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Analysis of the Role of the Bel and Bet Open Reading Frames of Human Foamy Virus by Using a New Quantitative Assay SHUYUARN F

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Analysis of the Role of the Bel and Bet Open Reading Frames of Human Foamy Virus by Using a New Quantitative Assay SHUYUARN F JOURNAL OF VIROLOGY, Nov. 1993, p. 6618-6624 Vol. 67, No. 11 0022-538X/93/1 16618-07$02.00/0 Copyright C 1993, American Society for Microbiology Analysis of the Role of the bel and bet Open Reading Frames of Human Foamy Virus by Using a New Quantitative Assay SHUYUARN F. YU AND MAXINE L. LINIAL* Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104-2092 Received 6 July 1993/Accepted 5 August 1993 We have constructed a BHK-21-derived indicator cell line containing a single integrated copy of the f-galactosidase (P-Gal) gene under control of the human foamy virus (HFV) long terminal repeat promoter (from -533 to +20). These foamy virus-activated ,8-Gal expression (FAB) cells can be used in a quantitative assay to measure the infectious titer of HFV. Our results show that the FAB assay is 50 times more sensitive than determination of the virus titer by the end-point dilution method. Using the FAB assay, we have found that HFV can productively replicate in several erythroblastoid cell lines as well as in the Jurkat T-cell line. We have also examined the roles of bel2, bet, and beI3 in viral replication by constructing proviral HFV clones in which the reading frame of Bel2, Bet, or Bel3 is disrupted by placement of translation stop codons. Analysis of these mutants reveals that while the beI3 gene is not required for viral replication in vitro, mutations in the beI2 or bet gene decrease cell-free viral transmission approximately 10-fold. The prototype spumavirus, human foamy virus (HFV) or activate gene expression directed by the HIV type 1 LTR, human spumaretrovirus, was originally isolated from a patient through a target site different from that of HIV Tat (16, 20). In with nasopharyngeal carcinoma (1). In culture, spumavirus is contrast to bell, the roles of bel2, bet, and bel3 in HFV highly cytopathic, inducing multinucleated syncytia with a replication remain unknown. characteristic vacuolated appearance in the cytoplasm. Al- Titers of foamy virus stocks have routinely been determined though it has been shown that in vitro HFV replicates produc- by examination of cytopathic effects (CPE) by the end-point tively in fibroblastic diploid cells but poorly in epithelium-like dilution method. Here, we present a quantitative method for cells (23, 24), viral replication in other cell types is still unclear. titration of HFV using an indicator cell line in which expres- There have been numerous searches for diseases associated sion of 3-galactosidase (,B-Gal) can be activated by HFV with HFV infection (37). The finding of both virus-related infection. This foamy virus-activated 1-gal expression (FAB) sequences and antigens in patients with Graves' disease sug- assay exploits the requirement for trans activation of the HFV gests an association with this autoimmune disease (19, 38). It LTR promoter by the viral Bell protein. It provides a simple has also been reported that expression of spumaviral genes in and rapid method for titration of HFV, which can be com- neurons and brain cells in transgenic mice is correlated with pleted in 2 days and is 50-fold more sensitive than titration of neurological disorders (2, 3, 5). virus by the end-point dilution method. Using this assay, we The genome of HFV contains gag, pol, and env, as well as have reexamined the spectrum of HFV cell tropism. We also several other genes, located between env and the 3' long report results of mutational analyses of the bel2, bet, and bel3 terminal repeat (LTR) (10, 25). At least four open reading genes and conclude that while bel3 is dispensable for viral frames (ORFs) are predicted to exist, including bell, bel2, bet, replication, the bel2 or bet gene is required for optimal cell-free and bel3, which result from singly and/or multiply spliced viral transmission in vitro. mRNAs (28). Analysis of viral proteins in infected cells has shown that while Bell accumulates in the nucleus, Bel2 is found in the cytoplasm (11, 21). Bet, an abundant viral protein in the cytoplasm, is encoded by a multiply spliced mRNA in MATERIALS AND METHODS which the N-terminal portion of Bell is spliced in frame with the Bel2 ORF. Thus far, expression of the Bel3 ORF has not Cells. Human embryonic lung (HEL) cells (ATCC CCL been reported, although bel3 transcripts have been found in 137) and human foreskin primary fibroblastic cells (obtained infected cells (28). from Adam Geballe, Fred Hutchinson Cancer Research Cen- The bell gene, like human immunodeficiency virus (HIV) tat ter) were grown in Dulbecco's modified Eagle medium sup- and human T-cell leukemia virus tax, encodes a potent tran- plemented with 10% fetal bovine serum (FBS). BHK-21 (baby scriptional transactivator of its own LTR promoter (17, 32). hamster kidney cells, ATCC CCL 10) and FAB cells were Deletion of bell completely abolishes the infectivity of HFV grown in Dulbecco's modified Eagle medium with 5% FBS. (21). The primary target sequence for Bell within the HFV Human cell lines, including B/N (an Epstein-Barr virus-trans- LTR has been mapped to -130 bp 5' to the cap site (36). A formed B lymphocytic cell line obtained from Mark Groudine, second region located -400 bp 5' to the cap site also contrib- Fred Hutchinson Cancer Research Center), 174xCEM (a T-B utes to optimal reactivity to Bell (27). Bell can also trans lymphoblastic hybrid cell line, NIH AIDS Reagent Program catalog no. 272), Jurkat (a CD4+ T-cell line, ATCC TIB 152), U937 (a histiocytic lymphoblastoid cell line, ATCC CRL 1593), K562 (an erythroblastoid cell line, ATCC CCL 243), * Corresponding author. and H92.1.7 (an erythroblastoid cell line originally called 6618 Vot-. 67, 1993 QUANTITATIVE ASSAY OF THE ROLE OF HFV bel AND bet ORFs 6619 HEL92.1.7, ATCC TIB 180), were grown in RPMI medium ance of CPE. In cocultivation experiments, HEL cells were first supplemented with 5% FBS. infected with HFV at a multiplicity of infection (MOI) of 3. Preparation of virus stocks. Virus stocks were originally Three days later, the cells were treated with 10 ,ug of mitomy- obtained by transfection of BHK-21 or HEL cells with the cin per ml for 2 h to inhibit cell division. After extensive full-length HFV proviral DNA. By the modified calcium washes, the treated HEL monolayers were trypsinized and phosphate method (6), the transfection efficiency of BHK-21 cocultivated with different hematopoietic suspension cells at a cells was much higher than that of HEL cells (data not shown). ratio of 1 to 20. The suspension cells were maintained in RPMI Transfected cells were then scraped from culture dishes and medium containing 5% FBS and subcultured every 3 to 5 days. removed along with the culture medium, and virus was re- After the second passage, adherent HEL cells would no longer leased by three cycles of freezing and thawing. These total cell be observed in the cultures. The virus titer in the culture lysates were then added to BHK-21 or HEL cells to obtain medium was periodically monitored by the FAB assay. high-titer virus stocks. Since the virus titer obtained from Site-directed mutagenesis. Translation stop codons were propagation of HFV in HEL cells was usually slightly higher introduced into the reading frame of bel2, bet, or bel3 without than in BHK-21 cells, HEL cells were used in all the infection affecting the coding sequence of the other overlapping genes experiments. To prepare cell-free virus stocks, culture super- (see Fig. 3), by using an oligonucleotide-directed mutagenesis natants were cleared by low-speed centrifugation (4,000 x g kit (Amersham) according to the manufacturer's protocol. The for 10 min) and filtered through a 0.2-,um-pore-size filter single-stranded DNA template derived from pHSRVAGPE membrane. was used in the mutagenesis reaction. A 21-mer, 5'-AGGGCT Plasmids. The infectious molecular clone of HFV, ACTAGAAGAGTCCAG-3', was used to create the Abel2 pHSRV13, and plasmids pdbell and pBCbell have been mutant, in which the Leu codon (TTG) at residue 34 was described previously (21) and were kindly provided by M. replaced with TAG. For the iXbel2C mutant, the Arg (AGA) Lochelt and R. Flugel (German Cancer Research Center, and Gln (CAG) codons at residues 246 and 248 were converted Heidelberg, Germany). Plasmid pdbell is derived from to TGA and TAG by a 30-mer, 5'-TGGAATGTCACTQGA pHSRV13 but contains a 208-bp deletion in the bell gene. AACTAGGGAAAACAA-3'. A 22-mer, 5'-CATAGAACCA Plasmid pBCbell is a eukaryotic bell expression vector using TAATGGCAGACT-3', was used to generate the Aibel3 mu- an immediate-early promoter from cytomegalovirus. Plasmid tant, in which the Leu codon (TTA) at residue 90 was replaced pHSRVAGPE was constructed by inserting the 2,488-bp ClaI with TAA. The Ser codon (TCA) at residue 130 was changed fragment of pHSRV13 into the 2,958-bp ClaI-digested frag- to TAA in the Abel3C mutant with a 23-mer, 5'-GGTGGAAT ment of phagemid pBluescriptll SK+ (Stratagene). Plasmid GTAACTAGAAACCAG-3'. After confirmation by DNA se- pCMVhph, as described previously (4), encodes resistance to quencing, each mutation was introduced into the full-length hygromycin B driven by the cytomegalovirus promoter. proviral cDNA by inserting the 2,488-bp ClaI fragment from Plasmid pEQJK was constructed by ligating a 2,065-bp each mutagenized pHSRVAGPE into the 12,919-bp ClaI HindlII-SacI fragment of pEQ3 (33), a promoterless [3-Gal fragment of pHSRV13. vector, with the 5,057-bp HindIII-SacI fragment of pJK2.
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