key: cord-0004998-ie4acutk authors: Norimine, J.; Miyazawa, T.; Kawaguchi, Y.; Tomonaga, K.; Shin, Y. -S.; Toyosaki, T.; Kohmoto, M.; Niikura, M.; Tohya, Y.; Mikami, T. title: Feline CD 4 molecules expressed on feline non-lymphoid cell lines are not enough for productive infection of highly lymphotropic feline immunodeficiency virus isolates date: 1993 journal: Arch Virol DOI: 10.1007/bf01319005 sha: 99556ef237004b9aff6368c7de7862079d06e513 doc_id: 4998 cord_uid: ie4acutk To investigate whether the feline CD 4 (fCD 4) molecules are involved in infections of highly lymphotropic feline immunodeficiency virus (FIV) isolates, we expressed fCD 4 stably on Crandell feline kidney cells andFelis catus whole foetus 4 cells by transfection of a cDNA encoding the fCD 4 glycoprotein, and then infected them with TM 1 and TM 2 strains of FIV, which are unable to infect these cells productively. In spite of fCD 4 being expressed on these cells, no virus production was observed. This result indicates that fCD 4 expression alone cannot induce a productive infection of the FIV TM 1 and TM 2 strains. To investigate whether the feline CD 4 (fCD 4) molecules are involved in infections of highly lymphotropic feline immunodeficiency virus (FIV) isolates, we expressed fCD 4 stably on Crandell feline kidney cells and Felis catus whole foetus 4 cells by transfection of a cDNA encoding the fCD 4 glycoprotein, and then infected them with TM 1 and TM 2 strains of FIV, which are unable to infect these cells productively. In spite of fCD 4 being expressed on these cells, no virus production was observed. This result indicates that fCD 4 expression alone cannot induce a productive infection of the FIV TM 1 and TM 2 strains. The CD 4 molecule is a major receptor for human immunodeficiency virus type 1 (HIV-1) [-6, 12, 13, 15] , but the other molecules are also implicated in CD4independent infection [-3, 7, 8, 26, 30] . Feline immunodeficiency virus (FIV) is an etiological agent of the acquired immunodeficiency-like diseases in cats [25] . Whether the FIV utilizes feline CD 4 (fCD 4) molecules as a receptor or not is unknown at present. However, the decrease of fCD 4/feline CD 8 (fCD 8) T cell ratio after experimental infection of cats with FIV was reported [2, 23] . In addition, our FIV isolates, TM 1 and TM 2 strains can productively infect fCD 4 + fCD 8-MYA-1 cells but not fCD 4 -fCD 8-FL 74 cells [ 14, [17] [18] [19] [20] . There are some other Japanese isolates which are also infectious to fCD4 + fCD 8-cell line (Fel-039 cells) [31] . These results imply that fCD4 is one of the candidate molecules for the FIV receptor. On the other hand, it has been reported that both CD 4 + and CD 8 + cells were productively infected with FIV in vitro [4] . Furthermore, Yamamoto et al. [32] reported that Crandell feline kidney (CRFK) cells were also productively infected by a Petaluma strain of FIV. However, we could not detect any virus production in fCD 4-negative CRFK and Felis catus whole foetus 4 (fcwf-4) cells by infection with cell-free FIV TM 1 or TM 2 strain. These contradictory results may be due to the difference in the host range between these virus strains. To clarify the role of fCD 4 on FIV infection, we used feline CD 4 cDNA, termed as FT 121 which had been cloned in a previous study [21] . In the present study, we established CRFK and fcwf-4 cells on which fCD 4 molecules were stably expressed by transfection of the expression plasmid containing FT 121 and selective marker gene. The purpose of the present study is to examine the role of fCD 4 on infection of FIV TM 1 and TM 2 strains, using these fCD 4 expressing cells. C R F K [5] and fcwf-4 [9] cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated foetal calf serum (FCS) and antibiotics. MYA-1 cells were cultured in RPMI 1640 growth medium supplemented with 10% FCS, antibiotics, 50 gM 2-mercaptoethanol, 2~tg/ml polybrene and 100 units/ml of recombinant human interleukin-2 at 37 °C in a humidified atmosphere of 5% CO2 in air. For preparation of virus stocks of FIV TM 1 and TM 2 strains, MYA-1 cells were infected with a low passaged FIV TM 1 or TM 2 strain, and then the supernate was harvested after 8 day-incubation when the virus titer reached a plateau [11] . These stock viruses were passed through a 0.45 gm Milipore filter and stored at -80 °C in 1 ml aliquots until use. The titration of the stock viruses was carried out as described previously [11] . To express the fCD4 (FT 121) in CRFK and fcwf-4 cells, we chose Rous sarcoma virus (RSV) long terminal repeat (LTR) for the promoter of the gene because of the strong activity of the RSV LTR in the cells [22] . Eco RI-linked FT 121 was blunted and inserted into blunted Hin dIII site of pRVSVneo [28] , which contained neo resistant gene under the control of simian virus 40 early gene promoter. This expression plasmid was designated as pRVSVfCD 4neo. The construction of the plasmid was shown in Fig. 1 . To establish the C R F K and fcwf-4 cells which stably express the fCD 4, CRFK, and fcwf-4 cells grown in six-well dishes were transfected with 5 gg of pRVSVfCD 4neo DNA by the phosphate calcium coprecipitation method [28] . Two days after transfection, the cell medium was replaced with the one containing 200 gg/ml G 418 (Geneticin; Gibco, BRL Life Technologies Inc., Gaithersburg, U.S.A.). The selected cells were maintained in the medium with the same concentration of G 418. For examination of fCD 4 expression, the cells (Fig. 2) . The fCD 4-expressed C R F K and fcwf-4 cells were referred to Cf4 and if4 cells, respectively. As shown in Fig. 2 (flow cytometric analysis) and Fig. 3 (indirect immunofluorescence assay, IFA), the efficiency of fCD 4 expression varied in each cell. This variation was thought to have been caused by the heterogenous population of the cells, because these cells had not been cloned. Maddon et al. reported the HeLa cells stably expressing CD 4, which were permissive for productive infections of HIV-1 [13] . In this case, the cells were cloned cells and CD 4 expression was very high. We did not clone the transfected cells, however, the expression efficiency of fCD 4 as observed in Cf4 and ff4 cells seemed to be sufficient for the infection assay for FIV, as long as the virus uses fCD 4 efficiently as the receptor. Next, we examined the virus susceptibility of these cells. CRFK, fcwf-4, Cf4 and if4 cells were infected with FIV TM 1 or TM 2 strain at an moi of 0.3 TCIDs0. The cells were passaged by 0.2% trypsin treatments and washed every 6 or 7 days. Twenty days after inoculation, the culture supernate was inoculated to MYA-1 cell cultures for the virus rescue. Simultaneously, these cells were cocultured with MYA-1 cells. Twenty day-incubation period was enough to avoid residual virus contamination from the inoculum used, because these isolates were known to be completely inactivated within a 14-day incubation at 37°C as previously reported [10] . The MYA-1 cells were periodically ex- Indirect stains were visualized with a rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate for 30 rain on ice amined for virus production by the IFA [16, 17] and the reverse transcriptase (RT) assay [24] . Though this experiment was repeated more than 5 times, we could not detect any evidence for virus production of FIV TM 1 or TM 2 strain (data not shown). Furthermore, we investigated the possibility of the cDNA synthesis of FIV in these cells. For detection of viral cDNA, we used the polymerase chain reaction (PCR) method using the primer flanking the part of gag gene of FIV. To avoid the viral DNA contamination, stock virus was treated with 200 gg/ ml of DNase (Boehringer Mannheim Yamanouchi, Co., Tokyo, Japan) for 1 h at 37 °C before inoculation. For virus infection, the cells were incubated with the DNase-treated virus for 15 h and washed in PBS three times. Five days after infection, FIV inoculated cells were harvested, and the cells were washed in PBS, lysed in proteinase K buffer (100 mM Tris-HC1 pH 7.5, 12.5 mM EDTA, 150M NaC1, 1% SDS, 200 gg/ml proteinase K) and then subjected to phenolchloroform extraction and ethanol precipitation. The resultant total DNAs were subjected to PCR amplification. For amplification of a gag fragment of FIV TM 1 and TM 2 strains, the primer 5'-CTGGTGATCCTACTTCTTGGCAGGC-3', nucleotides 1687--1663, was designed as an antisense primer, and the primer 5'-CTAGGAGGTGAGGAGGTCCAACTGTG-3', nucleotides 1126-.1151, Fig. 3 . Indirect immunofluorescence assay, a Cf4 cells, b CRFK cells, c ff4 cells, d fcwf-4 cells. Cells were reacted with the anti-feline CD 4 monoclonal antibody (Fel 7), and then with a a-mouse IgG conjugated with fluorescein isothiocyanate as a sense primer. The sequences of primers were derived from the sequence of FIV TM 2 strain [14] . PCR was carried out by the method of Saiki et al. [27] in a 50 lal volume overlaid with an equal volume of mineral oil. A GeneAmp PCR Reagent kit (Perkin Elmer Cetus, Norwalk, U.S.A.) was used for the reactions. Amplification proceeded for 30 cycles in a Thermal Cyclic Reactor Model TC-100 (Hoei Science Co., Tokyo, Japan). One cycle consisted of incubations at 94, 58, and 72°C for 1, 1, and 2min, respectively. After amplification, 10 gl of the 50 gl-reaction were electrophoresed on a 2% agarose gel (in Tris-borate-EDTA buffer). Fractionated DNA was transferred to a nylon membrane, and cross-linked by UV. Hybridization was carried out for 18 h at 50 °C in a solution containing 50% formamide, 6 x SSC (1 x SSC: 0.15M NaC1 and 0.015 M sodium citrate), 0.1% SDS, 5 x Denhardt's solution, 100 ~tg/ml denatured salmon sperm DNA and the 32p-labelled probe DNA. The amplified DNA from TM 2 clone was used as a probe DNA. Figure 4 shows the results of the PCR analysis. The cDNA of FIV gag gene was detected in the MYA-1 cells infected with FIV TM 1. However, we could not detect any positive band in the CRFK, fcwf-4, Cf4 and ff4 cells infected with the virus. Similar results were obtained for the FIV TM 2 (data not shown). The data obtained in this study revealed that FIV TM 1 and TM 2 strains cannot infect either of the cells irrespective of the fCD 4 expression on the cells. Therefore, we concluded that fCD 4 expression on CRFK and fcwf-4 cells is not sufficient for the induction of a productive infection of FIV TM 1 and TM 2 strains. An FIV DNA clone containing a full length of TM 1 or TM 2 strain produces the virus in CRFK and fcwf-4 cells by gene-transfection [14, 19; Miyazawa et al., unpubl, data] . Therefore, we considered that the failure of FIV TM 1 and TM 2 strains to infect the cells was due to the blockage at an early stage of infection, such as the viral adsorption, penetration, and cDNA synthesis. From our data, it is unlikely that fCD 4 alone is involved in the infection of highly lymphotropic FIV such as TM 1 or TM 2 isolate, and it is likely that some other molecules participate in virus-cell interaction, in addition to fCD 4. However, at the moment we cannot exclude the possibility that the FIV TM 1 and TM 2 strains can penetrate in the cells and the incomplete cDNA synthesis of the virus occurs as reported in the case of the HIV-1 infection in stationary cells [29] or quiescent primary lymphocytes [33, 34] . Identification of a CD 4 homologue in the cat Immunologic abnormalities in pathogen-free cats experimentally infected with feline immunodeficiency virus Complement mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD 4-and antibody-independent fashion Feline immunodeficiency virus infects both CD 4 + and CD 8 + T lymphocytes Development, characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK) The CD 4 (T 4) antigen is an essential component of the receptor for the AIDS retrovirus Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-1 induced syncitum formation The Fc and not CD4 receptor mediates antibody enhancement of HIV infection in human cells Expression of feline infectious peritonitis coronavirus antigens on the surface of feline macrophage-like cells Replicative difference in early-passage feline brain cells among feline immunodeficiency virus isolates Quantification of feline immunodeficiency virus in a newly established feline T-lymphoblastoid cell line (MYA-1 cells) T-lymphocyte T 4 molecule behaves as the receptor for human retrovirus LAV The T 4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain Molecular characterization and heterogeneity of feline immunodeficiency virus isolates Binding of HTLV-III/LAV to T4 + T cells by a complex of the l l 0 K viral protein and the T4 molecule Preliminary comparisons of the biological properties of two strains of feline imrnunodeficiency virus (FIV) isolated in Japan with FIV Petaluma strain isolated in the United States Establishment of a feline T-lymphoblastoid cell line highly sensitive for replication of feline immunodeficiency virus Continuous production of feline immunodeficiency virus in a feline T-lymphoblastoid cell line (MYA-1 cells) Molecular cloning of a novel isolate of feline immunodeficiency virus biologically and genetically different from the original U. S. isolate Feline CD4 molecules and FIV infection Further characterization of a feline T-lymphoblastoid cell line (MYA-1 cells) highly sensitive for feline immunodeficiency virus A cDNA encoding feline CD 4 has a unique repeat sequence downstream of the V-like region Comparison of the viral promoter activities in feline cell lines (CRFK and fcwf-4 cells) Lymphocyte population changes in cats naturally infected with feline immunodeficiency virus Isolation of simian immunodeficiency virus from African green monkeys and seroepidemiologic survey of the virus in various non-human primates Isolation ofa T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome Complement-mediated antibodydependent enhancement of H1V-1 infection requires CD 4 and complement receptors Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey HIV-1 replication is controlled at the level of T-cell activation and proviral integration Antibody-enhanced infection by HIV-I via Fc receptor-mediated entry Altered cell tropism and cytopathicity of feline immunodeficiency viruses in two different feline CD 4-positive, CD 8-negative cell lines Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle We are grateful to Dr. A. Adachi (Kyoto University, Kyoto, Japan) for providing pRVSVneo and we thank Drs. Momoi and Tsujimoto (The University of Tokyo, Tokyo, Japan) for supplying gag primers. This study was supported in part by grants from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare of Japan. J. Norimine was partly supported by the Supporting Organization of JOCV. T. Miyazawa and Y. Kawaguchi are supported by the Recruit Scholarship. Received August 26, 1992