Gibbon Ape Leukemia Virus and the Amphotropic Murine ... - NCBI - NIH

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Cancer 27:397-402. 16. McClure, M. O., M. A. Sommerfelt, M. Marsh, and R. A. Weiss. ... O'Hara, B. S., S. V. Johann, H. P. Klinger, D. G. Blair, H. Rubinson, K. J. ...
Vol. 67, No. 9

JOURNAL OF VIROLOGY, Sept. 1993, p. 5472-5477 0022-538X/93/095472-06$02.00/0 Copyright e 1993, American Society for Microbiology

Gibbon Ape Leukemia Virus and the Amphotropic Murine Leukemia Virus 4070A Exhibit an Unusual Interference Pattern on E36 Chinese Hamster Cells MARTIN A. EGLITIS,* MARIBETH V. EIDEN, AND CAROLYN A. WILSON Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892 Received 27 May 1993/Accepted 21 June 1993

The gibbon ape leukemia virus (GaLV), the amphotropic mouse leukemia virus (A-MLV) 4070A, and the xenotropic mouse leukemia virus (X-MLV) exhibit wide but not identical species host ranges. However, most Chinese hamster cells resist infection by all three viruses. We have now determined that the Chinese hamster cell line E36 differs from other Chinese hamster cell lines in that it is susceptible to infection by wild-type GaLV, A-MLV, and X-MLV. Surprisingly, analysis of the interference pattern of GaLV and A-MLV in E36 cells indicated that GaLV and A-MLV interfere in a nonreciprocal fashion. E36 cells productively infected with GaLV were resistant to superinfection by both GaLV and amphotropically packaged recombinant retroviral vectors. In contrast, E36 cells infected with A-MLV were resistant to superinfection with an amphotropic vector but could still be infected by a GaLV vector. These results imply the existence of a receptor on E36 cells that interacts with both GaLV and A-MLV. While the gibbon ape leukemia virus (GaLV), the amphotropic mouse leukemia virus (A-MLV), and the xenotropic mouse leukemia virus (X-MLV) possess broad species host range, they are in separate interference classes (26) and are believed to use different receptors to infect cells. The gene for the human GaLV receptor has been cloned (22) and mapped to chromosome 2 (12). The gene for the A-MLV receptor has not yet been cloned, but it is known to map to chromosome 8 in both humans (5) and mice (6). The gene for the X-MLV receptor has been mapped to mouse chromosome 1 (13). None of these viruses has been described as capable of efficiently infecting Chinese hamster cells (8, 29). Wild-type GaLV cannot infect Chinese hamster ovary (CHO-K1) cells. However, when an MLV core is packaged in the GaLV envelope, CHO-Kl cells are efficiently infected, demonstrating that CHO-Kl cells do express a functional GaLV receptor on their surface (29). Thus, the block to GaLV infection appears related to -events subsequent to the virus-receptor interaction (29). In contrast, neither CHO-Kl nor Don Chinese hamster cells are infected when the same MLV core and genome are packaged in an A-MLV envelope. Therefore, the block to infection by wild-type A-MLV in Chinese hamster cells is at the level of receptor. This block can be overcome by treatment with tunicamycin, an inhibitor of N-linked glycosylation. Presumably, this treatment affects glycosylated moieties that interfere with the interaction between the A-MLV envelope and the CHO-Kl A-MLV receptor (21). We have now found a Chinese hamster cell line, E36, which is efficiently infected by both GaLV and A-MLV. Since different mechanisms account for CHO-Kl cell resistance to infection by GaLV and A-MLV, it is likely that both changes in the receptor and relief of a postpenetration block account for the altered infection pattern in E36 cells. The acquisition of susceptibility to infection by two different retroviruses suggests that a common factor could be involved in this change. We have used recombinant retroviral

*

vectors differing only in their envelope to determine whether the factor(s) accounting for altered susceptibility to infection functions at the level of viral entry.

MATERIALS AND METHODS Cells and viruses. E36 cells are derived from the lung of a male Chinese hamster (7). CHO-Kl cells, derived from an adult Chinese hamster ovary (23), were obtained from the American Type Culture Collection (ATCC) (CCL 61). Rat-2 embryonic rat fibroblasts (27) and NIH 3T3 embryonic mouse cells (10) were obtained from the ATCC (CRL 1764 and CRL 1658, respectively). TblLu bat lung and MvlLu mink lung fibroblasts were also obtained from the ATCC (CCL 88 and CCL 64, respectively). HOS cells are a human osteogenic sarcoma (15) (ATCC CRL 1543). MDBK cells are derived from normal bovine kidney (14) (CCL 22). Cultured tail fibroblasts derived from the feral mouse Mus dunni (MDTF) (1) were provided by J. Hartley, National Institute of Allergy and Infectious Diseases. All cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, except for CHO-Kl cells, which were cultured in alpha minimum essential medium. GaLV of the SEATO subtype was collected from infected TblLu cells. A-MLV of the 4070A subtype was collected from infected MvlLu cells. X-MLV of the AKR 40-X subtype (4, 9) was collected from infected MvlLu cells. The recombinant retroviral vector GlBgSvN (17) carrying the bacterial lacZ and neomycin resistance genes was used in interference assays. The vector was collected from either PG13/ GlBgSvN or PA317/GlBgSvN cells. PG13 is a hybrid retroviral packaging cell line, expressing Moloney ecotropic MLV core proteins and the GaLV envelope (19) (ATCC CRL 10686). PA317 is a hybrid retroviral packaging cell line expressing Moloney ecotropic MLV core proteins and the envelope from the 4070A A-MLV (18) (ATCC CRL 9078). Titer of the vector produced by both packaging cells was approximately 105 G418-resistant CFU/ml, as assayed on HeLa cells. Wild-type virus infections. Medium containing wild-type

Corresponding author. 5472

RETROVIRAL INFECTION OF E36 CELLS

VOL. 67, 1993

virus was prepared by changing medium on the producer cells when they had reached approximately 50% confluence. Virus-containing medium was collected 24 to 48 h after the medium change, passed through a 0.22-,um-pore-size filter, and stored at -70°C until use. Target cells were seeded at 2 x 104 to 3 x 104 cells per well in 24-well dishes. Twenty-four hours after seeding, cells were infected with virus in medium containing 3 ,ug of Polybrene per ml. Twenty-four hours after exposure to virus, the cells were fed with fresh medium. After an additional 24 h of culture, the cells were passaged 1:10. Six days after splitting, the medium was collected and assayed for reverse transcriptase. Reverse transcriptase assays were performed as previously described (29). Interference analysis. For the interference assay, a series of virally infected cell lines were generated by infecting with GaLV-SEATO, 4070A, or AKR 40-X as described above. Cells tested were E36, HOS, and MDTF cells expressing the cloned GaLV receptor (22). Productive infection with a particular virus was confirmed by detection of significant levels of reverse transcriptase in the medium at levels 100 to 1,000 times above background. To assay the cells' susceptibility to superinfection with a recombinant hybrid retroviral vector, target cells were seeded in three individual wells of a 12-well culture plate at an initial density of 104 cells per well. The next day, the cells were exposed to the vector by replacing their medium with medium harvested from either PG13/GlBgSvN or PA317/GlBgSvN producers. Vectorcontaining medium was prepared as described above for wild-type virus. To facilitate infection with retroviral vectors, Polybrene at a final concentration of 8 ag/ml was added to the medium. Target cells were exposed to retroviral vector overnight, and then vector-containing medium was removed and replaced with fresh medium. After a further 24 h of culture, the cells were fixed and stained for the presence of ,B-galactosidase (3-gal) as described previously (25). Construction of MDTF cells expressing the human GaLV receptor cDNA. The pOJ9 plasmid containing the cDNA for the human GaLV receptor was obtained from Brian O'Hara (Lederle Laboratories, Pearl River, N.Y.). The 2.3-kb HindIII to EcoRV restriction fragment from pOJ9 was introduced into the pLNSX retroviral expression plasmid (20) at the HindIII to ClaI sites (the Clal site was filled in with T4 DNA polymerase) to make the pLNS-OJ9 plasmid. PA317 cells were transfected with 20 ,ug of pLNS-OJ9 plasmid (2). Transfected cells were selected with 400 pg of active G418 per ml. Supernatant medium from the stably transfected PA317 cells was used to infect MDTF cells. MDTF cells expressing the LNS-OJ9 vector were selected in 400 ,ug of active G418 per ml. RESULTS We used the wild-type GaLV, A-MLV, or X-MLV viruses to confirm their species host range and simultaneously determine whether E36 cells are susceptible to infection (Table 1). Productive infection of different target cells exposed to either GaLV, A-MLV, or X-MLV was assessed by a reverse transcriptase assay of the cell supernatant performed 8 days after the cells were exposed to filtered virus. The three retroviruses were able to infect human, mink, bat, and rat cells but differed in their ability to infect bovine MDBK, murine MDTF, and NIH 3T3 cells. Bovine cells were susceptible to infection by GaLV and X-MLV but were resistant to A-MLV. The murine cell line MDTF was capable of being infected by both murine retroviruses (A-MLV and X-MLV) but not the primate retrovirus GaLV. Murine

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TABLE 1. Susceptibility of cells to infection with GaLV, A-MLV, and X-MLV

Susceptibility' with Target cell

HOS

MvlLu TblLu Rat-2 MDBK MDTF NIH 3T3 CHO-Kl E36

challenge virus

Ongin

Human Mink Bat Rat Bovine Mus dunni Mus musculus Chinese hamster Chinese hamster

GaLV

A-MLV

X-MLV

+ + + + + +

+ + + +

+ + + + + +

+ +

+

+ +

a Susceptibility to infection was determined by assay for reverse tran-

scriptase in the medium. For reverse transcriptase assays: +, >2x background reverse transcriptase activity; -,