Focus Assay on FeLIX-Dependent Feline Leukemia Virus

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1 NOTE Virology Focus Assay on FeLIX-Dependent Feline Leukemia Virus Yuki NAKAYA 1), Takayuki SHOJIMA 1), Shigeki HOSHINO 1) and Takayuki MIYAZAWA 1) 1) Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto , Japan (Received 23 April 2009/Accepted 20 September 2009/Published online in J-STAGE 13 November 2009) ABSTRACT. T-lymphotropic feline leukemia virus (FeLV-T) induces immunodeficiency in cats. FeLV-T is fusion-defective and requires a cofactor, termed FeLIX, for infection. FeLIX is a truncated envelope glycoprotein of an endogenous FeLV and mediates infection by binding a phosphate transporter Pit-1. In this study, we established a feline sarcoma-positive leukemia-negative cell line expressing FeLIX, named QN/FeLIX cells. Upon infection, FeLV-T induced prominent foci with syncytia in QN/FeLIX cells and could be titrated by the focus assay. In addition, we established a FeLIX-expressing feline fibroblast cell line, named AH/FeLIX cells. FeLV-T productively infected AH/FeLIX cells and induced severe CPE with syncytia. QN/FeLIX and AH/FeLIX cells will be useful for the study of FeLIX-dependent mutants in FeLV-infected cats. KEY WORDS: AIDS, feline, feline leukemia virus, focus assay. J. Vet. Med. Sci. 72(1): , 2010 *CORRESPONDENCE TO: Dr. MIYAZAWA, T., Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto , Japan. takavet@goo.jp Feline leukemia virus (FeLV) belongs to a member of the genus gammaretrovirus and induces a variety of diseases, including leukemia, anemia and immunodeficiency [13]. FeLV is classified into three receptor interference subgroups: A, B and C [8, 21]. FeLV-A is a transmissible form of FeLV and is basically ecotropic [16]. FeLV-B and -C have an expanded host range that includes various mammalian cells. Besides these subgroups, Overbaugh et al. cloned a replication-defective mutant of FeLV that causes immunodeficiency in cats infected with a mixture of the mutant and a replication-competent FeLV-A [15, 17]. This variant was reconstructed to be a replication-competent form by the genetic engineering technique and the determinant of the disease was proved to reside in the env gene of the defective mutant [5, 17]. Later, the reconstructed virus was named T-lymphotropic FeLV (FeLV-T) because it does not infect feline fibroblast cell lines but infects feline T-lymphoma cell lines, such as 3201 cells [1, 9, 10]. Gammaretroviruses, including FeLV, have a consensus motif, termed the PHQ motif, which resides at the amino terminus of the surface unit (SU) of envelope protein (Env). Mutations of this motif, especially histidine residue, result in a defect in the membrane fusion process, although they do not affect the incorporation of Envs into virions and receptor binding (Fig. 1) [2, 11, 17]. FeLV-T also has a substitution of histidine with aspartate in this motif [5]; therefore, FeLV- T is fusion-defective. For the infection of FeLV-T, FeLIX, a truncated Env encoded by a defective endogenous FeLV, is required. FeLIX mediates the infection of FeLV-T by binding a phosphate transporter Pit-1 [1]. The schematic view of the FeLIX-dependent (cognate receptor-independent) infection is shown in Fig. 1. Binding of receptor binding domain (RBD) to the cognate receptor triggers the conformational change of SU and the disulfide isomerization enhanced by PHQ motif in N-terminus of SU (Fig. 1A). Release of SU from TM triggers the conformational change of TM, resulting in the membrane fusion. FeLV-T Env may bind its cognate receptor but lacks the ability to isomerize the disulfide bond. Although FeLV-T Env does not bind FeLIX receptor (Pit-1), FeLIX binds Pit1 and mediates the disulfide isomerization of FeLV-T Env, leading to the membrane fusion (Fig. 1B). Focus assays using sarcoma-positive leukemia-negative (S+L ) cells have been widely used to detect and quantitate infectious (i.e., replication-competent) gammaretroviruses. Feline S+L QN10S cells have been widely used to detect and quantitate FeLV subgroups A, B and C [6, 16]. QN10S cells express murine sarcoma virus (MSV) genome containing v-mos oncogene at a very low level. Once infectious FeLVs infect QN10S cells, mrna of MSV is rescued by packaging in the infectious viral particles. The virus containing MSV mrna re-infects the naïve QN10 cells in the vicinity of the initially infected cells, and then the infected cells are transformed by v-mos gene, leading to the formation of visible foci composed of the transformed cells. However, it was not possible to apply the QN10S cells to FeLV-T, because the cells do not express FeLIX at a detectable level. In this study, we established QN10S cells expressing FeLIX stably and developed a focus assay to quantitate infectious FeLV-T. In addition, we established a FeLIX-expressing feline fibroblast cell line which supports productive infection by FeLV-T. Human embryonic kidney (HEK) 293 cells [22], QN10S cells [6], AH927 cells (a feline fibroblast cell line) [12], and TELCeB6 cells (a derivative of TE671 cells (human rhabdomyosarcoma cells)) [4] were cultured in Dulbecco s modified Eagle s medium (Sigma-Aldrich, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 units/ml) and streptomycin (100 g/ml)

2 118 Y. NAKAYA, T. SHOJIMA, S. HOSHINO AND T. MIYAZAWA Fig. 1. Diagrams of conventional (receptor-dependent) (A) and receptor-independent (FeLIX-dependent) (B) entry mechanisms. White ovals: C-terminal region (C) and RBD of SU; grey oval: FeLIX; black box: membrane proximal region of TM; black triangle: fusion peptide of TM; dotted line: disulfide bond between SU and TM; double horizontal line: plasma membrane of the target cell; undulating lines: viral cognate receptor (black) and FeLIX receptor (grey). (Invitrogen, Carlsbad, CA, U.S.A.). These cells were cultured at 37 C in a humidified atmosphere of 5% CO 2 in air. To prepare lacz pseudotype viruses, the env expression plasmids of FeLV-A, B, C and T, and amphotropic murine leukemia virus (A-MLV), termed pfbfelv-a [16], pfbfelv-b [16], pfbfelv-c [16], pfbfelv-t [23] and pfbmosalf [4], were transfected using FuGENE6 (Roche, Basel, Switzerland) into TELCeB6 cells, which express large amounts of MLV core particles incorporating an MFGnlsLacZ vector [4]. The transfectants were selected with 50 g/ml phleomycin, and designated as TELCeB/ FBFeLV-A, TELCeB/FBFeLV-B, TELCeB/FBFeLV-C, TELCeB/FBFeLV-T, and TELCeB/SALF cells, respectively. Culture supernatants were harvested from the pooled phleomycin-resistant cell population, filtered through a 450- nm membrane filter, and used immediately in the pseudotype virus infection assay described below. The pseudotype viruses prepared from these cells were designated as lacz (FeLV-A), lacz (FeLV-B), lacz (FeLV-C), lacz (FeLV-T) and lacz (A-MLV), respectively. Titration of the lacz pseudotype viruses was performed as described by Takeuchi et al. [25]. Briefly, assay cells were seeded in 48-well plates 1 day before infection. After 4 hr of infection in the presence of 8 g/ml polybrene (hexadimethrine bromide) (Sigma-Aldrich) for viral adsorption, the virus solution was removed and cells were cultured in growth medium. Two days after infection, cells were stained with 5-bromo-4-chloro-3-indoyl- -D-galactopyranoside in situ, and lacz-positive foci were counted. To prepare stock viruses of FeLVs, we transfected infectious molecular clones of FeLV into human embryonic kidney (HEK) 293 cells to avoid recombination with the endogenous FeLV genome present in feline cells [18]. One g each of infectious molecular clones of FeLV-A, B, C, and T, named p61e [17], pgahf [24], pfsc [19], and peecc [17], respectively, were transfected twice into HEK293 cells using FuGENE6. Two weeks after transfection, culture supernatants were harvested, filtered through a 450-nm membrane filter, and stored at 80 C as stock viruses. Firstly, QN10S and AH927 cells were transfected with 1 g pfbfelix (an expression plasmid of FeLIX) [23] using FuGENE6. After the selection of transfectants with 50 g/ ml phleomycin, the cells were designated as QN/FeLIX and AH/FeLIX cells, respectively. Infectivities of lacz pseudotype viruses of FeLV-A, B, C, and T, and A-MLV were then compared in QN10, QN/FeLIX, AH927 and AH/FeLIX cells. lacz (FeLV-A), lacz (FeLV-B), lacz (FeLV-C) and lacz (A-MLV) infected all these cell lines efficiently (Fig. 2). On the other hand, lacz (FeLV-T) did not infect QN10S and AH927 cells efficiently but infected QN/FeLIX and AH/FeLIX cells as efficiently as the other pseudotype viruses (Fig. 2). Although QN10S and AH927 cells do not express FeLIX proteins examined by immunoblot analysis (data not shown), both cells were susceptible to lacz (FeLV- T) marginally. Precise mechanism of the infection is

3 FOCUS ASSAY ON FELIX-DEPENDENT FELV 119 Fig. 2. Sensitivity to lacz pseudotype viruses. lacz pseudotype viruses were inoculated into (A) AH927 ( ) and AH/FeLIX cells (+) and (B) QN10 ( ) and QN/FeLIX cells (+). Three independent experiments were conducted and averages of viral titers with standard deviations determined by counting blue foci are shown Fig. 3. Foci induced by FeLV-T in QN/FeLIX cells. QN10S (A) and QN/FeLIX cells (B) were inoculated with FeLV-T and prominent foci with syncytia were observed in QN/FeLIX cells at 7 d.p.i.. The cells were observed with a phase-contrast microscope. unknown at present; however, it is supposed that both cells express FeLIX protein at an undetectable level. McDougall et al. reported that FeLIX (referred to as 35kDa protein) produced from 3201 cells (feline thymic lymphoma) blocked infection by FeLV-B on C61 cells by the receptor interference mechanism [12]. In this study, however, both QN/ FeLIX and AH/FeLIX cells were highly susceptible to lacz (FeLV-B). Similarly, Anderson et al. observed that FeLIX did not block infection by FeLV-B [1]. To block FeLV-B infection in QN10S and AH927 cells, a higher expression of FeLIX may be needed. Next, the focus-inducing activity of FeLV-T was examined in QN10S and QN/FeLIX cells. The cells were seeded at a density of per well in 24-well plates one day before infection. Serially diluted stock virus of FeLV-T in 500 l medium was inoculated into cells in the presence of 8 g/ml polybrene. The cells were incubated at 37 C for 2 hr for viral adsorption and the inoculum was then replaced with fresh medium. No focus was observed in QN10S cells, whereas prominent foci with syncytia were observed in QN/ FeLIX cells 7 days post-infection (d.p.i.) (Fig. 3). These data indicate that QN/FeLIX cells can be applied for the focus assay to detect and titrate FeLV-T. Stock viruses of various subgroups of FeLV were then titrated on QN10S and QN/FeLIX cells by the focus assay as described previously [20]. Consequently, we succeeded in titrating FeLV- T in QN/FeLIX cells and could not find any significant differences between QN10S and QN/FeLIX cells in the titers of stock viruses of FeLV-A, B and C (Fig. 4). These data also indicated that QN/FeLIX cells will be useful for the titration of all subgroups (A, B, and C) in addition to FeLV- T by the focus assay. To examine whether AH/FeLIX cells support productive infection by FeLV-T, AH927 and AH/FeLIX cells were inoculated with FeLV-T. No CPE was observed in AH927 cells; however, severe CPE with syncytia was observed in AH/FeLIX cells at 8 d.p.i. and almost all infected cells died at 12 d.p.i.. The FeLIX produced from AH/FeLIX cells may have induced a conformational change of FeLV-T Env

4 120 Y. NAKAYA, T. SHOJIMA, S. HOSHINO AND T. MIYAZAWA Fig. 4. Viral titers of stock viruses of FeLV subgroups A, B, C and T. Three independent experiments were conducted and averages of viral titers with standard deviations determined by the focus assay using QN10S ( ) and QN/FeLIX cells (+) are shown. expressed on the cells, leading to cell fusion. Finally, the growth kinetics of FeLV-T in AH/FeLIX cells was examined by the focus assay using QN/FeLIX cells. At 8 d.p.i., the viral titer peaked, but drastically dropped at 12 d.p.i. due to severe CPE in AH/FeLIX cells (Fig. 5). On the other hand, no incidence of productive infection of FeLV-T was observed in AH927 cells. In this study, using QN/FeLIX cells, we succeeded in titrating infectious FeLV-T by the focus assay. In FeLVinfected cats, Env mutants in the PHQ motif were identified by polymerase chain reaction [3]; however there has been no report so far on the isolation of FeLIX-dependent and replication-competent FeLV, because FeLV has been isolated by using FeLIX-negative cell lines, such as FEA cells [7]. It was reported that about 10% of cats diagnosed as positive for the FeLV antigen were negative in the virus isolation test, and these cases were called discordant cats. [7, 14]. It is possible that replication-competent FeLV mutants in the PHQ motif are present in a portion of discordant cats and the mutants grow efficiently in cats by infecting cells expressing FeLIX. QN/FeLIX and AH/FeLIX cells will be useful for the study of FeLIX-dependent mutants in FeLV-infected cats. ACKNOWLEDGMENTS. We are grateful to Dr. Y. Takeuchi (University College London, London, U.K.) for providing TELCeB6 cells and pfbmosalf. We thank Prof. E. A. Hoover (Colorado State University, CO, U.S.A.) Fig. 5. Growth kinetics of FeLV-T in AH/FeLIX cells. FeLV-T prepared from HEK293 cells transfected with peecc was inoculated into AH/FeLIX ( ) and AH927 cells ( ). As controls, viral titers in the culture supernatants of mock-infected AH/ FeLIX ( ) and AH927 ( ) are also shown. Three independent experiments were conducted and averages of viral titers with standard deviations determined by the focus assay using QN/ FeLIX cells are shown. for providing p61e and peecc. We are also grateful to Prof. J. C. Neil and Prof. O. Jarrett (Glasgow University, Glasgow, U.K.) for providing QN10S cells, AH927 cells, pfsc and pgahf. This study was supported by grants from the Ministry of Education, Culture, Science and Sports of Japan and from the Bio-oriented Technology Research Advancement Institution. REFERENCES 1. Anderson, M. M., Lauring A. S., Burns, C. C. and Overbaugh, J Identification of a cellular cofactor required for infection by feline leukemia virus. Science 287: Bae, Y., Kingsman, S. M. and Kingsman, A. J Functional dissection of the Moloney murine leukemia virus envelope protein gp 70. J. Virol. 71: Chandhasin, C., Coan, P. N. and Levy, L. S Subtle mutational changes in the SU protein of a natural feline leukemia virus subgroup A isolate alter disease spectrum. J. Virol. 79: Cosset, F. L., Takeuchi. Y., Battini, J. L., Weiss, R. A. and Collins, M. K High-titer packaging cells producing recombinant retroviruses resistant to human serum. J. Virol. 69: Donahue, P. R., Quackenbush, S. L., Gallo, M. V., denoronha, C. M., Overbaugh, J., Hoover, E. A. and Mullins, J. I Viral genetic determinants of T-cell killing and immunodeficiency disease induction by the feline leukemia virus FeLV- FAIDS. J. Virol. 65: Jarrett, O. and Ganière, J. P Comparative studies of the efficacy of a recombinant feline leukemia virus vaccine. Vet. Rec. 138: 7 11.

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