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Induction of feline immunodeficiency virus from a chronically infected feline T-lymphocyte cell line
Research in Veterinary Science 92 (2012) 327–332
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Induction of feline immunodeficiency virus from a chronically infected feline T-lymphocyte cell line Tadafumi S. Tochikura a,⇑, Yuko Naito b, Yasunori Kozutsumi b, Tsutomu Hohdatsu c a
Center for Integrative Education of Pharmacy Frontier, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan c Department of Veterinary Infectious Diseases, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan b
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Article history: Received 8 October 2010 Accepted 18 February 2011
Keywords: Feline immunodeficiency virus FIV latency FeT-J 3201 Cell cloning Chemical inducers
a b s t r a c t The infection of the feline T-lymphocyte cell line FeT-J with the feline immunodeficiency virus (FIV) Petaluma strain led to the establishment of nonvirus-producing cells. One clone (C15) obtained by limiting dilution was found to express FIV in response to chemical inducers of retroviruses. The chemical treatment of C15 cells led to not only FIV protein synthesis but also an augmentation of viral production. Examination of the C15 cell derivatives obtained by recloning revealed that 10–40% of treated cells constitutively expressed FIV antigens, whereas 100% with expressed FIV antigen in response to the inducer. Chemical induction resulted in more than a 100-fold increase in infectious viral production. The results suggest that a majority of FeT-J cells that are infected with FIV exist in a non-productive state. Establishing a cell line that can be non-productively infected by FIV may help determine the mechanisms of FIV latency. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Feline immunodeficiency virus (FIV) (Pedersen et al., 1987), a retrovirus belonging to the lentivirus subfamily, commonly infects cats and can result in immunosuppression. Infected cats generally remain healthy for several years (Hosie et al., 2009). Such a lengthy asymptomatic period without clinical signs is common in many lentiviral diseases, including human acquired immunodeficiency syndrome (AIDS) (Levy, 1993). FIV establishes a nonproductive or latent infection, with no active viral shedding, and latently infected peritoneal macrophages can be induced to produce virions through viral reactivation leading to the expression of integrated proviral DNA via external activators (i.e. phorbol myristate acetate) (Brunner and Pedersen, 1989). Latently infected cells represent a reservoir of infection, and are a major barrier to eradication of the virus. Thus, a better understanding of the underlying mechanisms of FIV latency and reactivation is needed in order to design treatments to control or eradicate latently infected cells. It would be useful to generate an in vitro system to model the establishment and reactivation of FIV latency. However, there has been little information on these systems presumably owing to a lack of appropriate feline cell lines harboring a latent form of FIV, as opposed to studies of HIV for which there are already models
⇑ Corresponding author. Tel.: +81 75 753 4564; fax: +81 75 761 2698. E-mail address: [email protected] (T.S. Tochikura). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.02.013
available (Folks et al., 1986, 1988; Clouse et al., 1989). Primary feline cultures (Brunner and Pedersen, 1989; Joshi et al., 2005) have been of relatively limited utility probably due to their short-lived nature, and the laborious procedures necessary to establish the cultures. To this end we have established a model of FIV latency in vitro with a feline T-lymphocyte cell line, FeT-J. This model supports reactivation of the virus after chemical treatment. 2. Materials and methods 2.1. Cell cultures The Interleukin 2 (IL-2) – independent feline T-lymphocyte cell line FeT-J, derived from feline peripheral blood mononuclear cells (PBMCs) of a specific-pathogen-free (SPF) cat and shown to be susceptible to FIV infection (Hohdatsu et al., 1996), was obtained from the American Type Culture Collection (ATCC #CRL-11967), and maintained in RPMI 1640 medium with 2 mM L-glutamine, 1 mM sodium pyruvate and 0.05 mM 2-mercaptoethanol, supplemented with 10% heat-inactivated fetal bovine serum (FBS). Another FIVsusceptible T-lymphocyte cell line, 3201 (Tochikura et al., 1990, 2010) which is also IL-2-independent, was established from a feline thymic lymphoma of a SPF cat (Snyder et al., 1978), and was maintained in a medium consisting of equal parts Leibovitz L-15 medium and RPMI 1640 medium supplemented with 10% FBS. Both T-lymphocyte cell lines were grown in the presence of
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penicillin (100 IU/ml) and streptomycin (100 lg/ml). Feline PBMCs were obtained from a SPF adult cat (Shimizu Laboratory Supplies, Kyoto, Japan). The PBMCs were initially stimulated with 5 lg of phytohemagglutinin (PHA) per ml for 3 days, maintained on 5 ng of recombinant human IL-2 per ml, and replenished with fresh PHA and IL-2-stimulated PBMCs in a complete RPMI 1640 medium with 20% FBS every 5–7 days. 2.2. Virus A cell culture-adapted strain of FIV-Petaluma (FIV-Pet) was obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, MD, USA). Tissue culture supernatant from 3201 cells persistently infected with FIV was used as a source of infectious virus (Tochikura et al., 1990). After more than 80% of the 3201 cells became positive for FIV antigens, as detected with an indirect immunofluorescence assay (IFA), the culture supernatant was filtered through a 0.45 lm Millipore membrane filter and stored at 82 °C in small aliquots until used. The virus titer was 103.8 50% tissue culture infectious dose (TCID50) per ml, assayed in 3201 cells as calculated by the method of Reed and Muench (1938).
A
(7.8)
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FeT-J cells were infected with FIV-Pet at a multiplicity of infection (M.O.I.) of 0.04. After the adsorption of FIV at 37 °C for 1 h, cells were cultured in complete medium at 37 °C in 5% CO2. The cells were subcultured every 3–4 days and maintained for more than 50 days before cloning. The FIV-infected cells were then cloned in five round-bottom plates at 0.2 cells/well in a final volume of 0.1 ml of complete medium. Of the 28 clones subsequently obtained, No. 15 (C15) was selected for the present study based on its frequency of viral antigen positivity as determined by IFA. 2.4. Chemicals and detection of virus Phorbol myristate acetate (PMA; Sigma–Aldrich, St. Louis, MO, USA) and 5-iodo-deoxyuridine (IUdR; Sigma–Aldrich) were dissolved in dimethylsulfoxide (DMSO), and sodium butyrate (NaB; Wako, Osaka, Japan) in RPMI 1640 medium. Stock chemicals were diluted in complete RPMI 1640 medium and used immediately. Concentrations used in the experiments are optimal for inducing the release of virions (Brunner and Pedersen, 1989; Folks et al., 1986; Golub et al., 1991). Supernatant from chemically treated
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2.3. FIV infection and cell cloning
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Fig. 1. Flow cytometric analysis of CD134 (A) and CXCR4 (B) expression on FeT-J cells, 3201 cells and PBMCs. Expression of CD134 and CXCR4 on FeT-J cells, 3201 cells and peripheral blood mononuclear cells (PBMCs) was determined by using an anti-CD134 monoclonal antibody (MAb) or anti-CXCR4 MAb, followed by an fluorescein isothiocyanate-conjugated anti-mouse IgG. For all panels, data are shown as the relative cell number (y-axis) plotted against the relative fluorescence intensity (x-axis). The background staining is the signal derived from incubation of the cells with an isotype-matched control antibody as a primary antibody. Mean fluorescence intensity is indicated in parentheses.
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A 100
2.6. Flow cytometry Flow cytometric analyses were performed as reported previously (Tochikura et al., 2010), using monoclonal antibodies (MAbs) against CD134 (anti-CD134 MAb [Affinity BioReagents, Golden, CO, USA]) and CXCR4 (anti-CXCR4 MAb clone 44717 [R&D Systems, Minneapolis, MN, USA]), as a possible primary receptor and co-receptor for FIV, respectively, and a fluorescein isothiocyanate-conjugated anti-mouse IgG (ICN/Cappel, Aurora, OH, USA), as a secondary antibody. Appropriate isotype-matched antibody controls were conducted with mouse IgG2b (Invitrogen, Camarillo, CA, USA). 3. Results 3.1. Surface expression of FIV receptors and viral susceptibility of cell lines As shown in Fig. 1A, the flow cytometric experiments demonstrated the FeT-J cells to be CD134 , indicating that CD134 is not required for infection by FIV-Pet. Comparison of the mean fluorescence intensity (MFI) of CXCR4 expression on 3201 and FeT-J cells revealed that FeT-J cells expressed inherently much lower levels of CXCR4 (MFI of 254 versus 21.3) (Fig. 1B). When infected with FIVPet, the two lines differed in their response to the cytopathic changes of FIV (Fig. 2). The maximum decrease in cell viability (15% viability in FIV-infected 3201 cells [Fig. 2A] termed 3201/ FIV versus 43% viability in FIV-infected FeT-J cells [Fig. 2B] termed FeT-J/FIV) as determined by trypan blue dye exclusion was seen at 15 days post-inoculation (dpi).
Cell viability (%) Fluorescent cells (%)
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The expression of FIV antigen in FIV-infected cells was detected by indirect immunofluorescence assay (IFA) as described (Tochikura et al., 1990).
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2.5. Immunofluorescence assay
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FIV-infected cells was filtered, and the infectivity of the virus was assayed in 3201 cells as described above.
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Days post-inoculation Fig. 2. The kinetics of the appearance of viral antigens and cell viability after infection of 3201 (A) and FeT-J (B) cells with FIV. Cells were infected with the Petaluma isolate of FIV as described in the text. Viral antigens were examined by indirect immunofluorescence assay using an FIV-positive cat serum as the primary antibody, and cell viability was determined by trypan blue dye exclusion at the indicated times. More than 500 cells were counted under a fluorescence microscope, and the percentages of FIV-antigen-positive cells were calculated. Data points represent the mean ± standard deviation of triplicate cultures.
3.3. Cloning and activation of FeT-J cell derivatives chronically infected with FIV Since the proportion of viral antigen-positive cells did not reach 100% and remained at 50–60% beginning 21 days after the infection, the FeT-J/FIV cells at 58 dpi were cloned at 0.2 cells/well in 0.1 ml of culture medium by limiting dilution. Ten individual clones were tested for viral expression by IFA, and three were found to be highly positive (>90%) for viral antigen, as compared with the uncloned parental FeT-J/FIV cells (data not shown). The expression of FIV antigens was most stable in clone 15 designated as C15, which consequently was selected for further study.
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Both FeT-J and 3201 cells express CXCR4, but not CD134. This common antigen expression, although the expression levels in FeT-J cells was much lower than those in 3201 cells, prompted us to investigate the fate of these two cell lines after FIV infection. In FeT-J cells infected with FIV, replication of the virus was first evident by 6 dpi as demonstrated by an IFA, and FIV became cytopathic at 9 dpi (Fig. 3). The maximum decrease in cell viability to 38% due to cell killing was seen at 15 dpi, however, the cell viability in the FIV-infected culture which became free of cytopathic changes returned to normal levels (>90%) at 30 dpi.
Viable cells (%)
3.2. Establishment of cells free of cytopathic changes after FIV infection
0
Days post-inoculation Fig. 3. Establishment of FeT-J /FIV cells. FeT-J cells were infected with FIV, and the expression of FIV antigens was examined as above. At the same time, cells were processed in parallel for trypan blue dye exclusion.
Although C15 cells continued to produce viral proteins for 30 days in culture after cloning, the frequency of FIV antigen-positive cells began to decrease, and by day 106 was 10–30% (Fig. 4A). To know if the FIV genome was present in FeT-J cells that had survived infection, C15 cells were exposed for 48 h to PMA at 10 7 M, IUdR at 100 lg/ml, and NaB at 0.05–5 mM, which are
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Fig. 4. Immunofluorescent analysis of FIV protein expression in chemically treated and untreated FIV-infected FeT-J cells. C15 cells were cultured in complete medium alone (A) or with IUdR (B: 100 lg/ml), PMA (C: 10 7 M), or NaB (D: 5 mM; E: 0.5 mM; F: 0.05 mM) for 48 h. Cells were processed for immunofluorescence assays as described above. Magnification, 100.
well-known inducers of retroviruses. As shown in Fig. 4, 100% of cells became positive in response to each of the agents except NaB at 5 mM which had considerable cytotoxic effects. The release of virus by those chemicals, as determined using the TCID50, resulted in an approximately 50- to 160-fold increase in the production of infectious particles, depending on the inducer (Table 1). Since heterogeneity of FIV expression in C15 cultures under uninduced conditions was still observed, limiting-dilution techniques were re-employed twice to derive single-cell clones from C15 cells. Twenty moderately growing clones were obtained at 22 days after the first recloning, and examined for FIV antigen immunofluorescence. All clones were accompanied by a minor cell population constitutively expressing FIV antigens, although a majority of the cells did not express viral antigens (data not shown). We selected one clone designated as C15-13 for a second recloning. Fifteen clones were obtained at 25 days after the recloning, but no clonal variation with regard to virus expression was apparent (data not shown). C15-13 and C15-13-10 cells obtained after the first and second recloning, respectively, were monitored after exposure for 48 h to NaB at 0.5 mM for FIV antigen immunofluorescence and viral infectivity. Examination of those subclones derived from C15 cells revealed that 100% of the cells became positive in response to NaB treatment, however, 10–40% of uninduced cells constitutively expressed FIV antigens (data not shown). The production of infectious virus was found to be
Table 1 Effect of various chemicals on the release of infectious FIV in FeT-J/FIV C15 cells.a Treatment with
Virus yieldb (TCID50/20 ll)
Ratio
Medium alone IUdR PMA NaB (5 mM) NaB (0.5 mM) NaB (0.05 mM)
100.6 102.4 102.3 NDc 102.8 101.3
1 63 50 NAd 158 5
a C15 cells were cultured with or without chemicals for 48 h, and the supernatants harvested from each culture were tested for viral infectivity in 3201 cells. b Titers are expressed as log 10 TCID50/20 ll, based on the assay using immunofluorescence. c ND: not done. d NA: not applicable.
increased by 30- to 80-fold after induction, whereas FIV-positive cells and the infectious virus were detectable at a minimum level in the culture if left uninduced (data not shown). These results suggest that a majority of the cells are likely to exist in a non-productive state. Although these cells reverted to be low expressors when NaB was removed from cultures, their response to the drug has been found to be stable for 2 months in culture (data not shown).
4. Discussion One outstanding clinical feature of infections by lentiviruses is a relatively long period of latency characterized by a low to undetectable level of virus following an early acute phase of infection with viremia (Fauci, 1988; Hosie et al., 2009). However, especially for FIV, the mechanisms responsible for maintaining clinical latency and for the activation of latently infected cells are still poorly understood, because of a lack of appropriate in vitro models of FIV latency. We have described earlier that the feline T-lymphocyte cell line 3201 established a chronic FIV-producer cell line (3201/FIV) (Tochikura et al., 1990). In the present study, we first repeated the same experiment. It may be worth mentioning here that the cytopathic effects differed between the previous and the present experiments, although the cell lines were derived from the same source. The 3201 cells used in the previous study showed maximum kill (40% dead) at 16 dpi (Tochikura et al., 1990), whereas maximum kill (85% dead) was observed at 12 dpi in the present study. The cytopathogenic difference between the two cell lines remains to be explained, however, it might be due to differences in culture conditions across laboratories, thereby affecting the viral replication. The other T-lymphocyte cell line, FeT-J, used in the present study has been reported to be susceptible to FIV isolates and also established a chronic FIV-producer cell line (Hohdatsu et al., 1996). It has been shown that FeT-J and 3201 cells share some features: (1) both cell lines are lymphoid; (2) both are IL-2independent; and (3) both are positive for CXCR4 (Willett et al., 1997), the entry receptor for FIV, but not for CD134 (Shimojima et al., 2004), the binding receptor. Interestingly, in the present study, comparison of the MFI of CXCR4 expression on 3201 and FeT-J cells revealed that FeT-J cells expressed inherently much
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lower levels of CXCR4. This might be relevant to the finding that the initial expression of viral antigen was more rapid in 3201 cells than in FeT-J cells. A recent report (Tochikura et al., 2010) described that the infection of 3201 cells with a well-known laboratory strain, Petaluma (FIV-Pet), that does not require CD134 for adsorption (Lerner et al., 1998) led to the establishment of cells, designated as 3201-S, which were free of FIV DNA after a productive infection associated with considerable cell killing. These observations encourage further studies on how FeT-J cells behave after FIV infection. In this study, we have demonstrated that three subclones (C15, C15-13 and C15-13-10) derived from FeT-J cells chronically infected with FIV-Pet produced little virus in the uninduced state but showed markedly increased viral expression and production on stimulation with chemicals. The majority of cell populations are likely to be in a non-productive state. We are not sure why each clone was accompanied by a minor population of cells in a productive state, although the C15 cells were recloned twice to obtain C15-13 and then C15-13-10. However, this might be solely because C15 cells are unstable in terms of viral expression. It is of particular interest that FeT-J cells entered a non-productive state after infection with FIV, but 3201 cells did not. The reason 3201 cells did not enter a non-productive state remains to be elucidated. However, one possible explanation would be that another feline retrovirus, designated RD114, could be involved in the cytopathogenicity observed in 3201 cells. An etiologic role for RD114 has not been demonstrated, however, the endogenous viral sequences are rarely expressed in adult feline tissues but are expressed in some lymphomas (Niman et al., 1977; Cheney et al., 1990). In a previous study, both Mg2+ and Mn2+-dependent reverse transcriptase activities associated with FIV and RD114 virus, respectively, were induced in FIV-infected 3201 cells as compared to uninfected 3201 cells (Tochikura et al., 1990). Similar results were noted with HIV infections of human cell lines carrying human T-cell leukemia virus type 1, another human retrovirus (Harada and Yamamoto, 1987). As a result, HIV-infected cells showed marked cytopathic changes preventing further passage. Further study will be needed to ascertain whether the profound cytopathogenicity observed in FIV-infected 3201 cells is associated with the endogenous feline retrovirus, as opposed to the phenomenon in FIV-infected FeT-J cells. So far, there has been only one report regarding FIV latency in a cell line: FIV established a latent infection in the human leukemic T-lymphocyte cell line MOLT-4, and the cells were capable of producing infectious virions following treatment with PMA (Ikeda et al., 1996). However, these results contradict our previous finding that surviving MOLT-4 cells after cocultivation with 3201/FIV cells did not produce any infectious virus even after treatment with PMA (Tochikura et al., 1993 and unpublished observations). In that study, Southern blot analysis failed to detect proviral DNA and the polymerase chain reaction technique barely detected FIV-specific DNA in MOLT-4 cells. The discrepancies between these two studies, using similar materials and methods, are as yet unexplained. To our knowledge, this is the first description of FIV latency, at least in vitro in a feline cell line. The feline T-lymphocyte cell lines 3201 and FeT-J are expected to provide a useful experimental model for investigating the mechanisms of FIV latency.
5. Conclusions We have established nonvirus-producing feline T-lymphocyte cells. The results of the present study show that chemical treatment of the cells led to not only FIV protein synthesis but also an
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augmentation of viral production. The treatment resulted in more than a 100-fold increase in the production of infectious virions. The cloned cells described here provide an attractive in vitro model for studying the mechanisms of FIV latency. 6. Conflict of interest statement No author has had any financial or personal relationship with people or organizations that could inappropriately influence their work. Acknowledgements We are grateful to Dr. Lawrence E. Mathes for providing the 3201 cell line, Dr. Makoto Hitomi and Dr. Hirokazu Seo for serum samples from cats, and Hiroyuki Miwa and Ryo Higashiyama for graphics and photographic assistance. We also wish to thank Dr. Nobuyuki Itoh, Dr. Hideo Saji and Dr. Nobutaka Fujii for their constant support and encouragement. We would like to dedicate this study to the late Dr. James R. Blakeslee, Jr. The feline immunodeficiency virus (Petaluma strain) was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS (DAIDS), NIAID, NIH, from contributors Dr. Niels C. Pedersen and Dr. Janet K. Yamamoto. References Brunner, D., Pedersen, N.C., 1989. Infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus. Journal of Virology 63, 5483–5488. Cheney, C.M., Rojko, J.L., Kociba, G.J., Wellman, M.L., Di Bartola, S.P., Rezanka, L.J., Forman, L., Mathes, L.E., 1990. A feline large granular lymphoma and its derived cell line. In Vitro Cellular & Developmental Biology 26, 455–463. Clouse, K.A., Powell, D., Washington, I., Poli, G., Strebel, K., Farrar, W., Barstad, P., Kovacs, J., Fauci, A.S., Folks, T.M., 1989. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. Journal of Immunology 142, 431–438. Fauci, A.S., 1988. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239, 617–622. Folks, T.M., Justement, J., Kinter, A., Schnittman, S., Orenstein, J., Poli, G., Fauci, A.S., 1988. Characterization of a promonocyte clone chronically infected with HIV and inducible by 13-phorbol-12-myristate acetate. Journal of Immunology 140, 1117–1122. Folks, T.M., Powell, D.M., Lightfoote, M.M., Benn, S., Martin, M.A., Fauci, A.S., 1986. Induction of HTLV-III/LAV from a nonvirus-producing T-cell line: implications for latency. Science 231, 600–602. Golub, E.I., Li, G., Volsky, D.J., 1991. Induction of dormant HIV-1 by sodium butyrate: involvement of the TATA box in the activation of the HIV-1 promotor. AIDS 5, 663–668. Harada, S., Yamamoto, N., 1987. AIDS studies in Japan. Japanese Journal of Cancer Research (Gann) 78, 415–427. Hohdatsu, T., Hirabayashi, H., Motokawa, K., Koyama, H., 1996. Comparative study of the cell tropism of feline immunodeficiency virus isolates of subtypes A, B and D classified on the basis of the env gene V3–V5 sequence. Journal of General Virology 77, 93–100. Hosie, M.J., Addie, D., Belák, S., Boucraut-Baralon, C., Egberink, H., Frymus, T., Gruffydd-Jones, T., Hartmann, K., Lloret, A., Lutz, H., Marsilio, F., Pennisi, M.G., Radford, A.D., Thiry, E., Truyen, U., Horzinek, M.C., 2009. Feline immunodeficiency. ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 575–584. Ikeda, Y., Tomonaga, K., Kawaguchi, Y., Kohmoto, M., Inoshima, Y., Tohya, Y., Miyazawa, T., Kai, C., Mikami, T., 1996. Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells. Journal of General Virology 77, 1623–1630. Joshi, A., Garg, H., Tompkins, M.B., Tompkins, W.A., 2005. Different thresholds of T cell activation regulate FIV infection of CD4+CD25+ and CD4+CD25 cells. Virology 335, 212–221. Lerner, D.L., Gran, C.K., de Parseval, A., Elder, J.H., 1998. FIV infection of IL-2dependent and – independent feline lymphocyte lines: host cells range distinctions and specific cytokine upregulation. Veterinary Immunology and Immunopathology 65, 277–297. Levy, J.A., 1993. HIV pathogenesis and long-term survival. AIDS 7, 1401–1410. Niman, H.L., Gardner, M.B., Stephenson, J.R., Roy-Burman, P., 1977. Endogenous RD114 virus genome expression in malignant tissues of domestic cats. Journal of Virology 23, 578–586. Pedersen, N.C., Ho, E., Brown, M.L., Yamamoto, J.K., 1987. Isolation of a Tlymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science 235, 790–793.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Induction of feline immunodeficiency virus from a chronically infected feline T-lymphocyte cell line Tadafumi S. Tochikura a,⇑, Yuko Naito b, Yasunori Kozutsumi b, Tsutomu Hohdatsu c a
Center for Integrative Education of Pharmacy Frontier, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan c Department of Veterinary Infectious Diseases, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan b
a r t i c l e
i n f o
Article history: Received 8 October 2010 Accepted 18 February 2011
Keywords: Feline immunodeficiency virus FIV latency FeT-J 3201 Cell cloning Chemical inducers
a b s t r a c t The infection of the feline T-lymphocyte cell line FeT-J with the feline immunodeficiency virus (FIV) Petaluma strain led to the establishment of nonvirus-producing cells. One clone (C15) obtained by limiting dilution was found to express FIV in response to chemical inducers of retroviruses. The chemical treatment of C15 cells led to not only FIV protein synthesis but also an augmentation of viral production. Examination of the C15 cell derivatives obtained by recloning revealed that 10–40% of treated cells constitutively expressed FIV antigens, whereas 100% with expressed FIV antigen in response to the inducer. Chemical induction resulted in more than a 100-fold increase in infectious viral production. The results suggest that a majority of FeT-J cells that are infected with FIV exist in a non-productive state. Establishing a cell line that can be non-productively infected by FIV may help determine the mechanisms of FIV latency. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Feline immunodeficiency virus (FIV) (Pedersen et al., 1987), a retrovirus belonging to the lentivirus subfamily, commonly infects cats and can result in immunosuppression. Infected cats generally remain healthy for several years (Hosie et al., 2009). Such a lengthy asymptomatic period without clinical signs is common in many lentiviral diseases, including human acquired immunodeficiency syndrome (AIDS) (Levy, 1993). FIV establishes a nonproductive or latent infection, with no active viral shedding, and latently infected peritoneal macrophages can be induced to produce virions through viral reactivation leading to the expression of integrated proviral DNA via external activators (i.e. phorbol myristate acetate) (Brunner and Pedersen, 1989). Latently infected cells represent a reservoir of infection, and are a major barrier to eradication of the virus. Thus, a better understanding of the underlying mechanisms of FIV latency and reactivation is needed in order to design treatments to control or eradicate latently infected cells. It would be useful to generate an in vitro system to model the establishment and reactivation of FIV latency. However, there has been little information on these systems presumably owing to a lack of appropriate feline cell lines harboring a latent form of FIV, as opposed to studies of HIV for which there are already models
⇑ Corresponding author. Tel.: +81 75 753 4564; fax: +81 75 761 2698. E-mail address: [email protected] (T.S. Tochikura). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.02.013
available (Folks et al., 1986, 1988; Clouse et al., 1989). Primary feline cultures (Brunner and Pedersen, 1989; Joshi et al., 2005) have been of relatively limited utility probably due to their short-lived nature, and the laborious procedures necessary to establish the cultures. To this end we have established a model of FIV latency in vitro with a feline T-lymphocyte cell line, FeT-J. This model supports reactivation of the virus after chemical treatment. 2. Materials and methods 2.1. Cell cultures The Interleukin 2 (IL-2) – independent feline T-lymphocyte cell line FeT-J, derived from feline peripheral blood mononuclear cells (PBMCs) of a specific-pathogen-free (SPF) cat and shown to be susceptible to FIV infection (Hohdatsu et al., 1996), was obtained from the American Type Culture Collection (ATCC #CRL-11967), and maintained in RPMI 1640 medium with 2 mM L-glutamine, 1 mM sodium pyruvate and 0.05 mM 2-mercaptoethanol, supplemented with 10% heat-inactivated fetal bovine serum (FBS). Another FIVsusceptible T-lymphocyte cell line, 3201 (Tochikura et al., 1990, 2010) which is also IL-2-independent, was established from a feline thymic lymphoma of a SPF cat (Snyder et al., 1978), and was maintained in a medium consisting of equal parts Leibovitz L-15 medium and RPMI 1640 medium supplemented with 10% FBS. Both T-lymphocyte cell lines were grown in the presence of
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penicillin (100 IU/ml) and streptomycin (100 lg/ml). Feline PBMCs were obtained from a SPF adult cat (Shimizu Laboratory Supplies, Kyoto, Japan). The PBMCs were initially stimulated with 5 lg of phytohemagglutinin (PHA) per ml for 3 days, maintained on 5 ng of recombinant human IL-2 per ml, and replenished with fresh PHA and IL-2-stimulated PBMCs in a complete RPMI 1640 medium with 20% FBS every 5–7 days. 2.2. Virus A cell culture-adapted strain of FIV-Petaluma (FIV-Pet) was obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, MD, USA). Tissue culture supernatant from 3201 cells persistently infected with FIV was used as a source of infectious virus (Tochikura et al., 1990). After more than 80% of the 3201 cells became positive for FIV antigens, as detected with an indirect immunofluorescence assay (IFA), the culture supernatant was filtered through a 0.45 lm Millipore membrane filter and stored at 82 °C in small aliquots until used. The virus titer was 103.8 50% tissue culture infectious dose (TCID50) per ml, assayed in 3201 cells as calculated by the method of Reed and Muench (1938).
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FeT-J cells were infected with FIV-Pet at a multiplicity of infection (M.O.I.) of 0.04. After the adsorption of FIV at 37 °C for 1 h, cells were cultured in complete medium at 37 °C in 5% CO2. The cells were subcultured every 3–4 days and maintained for more than 50 days before cloning. The FIV-infected cells were then cloned in five round-bottom plates at 0.2 cells/well in a final volume of 0.1 ml of complete medium. Of the 28 clones subsequently obtained, No. 15 (C15) was selected for the present study based on its frequency of viral antigen positivity as determined by IFA. 2.4. Chemicals and detection of virus Phorbol myristate acetate (PMA; Sigma–Aldrich, St. Louis, MO, USA) and 5-iodo-deoxyuridine (IUdR; Sigma–Aldrich) were dissolved in dimethylsulfoxide (DMSO), and sodium butyrate (NaB; Wako, Osaka, Japan) in RPMI 1640 medium. Stock chemicals were diluted in complete RPMI 1640 medium and used immediately. Concentrations used in the experiments are optimal for inducing the release of virions (Brunner and Pedersen, 1989; Folks et al., 1986; Golub et al., 1991). Supernatant from chemically treated
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Fig. 1. Flow cytometric analysis of CD134 (A) and CXCR4 (B) expression on FeT-J cells, 3201 cells and PBMCs. Expression of CD134 and CXCR4 on FeT-J cells, 3201 cells and peripheral blood mononuclear cells (PBMCs) was determined by using an anti-CD134 monoclonal antibody (MAb) or anti-CXCR4 MAb, followed by an fluorescein isothiocyanate-conjugated anti-mouse IgG. For all panels, data are shown as the relative cell number (y-axis) plotted against the relative fluorescence intensity (x-axis). The background staining is the signal derived from incubation of the cells with an isotype-matched control antibody as a primary antibody. Mean fluorescence intensity is indicated in parentheses.
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2.6. Flow cytometry Flow cytometric analyses were performed as reported previously (Tochikura et al., 2010), using monoclonal antibodies (MAbs) against CD134 (anti-CD134 MAb [Affinity BioReagents, Golden, CO, USA]) and CXCR4 (anti-CXCR4 MAb clone 44717 [R&D Systems, Minneapolis, MN, USA]), as a possible primary receptor and co-receptor for FIV, respectively, and a fluorescein isothiocyanate-conjugated anti-mouse IgG (ICN/Cappel, Aurora, OH, USA), as a secondary antibody. Appropriate isotype-matched antibody controls were conducted with mouse IgG2b (Invitrogen, Camarillo, CA, USA). 3. Results 3.1. Surface expression of FIV receptors and viral susceptibility of cell lines As shown in Fig. 1A, the flow cytometric experiments demonstrated the FeT-J cells to be CD134 , indicating that CD134 is not required for infection by FIV-Pet. Comparison of the mean fluorescence intensity (MFI) of CXCR4 expression on 3201 and FeT-J cells revealed that FeT-J cells expressed inherently much lower levels of CXCR4 (MFI of 254 versus 21.3) (Fig. 1B). When infected with FIVPet, the two lines differed in their response to the cytopathic changes of FIV (Fig. 2). The maximum decrease in cell viability (15% viability in FIV-infected 3201 cells [Fig. 2A] termed 3201/ FIV versus 43% viability in FIV-infected FeT-J cells [Fig. 2B] termed FeT-J/FIV) as determined by trypan blue dye exclusion was seen at 15 days post-inoculation (dpi).
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Days post-inoculation Fig. 2. The kinetics of the appearance of viral antigens and cell viability after infection of 3201 (A) and FeT-J (B) cells with FIV. Cells were infected with the Petaluma isolate of FIV as described in the text. Viral antigens were examined by indirect immunofluorescence assay using an FIV-positive cat serum as the primary antibody, and cell viability was determined by trypan blue dye exclusion at the indicated times. More than 500 cells were counted under a fluorescence microscope, and the percentages of FIV-antigen-positive cells were calculated. Data points represent the mean ± standard deviation of triplicate cultures.
3.3. Cloning and activation of FeT-J cell derivatives chronically infected with FIV Since the proportion of viral antigen-positive cells did not reach 100% and remained at 50–60% beginning 21 days after the infection, the FeT-J/FIV cells at 58 dpi were cloned at 0.2 cells/well in 0.1 ml of culture medium by limiting dilution. Ten individual clones were tested for viral expression by IFA, and three were found to be highly positive (>90%) for viral antigen, as compared with the uncloned parental FeT-J/FIV cells (data not shown). The expression of FIV antigens was most stable in clone 15 designated as C15, which consequently was selected for further study.
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Both FeT-J and 3201 cells express CXCR4, but not CD134. This common antigen expression, although the expression levels in FeT-J cells was much lower than those in 3201 cells, prompted us to investigate the fate of these two cell lines after FIV infection. In FeT-J cells infected with FIV, replication of the virus was first evident by 6 dpi as demonstrated by an IFA, and FIV became cytopathic at 9 dpi (Fig. 3). The maximum decrease in cell viability to 38% due to cell killing was seen at 15 dpi, however, the cell viability in the FIV-infected culture which became free of cytopathic changes returned to normal levels (>90%) at 30 dpi.
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Days post-inoculation Fig. 3. Establishment of FeT-J /FIV cells. FeT-J cells were infected with FIV, and the expression of FIV antigens was examined as above. At the same time, cells were processed in parallel for trypan blue dye exclusion.
Although C15 cells continued to produce viral proteins for 30 days in culture after cloning, the frequency of FIV antigen-positive cells began to decrease, and by day 106 was 10–30% (Fig. 4A). To know if the FIV genome was present in FeT-J cells that had survived infection, C15 cells were exposed for 48 h to PMA at 10 7 M, IUdR at 100 lg/ml, and NaB at 0.05–5 mM, which are
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Fig. 4. Immunofluorescent analysis of FIV protein expression in chemically treated and untreated FIV-infected FeT-J cells. C15 cells were cultured in complete medium alone (A) or with IUdR (B: 100 lg/ml), PMA (C: 10 7 M), or NaB (D: 5 mM; E: 0.5 mM; F: 0.05 mM) for 48 h. Cells were processed for immunofluorescence assays as described above. Magnification, 100.
well-known inducers of retroviruses. As shown in Fig. 4, 100% of cells became positive in response to each of the agents except NaB at 5 mM which had considerable cytotoxic effects. The release of virus by those chemicals, as determined using the TCID50, resulted in an approximately 50- to 160-fold increase in the production of infectious particles, depending on the inducer (Table 1). Since heterogeneity of FIV expression in C15 cultures under uninduced conditions was still observed, limiting-dilution techniques were re-employed twice to derive single-cell clones from C15 cells. Twenty moderately growing clones were obtained at 22 days after the first recloning, and examined for FIV antigen immunofluorescence. All clones were accompanied by a minor cell population constitutively expressing FIV antigens, although a majority of the cells did not express viral antigens (data not shown). We selected one clone designated as C15-13 for a second recloning. Fifteen clones were obtained at 25 days after the recloning, but no clonal variation with regard to virus expression was apparent (data not shown). C15-13 and C15-13-10 cells obtained after the first and second recloning, respectively, were monitored after exposure for 48 h to NaB at 0.5 mM for FIV antigen immunofluorescence and viral infectivity. Examination of those subclones derived from C15 cells revealed that 100% of the cells became positive in response to NaB treatment, however, 10–40% of uninduced cells constitutively expressed FIV antigens (data not shown). The production of infectious virus was found to be
Table 1 Effect of various chemicals on the release of infectious FIV in FeT-J/FIV C15 cells.a Treatment with
Virus yieldb (TCID50/20 ll)
Ratio
Medium alone IUdR PMA NaB (5 mM) NaB (0.5 mM) NaB (0.05 mM)
100.6 102.4 102.3 NDc 102.8 101.3
1 63 50 NAd 158 5
a C15 cells were cultured with or without chemicals for 48 h, and the supernatants harvested from each culture were tested for viral infectivity in 3201 cells. b Titers are expressed as log 10 TCID50/20 ll, based on the assay using immunofluorescence. c ND: not done. d NA: not applicable.
increased by 30- to 80-fold after induction, whereas FIV-positive cells and the infectious virus were detectable at a minimum level in the culture if left uninduced (data not shown). These results suggest that a majority of the cells are likely to exist in a non-productive state. Although these cells reverted to be low expressors when NaB was removed from cultures, their response to the drug has been found to be stable for 2 months in culture (data not shown).
4. Discussion One outstanding clinical feature of infections by lentiviruses is a relatively long period of latency characterized by a low to undetectable level of virus following an early acute phase of infection with viremia (Fauci, 1988; Hosie et al., 2009). However, especially for FIV, the mechanisms responsible for maintaining clinical latency and for the activation of latently infected cells are still poorly understood, because of a lack of appropriate in vitro models of FIV latency. We have described earlier that the feline T-lymphocyte cell line 3201 established a chronic FIV-producer cell line (3201/FIV) (Tochikura et al., 1990). In the present study, we first repeated the same experiment. It may be worth mentioning here that the cytopathic effects differed between the previous and the present experiments, although the cell lines were derived from the same source. The 3201 cells used in the previous study showed maximum kill (40% dead) at 16 dpi (Tochikura et al., 1990), whereas maximum kill (85% dead) was observed at 12 dpi in the present study. The cytopathogenic difference between the two cell lines remains to be explained, however, it might be due to differences in culture conditions across laboratories, thereby affecting the viral replication. The other T-lymphocyte cell line, FeT-J, used in the present study has been reported to be susceptible to FIV isolates and also established a chronic FIV-producer cell line (Hohdatsu et al., 1996). It has been shown that FeT-J and 3201 cells share some features: (1) both cell lines are lymphoid; (2) both are IL-2independent; and (3) both are positive for CXCR4 (Willett et al., 1997), the entry receptor for FIV, but not for CD134 (Shimojima et al., 2004), the binding receptor. Interestingly, in the present study, comparison of the MFI of CXCR4 expression on 3201 and FeT-J cells revealed that FeT-J cells expressed inherently much
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lower levels of CXCR4. This might be relevant to the finding that the initial expression of viral antigen was more rapid in 3201 cells than in FeT-J cells. A recent report (Tochikura et al., 2010) described that the infection of 3201 cells with a well-known laboratory strain, Petaluma (FIV-Pet), that does not require CD134 for adsorption (Lerner et al., 1998) led to the establishment of cells, designated as 3201-S, which were free of FIV DNA after a productive infection associated with considerable cell killing. These observations encourage further studies on how FeT-J cells behave after FIV infection. In this study, we have demonstrated that three subclones (C15, C15-13 and C15-13-10) derived from FeT-J cells chronically infected with FIV-Pet produced little virus in the uninduced state but showed markedly increased viral expression and production on stimulation with chemicals. The majority of cell populations are likely to be in a non-productive state. We are not sure why each clone was accompanied by a minor population of cells in a productive state, although the C15 cells were recloned twice to obtain C15-13 and then C15-13-10. However, this might be solely because C15 cells are unstable in terms of viral expression. It is of particular interest that FeT-J cells entered a non-productive state after infection with FIV, but 3201 cells did not. The reason 3201 cells did not enter a non-productive state remains to be elucidated. However, one possible explanation would be that another feline retrovirus, designated RD114, could be involved in the cytopathogenicity observed in 3201 cells. An etiologic role for RD114 has not been demonstrated, however, the endogenous viral sequences are rarely expressed in adult feline tissues but are expressed in some lymphomas (Niman et al., 1977; Cheney et al., 1990). In a previous study, both Mg2+ and Mn2+-dependent reverse transcriptase activities associated with FIV and RD114 virus, respectively, were induced in FIV-infected 3201 cells as compared to uninfected 3201 cells (Tochikura et al., 1990). Similar results were noted with HIV infections of human cell lines carrying human T-cell leukemia virus type 1, another human retrovirus (Harada and Yamamoto, 1987). As a result, HIV-infected cells showed marked cytopathic changes preventing further passage. Further study will be needed to ascertain whether the profound cytopathogenicity observed in FIV-infected 3201 cells is associated with the endogenous feline retrovirus, as opposed to the phenomenon in FIV-infected FeT-J cells. So far, there has been only one report regarding FIV latency in a cell line: FIV established a latent infection in the human leukemic T-lymphocyte cell line MOLT-4, and the cells were capable of producing infectious virions following treatment with PMA (Ikeda et al., 1996). However, these results contradict our previous finding that surviving MOLT-4 cells after cocultivation with 3201/FIV cells did not produce any infectious virus even after treatment with PMA (Tochikura et al., 1993 and unpublished observations). In that study, Southern blot analysis failed to detect proviral DNA and the polymerase chain reaction technique barely detected FIV-specific DNA in MOLT-4 cells. The discrepancies between these two studies, using similar materials and methods, are as yet unexplained. To our knowledge, this is the first description of FIV latency, at least in vitro in a feline cell line. The feline T-lymphocyte cell lines 3201 and FeT-J are expected to provide a useful experimental model for investigating the mechanisms of FIV latency.
5. Conclusions We have established nonvirus-producing feline T-lymphocyte cells. The results of the present study show that chemical treatment of the cells led to not only FIV protein synthesis but also an
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augmentation of viral production. The treatment resulted in more than a 100-fold increase in the production of infectious virions. The cloned cells described here provide an attractive in vitro model for studying the mechanisms of FIV latency. 6. Conflict of interest statement No author has had any financial or personal relationship with people or organizations that could inappropriately influence their work. Acknowledgements We are grateful to Dr. Lawrence E. Mathes for providing the 3201 cell line, Dr. Makoto Hitomi and Dr. Hirokazu Seo for serum samples from cats, and Hiroyuki Miwa and Ryo Higashiyama for graphics and photographic assistance. We also wish to thank Dr. Nobuyuki Itoh, Dr. Hideo Saji and Dr. Nobutaka Fujii for their constant support and encouragement. We would like to dedicate this study to the late Dr. James R. Blakeslee, Jr. The feline immunodeficiency virus (Petaluma strain) was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS (DAIDS), NIAID, NIH, from contributors Dr. Niels C. Pedersen and Dr. Janet K. Yamamoto. References Brunner, D., Pedersen, N.C., 1989. Infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus. Journal of Virology 63, 5483–5488. Cheney, C.M., Rojko, J.L., Kociba, G.J., Wellman, M.L., Di Bartola, S.P., Rezanka, L.J., Forman, L., Mathes, L.E., 1990. A feline large granular lymphoma and its derived cell line. In Vitro Cellular & Developmental Biology 26, 455–463. Clouse, K.A., Powell, D., Washington, I., Poli, G., Strebel, K., Farrar, W., Barstad, P., Kovacs, J., Fauci, A.S., Folks, T.M., 1989. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. Journal of Immunology 142, 431–438. Fauci, A.S., 1988. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239, 617–622. Folks, T.M., Justement, J., Kinter, A., Schnittman, S., Orenstein, J., Poli, G., Fauci, A.S., 1988. Characterization of a promonocyte clone chronically infected with HIV and inducible by 13-phorbol-12-myristate acetate. Journal of Immunology 140, 1117–1122. Folks, T.M., Powell, D.M., Lightfoote, M.M., Benn, S., Martin, M.A., Fauci, A.S., 1986. Induction of HTLV-III/LAV from a nonvirus-producing T-cell line: implications for latency. Science 231, 600–602. Golub, E.I., Li, G., Volsky, D.J., 1991. Induction of dormant HIV-1 by sodium butyrate: involvement of the TATA box in the activation of the HIV-1 promotor. AIDS 5, 663–668. Harada, S., Yamamoto, N., 1987. AIDS studies in Japan. Japanese Journal of Cancer Research (Gann) 78, 415–427. Hohdatsu, T., Hirabayashi, H., Motokawa, K., Koyama, H., 1996. Comparative study of the cell tropism of feline immunodeficiency virus isolates of subtypes A, B and D classified on the basis of the env gene V3–V5 sequence. Journal of General Virology 77, 93–100. Hosie, M.J., Addie, D., Belák, S., Boucraut-Baralon, C., Egberink, H., Frymus, T., Gruffydd-Jones, T., Hartmann, K., Lloret, A., Lutz, H., Marsilio, F., Pennisi, M.G., Radford, A.D., Thiry, E., Truyen, U., Horzinek, M.C., 2009. Feline immunodeficiency. ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 575–584. Ikeda, Y., Tomonaga, K., Kawaguchi, Y., Kohmoto, M., Inoshima, Y., Tohya, Y., Miyazawa, T., Kai, C., Mikami, T., 1996. Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells. Journal of General Virology 77, 1623–1630. Joshi, A., Garg, H., Tompkins, M.B., Tompkins, W.A., 2005. Different thresholds of T cell activation regulate FIV infection of CD4+CD25+ and CD4+CD25 cells. Virology 335, 212–221. Lerner, D.L., Gran, C.K., de Parseval, A., Elder, J.H., 1998. FIV infection of IL-2dependent and – independent feline lymphocyte lines: host cells range distinctions and specific cytokine upregulation. Veterinary Immunology and Immunopathology 65, 277–297. Levy, J.A., 1993. HIV pathogenesis and long-term survival. AIDS 7, 1401–1410. Niman, H.L., Gardner, M.B., Stephenson, J.R., Roy-Burman, P., 1977. Endogenous RD114 virus genome expression in malignant tissues of domestic cats. Journal of Virology 23, 578–586. Pedersen, N.C., Ho, E., Brown, M.L., Yamamoto, J.K., 1987. Isolation of a Tlymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science 235, 790–793.
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