Journal of General Virology (1992), 73, 1543-1546. Printed in Great Britain
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Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone Takayuki Miyazawa, 1 Yasushi Kawaguchi, 1 Mariko Kohmoto, 1 Jun-ichi Sakuragi, 2 Akio Adachi, 2 Masashi Fukasawa 3 and Takeshi MikamP* 1Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, 1-1-1 YayoL Bunkyo-ku, Tokyo 113, 2Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606 and 3Department of Preventive Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan
An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) nonlymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when
the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAG M LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.
Human and animal lentiviruses cause a persistent infection of their host (for a review see Narayan & Clements, 1990). The feline lentivirus, feline immunodeficiency virus (FIV), also causes persistent infection and induces immunodeficiency-like diseases in cats (Pedersen et al., 1987; Yamamoto et al., 1988, 1989; Ishida et al., 1988, 1989). FIV is considered to be highly species-specific (Yamamoto et al., 1988), infecting feline T lymphocytes (Pedersen et al., 1987; Yamamoto et al., 1988; Miyazawa et al., 1989b), macrophages (Brunner & Pedersen, 1989) and brain cells (Dow et al., 1990), but not human, rabbit, mouse or dog lymphocytes in vitro (Yamamoto et al., 1988). Furthermore, monkey (COS and Vero) and human (HeLa, SW480 and Molt-4) cell lines can not be infected using cell-free FIV strain TM 1 (unpublished data). Transfection of cells with an infectious molecular clone avoids any barrier imposed by the interaction of virus particles with their receptors during infection, allowing the intracellular restriction affecting expression of viral genes to be examined readily. The purpose of this study was to evaluate the potential restriction of FIV replication in various non-lymphoid cell lines, including Crandell feline kidney (CRFK), Vero, COS, HeLa and
SW480, and the possible involvement of the basal promoter activity of the FIV long terminal repeat (LTR) in this restriction. The construction of infectious molecular clones of four different strains of FIV has been reported (Olmsted et al., 1989; Phillips et al., 1990; Miyazawa et al., 1991; Maki et al., 1992). The FIV Petaluma clone can infect and replicate in non-lymphoid CRFK cells (Olmsted et al., 1989; Phillips et al., 1990), whereas the other clones (FIV PPR, TM1 and TM2) can replicate in CRFK cells only after gene transfection, and not after inoculation of virions derived from the transfected cells (Phillips et al., 1990; Miyazawa et al., 1991 ; Maki et al., 1992). These data suggest that there are differences in cell tropism among various FIV strains, FIV Petaluma seemingly having a wider host cell range. In this study, we used an infectious molecular clone of FIV strain TM1, pFTM191 CG, isolated and cloned in our laboratory (Miyazawa et al., 1989a, 1991). The HeLa (human epithelioid carcinoma cells), SW480 (human colon carcinoma cells) (Adachi et al., 1986), COS (monkey kidney cells transformed by simian virus 40) (Gluzman, 1981), Vero (monkey kidney cells) and CRFK cells (feline kidney cells) (Crandell et al.,
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1973) used were grown in Dulbecco's modified Eagle's medium supplemented with 10 ~ foetal calf serum (FCS) and antibiotics. The MYA-1 cells (Miyazawa et al., 1989b) were grown in R P M I 1640 growth medium supplemented with 1 0 ~ FCS, antibiotics, 50 laM2-mercaptoethanol, 2 lag/ml polybrene and 100 units/ml of recombinant human interleukin-2 (IL-2) at 37 °C in a humidified atmosphere of 5 ~ CO2 in air. For transfection of plasmids, cells grown to 9 0 ~ confluence in six-well dishes were used. Five micrograms of pFTM191 CG or 2 to 10 lag of L T R reporter plasmids carrying a bacterial chloramphenicol acetyltransferase (CAT) gene was transfected into cells by the calcium phosphate coprecipitation method (Graham & van der Eb, 1973). CAT constructs under the control of the L T R of each of three primate lentiviruses and FIV have been described previously (Shibata et al., 1990a; Kawaguchi et al., 1991). They were made by placing L T R fragments in front of the CAT gene of p H d C A T (Shibata et al., 1990 a). Each construct contains a complete L T R derived from the infectious molecular clones pNL432 (Adachi et al., 1986), pS'A212 (Shibata et al., 1990b), pGH123 (Shibata et al., 1990 a) or p F T M 191 C G (Miyazawa et al., 1991). Expression of pH 1CAT, pH2CAT, pSACAT and p T M 1 C A T is directed by the L T R of human immunodeficiency virus type 1 (HIV-1), HIV-2, simian immunodeficiency virus of African green monkey (SIVA~M) and FIV, respectively. The p H d C A T , which has a CAT gene and poly(A) signal, was used as a negative control plasmid. For the CAT assay, cell monolayers in each well of sixwell dishes were harvested by scraping 48 h after transfection. After being washed once with cold PBS, the cells were lysed by freezing and thawing four times in 100 ~1250 mM-Tris-HC1 pH 7-8. Cell debris was pelleted by centrifugation, and various amounts of each extract were assayed for CAT activity (Gorman et al., 1982) by the solvent partition method (Neumann et al., 1987). In brief, a 240 lal reaction mixture containing 100 mMTris-HC1 pH 7.8, 1.0 mM-chloramphenicol, 3-7 kBq [14C]acetyl coenzyme A (Du Pont, N E N ) and cell extract was overlaid with 5 ml of scintillation fluid (Econofluor; Du Pont, NEN). Reactions were carried out at 37 °C and production of radioactively labelled acetylchloramphenicol was monitored by counting in a liquid scintillation counter. All the CAT assay data reported in this paper are from points in the linear range of the assay. The CAT activity of each reporter plasmid, expressed as the net d.p.m, product formed/h, was calculated after subtraction of the background level obtained with pHdCAT. For measurement of virus production, the Mg 2+dependent reverse transcriptase (RT) activity assay was conducted as described previously (Ohta et al., 1988).
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Fig. 1. (a) Kinetics of RT activity in feline and non-feline cells transfected with pFTM191 CG. The cells were grown to 90~ confluence in 60 mm diameter dishes and transfected with 5 ktg of uncleavedpFTM 191 CG. (b) Kinetics of RT activity in MYA-1 cellsto which the culture supernatant of transfectedcellshad been transferred. Three days p.t., 1 ml of culture supernatant from transfected cells was transferredonto 1 × 106 MYA-1 cells. (a and b) CRFK (e), HeLa (11), SW480 (D), COS (A) and Vero (A) cells.
FIV-specific antigen in cells was detected by the indirect immunofluorescence assay (IFA), also described previously (Miyazawa et al., 1989b). First, we transfected pFTM191 C G into feline (CRFK), human (HeLa and SW480) and simian (Vero and COS) cells, and assayed virus in the culture supernatant by measuring RT activity (Fig. l a). In C R F K cell cultures, RT activity was detected 3 days post-transfection (p.t.) and then decreased gradually, disappearing 20 days p.t., as reported previously (Miyazawa et al., 1991). In non-feline cell lines, weak RT activity was detected only in Vero cell supernatants at 3 days p.t. ; no RT activity was found in HeLa, COS or SW480 cell cultures.
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To confirm virus production by transfected cells, 1 ml of culture supernatant from the transfected cells (3 days p.t.) was transferred to 1 × 106 feline T lymphoblastoid MYA-1 cells, which are highly sensitive to FIV (Miyazawa et al., 1989b). Virus production in the culture was monitored by detecting R T activity and confirmed by IFA. Both RT activity and FIV-specific antigen were found in MYA-1 cells inoculated with the culture supernatants of p F T M 191 CG-transfected C R F K , Vero and HeLa cells, but not in those inoculated with the culture supernatants of transfected COS and SW480 cells (Fig. l b). These results indicated that HeLa cells transfected with pFTM191 C G produce virus with undetectable levels of RT activity. The RT activity assay is known not to be applicable for the detection of FIV strain TM1 with a titre of less than 10 2 TCIDs0/ml (Kawaguchi et al., 1990), so it is perhaps not surprising that a small amount of virus released from HeLa cells became detectable on MYA-1 cells. Furthermore, the RT activity did not increase in transfected C R F K , HeLa and Vero cells, indicating that the virus produced was not infectious for these cells, even if direct cell-to-cell contact and one-cycle replication of FIV occurred. The reason that virus produced from Vero cells (with detectable RT activity) is less infectious than that produced from HeLa cells (undetectable RT) is not clear at present. From these data, we concluded that the interaction between FIV particles and their receptor(s) is the principal determinant of cell tropism. Once cell surface restriction is bypassed, viral R N A and proteins are synthesized and assembled into infectious virus, even in non-feline cells. However, replication of FIV was undetectable in SW480 and COS cells, suggesting that it is also controlled by intracellular factors. Next, to examine one of the intracellular mechanisms controlling FIV replication, the basal promoter activity of the FIV L T R was examined in these non-lymphoid cell lines by the CAT assay, and compared with those of three primate lentivirus LTRs (HIV-1, HIV-2 and SIVAGM) after transfection of the lentivirus L T R - C A T plasmids into these cells. Fig. 2 (a) shows measurements of CAT activity in cell extracts made 48 h after introduction of pTM 1CAT. The values for CAT activity were calculated as the C A T production in 10 ~tl of a 100 ktl cell extract prepared from each well of cells transfected with 5 ~tg of pTM 1CAT, although it can not be assumed that all the cell types have the same transfection efficiency. The relative CAT expression, which was highest in C R F K cells, followed by that in Vero, HeLa, SW480 and COS cells, correlated with the virus production shown in Fig. 1 (a). Fig. 2 (b) shows the CAT activity of the reporter plasmids expressed as the CAT expression relative to that of the S I V A G M L T R reporter plasmid, pSACAT, which had the highest activity in all
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Fig. 2. (a) CAT production by the FIV LTR-CAT plasmid pTM 1CAT in various cell lines. The CAT activity is expressed as the CAT productionin 10 ~tlof a 100~tlcellextract prepared from each well of cells transfected with 5 I~g of pTM1CAT. Three independent experimentswere performed and the average is presented. (b) Relative CAT productionby various LTR-CAT constructs in various celllines. The basal activityof the FIV, HIV-1 and HIV-2LTRs was expressedas a value relative to that of the SIVA~M LTR. Three independent experimentswereperformedand the averageis presented. The reporter LTR-CAT constructspTM 1CAT (FIV), pH 1CAT (HIV-1),pH2CAT (HIV-2), pSACAT (SIVAGM)were used.
the cell lines examined and showed a wide range of expression of the LTR. Based on the results shown in Fig. 2, it is possible that the failure of FIV production in COS and SW480 cells is due to the low activity of the FIV L T R in the cells, although we could not detect the absolute activity of the FIV L T R in each cell line. The different promoter activities of the FIV L T R in various cell lines might be ascribed to the enhancer proteins which bind to the L T R in the cells. The primate lentiviruses commonly possess N F x B and Spl binding
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sites in the U3 region of the LTR (Fukasawa et al., 1988). In contrast, FIV has many putative protein-binding sites, such as those for AP-1, AP-4, C/EBP and ATF, in this region of the LTR (Miyazawa et al., 1991). It will be necessary to study cellular proteins that regulate the promoter activity of the FIV LTR in each cell line to provide an explanation of these different promoter activities. We are grateful to Dr M. Hattori (Hokkaido University, Sapporo, Japan) for providing recombinant human IL-2-producing Ltk-IL-2.23 cells. This work was partly supported by grants from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare of Japan. T. Miyazawa is supported by the Recruit Scholarship.
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(Received 16 January 1992; Accepted 18 February 1992)