293 cells remained at a Sequence studies have shown that PERV carry gag, pol, lower level, which reflected less efficient productive infec- and env genes, and the subtypes PERV-A, -B and -C are tion. Whether PERV DNA can permanently present in closely related in gag and pol genes but differ in evn infected human cells in vitro and lead to efficient expression genes.11 It is reported that PERV derived from PK15 cells of PERV protein remains to be determined. may infect many nonporcine cells including human cells.5,6 Morphology and cell growth of infected HEK-293 cells We performed PCR using primers specific for PERV-gag and uninfected control showed no significant difference, and pol gene to identify PERV infection of HEK-293. To only infected HEK-293 cells doubled a little earlier than the eliminate the contaminating of PK15 cells, PCR with prim- control. A similar result was reported by Kuddus, et al9 But ers specific for pig mitochondrion cytochrome B oxidase the infected cells could not survive in serum-free medium, gene were performed. As we showed in the results section, which suggested that human cells needed better conditions there was no PK15 cells microchimerism and PERV DNA for growth in vitro after PERV infection. Whether this

Fig 3. (A) Total DNA contents of about 2 ϫ 106 cells. 1: Uninfected HEK-293; 2: Infected HEK-293. (B) Growth of cells in DH containing 10% NBS. (C) Growth of cells in DH-containing a different volume fraction of NBS. 中国科技论文在线 http://www.paper.edu.cn

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