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Virology 308 (2003) 83–91

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Molecular cloning, complete sequence, and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B-lymphoblastoid cell line DG-75 Kevin P. Raisch,a,1 Massimo Pizzato,b,2 Hai-Yuan Sun,a Yasuhiro Takeuchi,b L. William Cashdollar,a and Sidney E. Grossberga,* a

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA b Wohl Virion Centre, Windeyer Institute of Medical Sciences, University College London, London, W1P 6DB, UK Received 16 August 2002; returned to author for revision 2 October 2002; accepted 14 October 2002

Abstract A previously undetected retrovirus has been isolated from the human Epstein–Barr virus (EBV)-negative, B-lymphoblastoid DG-75 cell line, widely used for EBV gene transfection studies. The complete 8207-base genome of the DG-75 retrovirus was molecularly cloned from viral mRNA and sequenced (Accession No. AF221065). Northern blot analysis with probes specific for the putative RU-5, gag, pol, and env regions identified a full-length viral RNA and spliced env mRNA. DG-75 viral RNA was isolated from the DG-75 cell sublines UW and KAR, but not from the HAD subline. The DG-75 retrovirus was isolated with primer-binding sites that match tRNAThr and tRNAGln2. Homology searches revealed homology to (i) xenotropic NZB-9-1 env mRNA, (ii) Moloney-MLV pol region, and (iii) a truncated Evi-2 endogenous proviral sequence gag and pol region. Viral interference and infectivity assays confirmed the xenotropic nature of the DG-75 retrovirus. The DG-75 retrovirus is the first isolate of an exogenous xenotropic MLV in which the full-length genomic sequence has been characterized. © 2003 Elsevier Science (USA). All rights reserved. Keywords: DG-75 MLV; Retrovirus; MLV; Lymphoblastoid; Complete genome; Evi-2; Primer-binding site

Introduction The murine leukemia viruses (MLVs) comprise a group of C-type retroviruses that can produce leukemia, lymphoma, and neurologic disease in rodents. They are classified into four major host range subgroups based on the distribution of their cell-surface receptors among mammalian species and accordingly their ability to infect such species (Cunningham and Kim, 1994; Goff, 2001; Hunter, 1997). The receptor for ecotropic MLV strains, which are infectious for mice and rats, is restricted to cells of rodent * Corresponding author. Fax: ⫹1-414-456-6566. E-mail address: [email protected] (S.E. Grossberg). 1 Present address: Department of Radiation Oncology, University of Alabama–Birmingham, Birmingham, AL 35294. 2 Present address: Dana-Farber Cancer Institute, Harvard University, Boston, MA 02115.

origin. The receptor for xenotropic viruses is present on cells of a variety of species, but not on mouse cells, although xenotropic viruses are weakly infectious for feral mice. The amphotropic and dual or polytropic viruses utilize receptors found on the cells both of rodents and of other mammalian species. These different groups can be further identified by the use of viral interference assays (Weiss, 1993), which measure the inhibition of superinfection that depends on the interaction between the viral envelope (Env) proteins and the cell receptor. Xenotropic and polytropic MLVs are present as endogenous proviruses in inbred strains of mice, whereas amphotropic viruses are exogenous MLVs that can efficiently infect human cells (Hunter, 1997). The receptor for both polytropic and xenotropic MLVs has been cloned from cDNA libraries of human lymphocytes, human epithelial cells (HeLa), and murine NIH3T3

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K.P. Raisch et al. / Virology 308 (2003) 83–91

Fig. 1. Schema for cloning the DG-75 retrovirus. (A) R-U5 region using the tRNA primer-binding site to reverse-transcribe the viral RNA followed by amplification of the poly(C)-tailed cDNA by PCR. (B) pol region using degenerate pol primers to amplify a random primed viral cDNA. (C) The 5⬘ region using an oligo(dT)-primed viral cDNA amplified by PCR using primers from the pol region (B) and putative R region (A). (D) 3⬘ region using an oligo(dT)-primed viral cDNA amplified by PCR using primers from the pol region (B) and putative R region (A). (E) 3⬘ end of the viral genome amplified by PCR using the 3⬘ RACE technique with a primer from the putative U3 region (D).

fibroblasts (Battini et al., 1999; Tailor et al., 1999; Yang et al., 1999). The ecotropic MLV receptor was identified as the murine cationic amino acid transporter CAT-1, which belongs to a family of multiple, membrane-spanning, solute transporters (Albritton et al., 1989). A related family member, sodium phosphate symporter (Pit1), previously identified as the receptor for gibbon ape leukemia virus and feline leukemia virus subgroub B, is used by the distinctive MLV10A1 (Miller and Miller, 1994; Wilson et al., 1994); this virus can also use a structurally and functionally related receptor for amphotropic MLV, the sodium-dependant phosphate symporter Pit2 (Sommerfelt, 1999; van Zeijl et al., 1994). A previously unrecognized C-type retrovirus, which is constitutively produced and released by two sublines of Epstein–Barr virus (EBV)-negative, human B-lymphoblastoid DG-75 cells, widely used for EBV gene transfection studies (Fahraeus et al., 1990; Harada and Yanagi, 1992), was identified immunologically as MLV (Raisch et al., 1998). This DG-75 MLV was infectious for a third DG-75 subline designated Hadassah (HAD) (Ben-Bassat et al., 1977). The HAD subline was originally shown to be retrovirus-free by RT-PCR (Raisch et al., 1998). In this study, the DG-75 retrovirus was characterized by molecular cloning and sequencing of its entire 8207-base genome. The env gene of the DG-75 retrovirus has homology to the xenotropic NZB-9-1 proviral sequence (O’Neill et al., 1985), and its gag gene has homology to that of the defective Evi-2 provirus, which is integrated at the Evi-2 (ecotropic viral integration site-2) locus in the tumor-suppressor neurofibromatosis type I gene (Cho et al., 1995). The initial 1490-nucleotide (nt) portion of the pol region has high homology to Moloney MLV, whereas the final 1765-nt section has high homology to the truncated Evi-2 endogenous proviral sequence. Two primer-binding sites (PBS) have been identified for the DG-75 retrovirus clones. One PBS uses glutamine-2 tRNA, and the second PBS uses threonine tRNA, which corresponds to the PBS for the Evi-2 provirus. Two DG-75 viral mRNA molecules were identified, one full-length of about 8.2 kb and another for the spliced env mRNA of 2.9 kb. The viral mRNAs were found associated with the two constitutively productive sub-

lines, KAR and UW, whereas the HAD subline had none. The inability of DG-75 virus to infect murine cells and its infectivity for other mammalian species cells, especially some human cells, indicate its xenotropic nature, which was confirmed by retroviral interference assays.

Results Genomic structure of DG-75 viral RNA Five overlapping cDNA fragments of the DG-75 genome were cloned. Multiple clones for each cDNA fragment were sequenced in both directions and aligned to determine the complete genomic sequence (Fig. 1). Restriction enzyme digests of the multiple independent clones of the two large cDNA clones (Fig. 1C and D) produced expected fragments as predicted from the genomic sequence (Fig. 2). The viral RNA is predicted to be composed of 8207 bases with a structure resembling the C-type M-MLV. The genomic structure was deduced (Fig. 2) from comparative analysis with other MLV sequence data using BLASTN (National Center for Biotechnology Information genetic databases). The 5⬘-terminal region consists of a 73-nt identical repeat (R) region, also found at the 3⬘-terminal region (nt 8135 to 8207). Downstream of the 5⬘R region is the 62-nt U5 region followed by the primer-binding site (nt 146 to 163), which is complementary to the last 18 nucleotides at the 3⬘ end of a transfer RNA (tRNA) (Mak and Kleiman, 1997). Sixteen clones were obtained, of which 8 clones had one PBS sequence (TGGAGGTCCCACCGAGAT) that would be primed by threonine tRNA and the other 8 (TGGAGGCCCCAGCGAGAT) (base changes underscored) primed by glutamine-2 tRNA (Sprinzl et al., 1989). Two open-reading-frames (ORF) could be identified by sequence analysis using the GCG program (Madison, WI). The first ORF can encode a 536-amino acid (aa) precursor Gag polypeptide, followed by a read-through of an amber stop codon to encode a 112-aa putative protease protein and a 1084-aa polymerase protein. The second ORF indicates a spliced message of 2910 nt from the 5⬘ splice donor site to the 3⬘ splice acceptor site and can encode a 644-aa envelope


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Fig. 2. A map of the 8207-base genome of the DG-75 retrovirus, showing the start codon (AUG), termination codon (UAA), amber stop codon (UAG), and the splice donor (SD) and acceptor (SA) sites with their nucleotide locations. Two open reading frames are predicted, one encoding the Gag and Pol polyproteins, and the other, a 3-kb spliced mRNA encoding the Env polypeptides. Restriction enzyme sites are indicated by single letters: A, AccI; S, SmaI; and E, EcoRI. The R-U5 riboprobe (cross-hatched box) and the gag, pol, and env double-stranded DNA probes (stippled box) were used for Northern blot analyses.

polypeptide. Downstream from the env stop codon is the 337-nt U3 region followed by the R region and a polyadenylated tail. Analysis of viral homology A search of the current databases, EMBL and GenBank, using the complete DG-75 nucleotide sequence as query, showed 92.7% identity with the complete genomic sequence from a retrovirus in the genome of the mouse myeloma cell line SP2/0-Ag-14 (Accession No. X94150). Homology searches performed with individual regions of the DG-75 retrovirus were more informative. The R-U5 and R-U3 nucleotide sequence showed the highest homology, 98 and 97%, respectively, to a defective murine proviral sequence, Evi-2 (Accession No. S80082). The nucleotide sequence of the gag region showed 97% homology to Evi-2. The nucleotide sequence of the pol region showed an overall homology to the Retroviridae complete genome of 94%, with 95% homology of the initial 5⬘ 1490 bases of the pol region to Moloney MLV (Accession No. AF033811); and the remaining 1765 bases were 97% homologous to the truncated pol sequence of Evi-2. The nucleotide sequence of the env region showed a 98% homology to MLV-NZB-9-1 xenotropic DNA (Accession No. K02730) (Mosier et al., 1985).

tected. As a positive control, a GAPDH probe showed similar levels of nondegraded mRNA in each lane. To confirm that the two viral RNA bands were consistent with their predicted structure, specific probes for the gag, pol, and env sequences were designed (Fig. 2). The probes hybridized to the DG-75 (UW) mRNA to identify their respective viral RNA segments (Fig. 4). All three probes identified a full-length viral RNA of 8.2 kb, whereas only the env probe identified the 2.9-kb sequence to confirm the identity of the envelope mRNA.

Identification of viral genome The structure of the viral RNA predicted two viral mRNA species to be transcribed. The R-U5 clone (Fig. 2) was therefore used as a riboprobe for Northern blot analysis since all DG-75-specific viral RNAs should hybridize to this sequence. The predicted viral structure was confirmed when an antisense R-U5 riboprobe hybridized to two mRNA species from the DG-75 sublines UW and KAR (Fig. 3), a full-length viral mRNA of approximately 8.2 kb and a putative env mRNA of approximately 2.9 kb. No viral mRNA was identified in the HAD subline nor did the sense probe hybridize to any mRNA species (data not shown). Defective RNA species with large deletions were not de-

Fig. 3. Northern blot analysis of mRNA isolated from three sublines of DG-75 cells. (A) The blot was probed with a 32P-labeled antisense riboprobe from the R-U5 clone (Fig. 2). A full-length mRNA of approximately 8.2 kb and a spliced 2.9-kb mRNA were found in the KAR and UW sublines. (B) The blot was stripped and probed with a 32P-labeled DNA probe for GAPDH to show equal loading of the RNA in each lane.

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B-cells, B95-8 (data not shown). However, after infection of the human foreskin fibroblast FS-7 strain and rat sarcoma XC cells, as well as the DG-75 (HAD) subline, PCRamplifiable DG-75 virus sequences were found integrated in their cellular genomes 21 or 30 days after infection (data not shown). Unlike classical MLV-X, the DG-75 virus failed repeatedly to grow in Mv1.Lu mink lung cells. Following intraperitoneal injection of DG-75 virus into sixteen 3- to 4-week-old male C57BL/6 mice, no hepatosplenomegaly or lymphodenopathy was noted either in 3 mice sacrificed at 4 months or in the remaining mice at 9 months, all of which had remained healthy.

Discussion Fig. 4. Northern blot analysis of mRNA from the DG-75 (UW) cells. Each probe is a 32P-labeled DNA generated by PCR using primers specifically designed to amplify a portion of the gag, pol, or env genes (Fig. 2). All three probes identified a full-length viral mRNA of approximately 8.2 kb, while only the env probe identified the spliced mRNA of 2.9 kb. A GAPDH probe was used to show that there was no degraded mRNA in each lane (data not shown).

Host range and interference assays The host range of the DG-75 virus was examined by pseudotype formation. Human TE671 and murine NIH3T3 cells harboring an MLV vector coding for ␤-galactosidase, that is, TEL and 3T3L cells, respectively, were cocultivated with DG-75 (KAR) virus-infected cells for 2 weeks. Cell supernatants were harvested and tested for mobilization of the MLV vector on TE671 and NIH/3T3 cells. While no enzymatic activity was recovered from the 3T3L cells, the TEL cells produced a titer of 105 cfu/ml on TE671 cells. These results indicated that human cells, but not murine cells, have a receptor for the DG-75 virus and can support its replication. The results are consistent with the sequence data, indicating that the envelope sequence belongs to the xenotropic class of MLV. To characterize further the receptor for DG-75 virus, a cross-interference assay was carried out by challenging cells chronically infected with xenotropic and amphotropic MLV with lacZ vectors carrying the DG-75 envelope (Table 1). The MLV-X virus efficiently blocked infection of the lacZDG-75 virus on TE/MLV-X cells. The infection was blocked 500-fold when compared to uninfected TE671 cells, whereas no reduction of titer was observed on the TE/MLV-A cells. In the same experiment, efficient autointerference of MLV-X and MLV-A was shown as TE/ MLV-X and TE/MLV-A blocked lacZMLV-X and lacZMLV-A by 650- and 10,000-fold, respectively. These results indicate that the DG-75 virus and MLV-X (NZB) share the same receptor and confirm that DG-75 virus is a xenotropic MLV. Further, host-range infectivity experiments showed that the DG-75 virus was incapable of infecting murine NIH 3T3 and SC-1 fibroblasts, as well as marmoset EBV-transformed

The DG-75 retrovirus is a replication-competent, murine leukemia virus variant with an 8207-base genomic RNA. The full-length genomic RNA encodes a putative 536-aa precursor Gag polypeptide, followed by a read through of an amber stop codon to encode a 112-aa putative protease protein and a 1084-aa polymerase domain. Immunoblot analysis showed the p30 protein from DG-75 retrovirus to be immunoreactive with anti-MLV p30 antiserum (Raisch et al., 1998). The viral env gene, a 2910-base spliced mRNA, encodes a putative 644-aa Env polypeptide, which is cleaved to a predicted 70-kDa surface unit (SU) glycoprotein and a 15-kDa transmembrane (TM) domain. The putative aa sequence of the SU is highly homologous to other xenotropic Env sequences and identical in the variable regions A and B to the NZB-9-1 retroviral sequence (Fig. 5). Variable region A is considered to be important in specifying receptor recognition, whereas variable region B is used for stabilization of the virus to the receptor (Battini et al., 1992). Some characteristics of the DG-75 retrovirus distinguish it from other murine retroviruses. The most common primer tRNA for murine leukemia viruses is the one for proline (Harada et al., 1979), although murine retroviral PBS sequences have been reported that are complementary to tRNAs for glutamine, phenylalanine, theronine, and glycine Table 1 Cross-interference assay to confirm the receptor tropism of the DG-75 retrovirus Virus

MLV-A MLV-X DG-75

Mean of quadruplicate LacZ pseudotype titers (colony-forming units/ml)a in the cells indicated TE671b

TE/MLV-Ab

TE/MLV-Xb

1.3 ⫻ 107 6.5 ⫻ 105 9.3 ⫻ 104

1.5 ⫻ 103 6.0 ⫻ 105 1.2 ⫻ 105

1.2 ⫻ 107 1.0 ⫻ 103 1.9 ⫻ 102

Standard error of the mean for all titers ⬍15%. TE/MLV-A and TE/MLV-X are human rhabdomyosarcoma TE671 cells chronically infected with amphotropic MLV-A 1504 and xenotropic MLV-X NZB virus strains, respectively. a

b


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Fig. 5. Schematic diagram of a MLV gp70 protein with the designation of variable regions A (VRA) and B (VRB) which have been identified as important regions for the characterization of cellular tropism (Battini et al., 1992). Alignment of the putative amino acid sequence of DG-75 retrovirus for VRA and VRB with retroviral protein sequences from the following viruses: mlvenvx, xenotropic MLV NZB-9-1 (O’Neill et al., 1985); mcfenvp, polytropic MLV MCF 247 (Holland et al., 1983); mkvenva, amphotropic MLV 4070A (Ott et al., 1990); and mlvenve, ecotropic Mo-MLV (Shinnick et al., 1981). The standard one-letter code for amino acids is used: capital letters indicate conserved amino acids, small characters represent variable positions, and dots are used for proper spacing.

(Colicelli and Goff, 1986; Nikbakht et al., 1985; Policastro et al., 1989). Sequence analysis indicates that the PBS of DG-75 virions are complementary to the 3⬘ end of human tRNAThr and tRNAGln2. The cloning of the PBS for two tRNA species from the same population of virions is unusual. Lund et al. (1993) showed that progeny virions of cells transfected with an Akv-MLV-based retroviral vector containing the glutamine-1 tRNA PBS had an equal proportion of tRNAs for glutamine-1 and -2. The identification of two different PBS sequences, that is, for tRNA primers for threonine and glutamine-2, in DG-75 virus suggests that alternative tRNA molecules may also be used in the replication of this virus. A coding sequence for the Gag polypeptide is 96% homologous to a truncated retroviral sequence identified by its integration in the ecotropic viral integration site-2, Evi-2, a common integration site involved in murine myeloid leukemogenesis and located within the neurofibromatosis type 1 gene, a tumor suppressor gene (Cho et al., 1995). The endogenous Evi-2 retroviral sequence contains a complete ORF for the gag gene and a truncated pol and env gene bordered by a xenotropic LTR on the 5⬘ end. The truncated pol and env genes have 97 and 96% homology, respectively, with the DG-75 retrovirus sequence. The DG-75 R-U5 region is 98% identical to the same region of the Evi-2 sequence. However, the PBS for DG-75 virus corresponds to glutamine-2 and threonine tRNA, whereas only threonine tRNA has been identified for the Evi-2 provirus. The gag

gene for Evi-2 has high homology to the murine immunodeficiency (MAIDS) MLV variant, LP-BM5 (Mak and Kleiman, 1997; Miller et al., 1994), the truncated sequence of which has a functional gag gene followed by only partial sequences of the pol and env genes (Cho et al., 1995); to be infectious, the LP-BM5 truncated virus requires a helper virus (Chattopadhyay et al., 1991). The truncated genome of Evi-2 may also require a helper virus to be infectious. Based on the homology between Evi-2 and the LP-BM5 MAIDS virus, it is conceivable that DG-75 retrovirus infection may be able to induce immune suppression in an appropriate line of feral mice. Further studies are necessary to determine the pathogenesis of the DG-75 retrovirus, for example, if it would induce immunodeficiency or produce tumors in feral mice. The putative mRNA encoding the env gene of the DG-75 retrovirus is most closely related to the MLV NZB-9-1 xenotropic env mRNA (O’Neill et al., 1985) with a 98% sequence identity at the nucleotide level. Since the sequence of only the NZB-9-1 env mRNA has been reported, it is not possible to know whether the high homology to the env gene of the DG-75 retrovirus extends into the gag region and the remaining pol region. The complete proviral sequence of the NZB-9-1 retrovirus would be necessary to determine its relationship to the DG-75 retrovirus genome. It is of interest that the truncated Evi-2 retroviral sequence contains a U3-R region and fragments of the pol and env genes that have nearly identical homology to the NZB-9-1

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retroviral sequence and the DG-75 proviral sequence. The high homology of regions of the exogenous DG-75 retrovirus and the truncated Evi-2 sequence suggest that the latter may be a deletion mutant of the former. The origin of the constitutively produced retrovirus in the DG-75 KAR or UW sublines is not known. It is clear that an early passage of these cells, the HAD subline, is free of retroviruses as determined by Northern (Fig. 3) and Southern (data not shown) blot analyses, immunoblot, electron microscopy, and the PCR-based product-enhanced reverse transcriptase assay (Raisch et al., 1998). Propagating human tumor cells in immunodeficient mice has resulted in infection of the cells with xenotropic murine viruses (Levy, 1973; Todaro et al., 1973); and retroviral contamination has been reported of the human B-lymphoblastoid cell line, Ramos, after its passage in nude mice (Kotler et al., 1977). The investigators who have carried the DG-75 sublines in their laboratories have indicated that no attempts were made either to passage the cells in nude mice, to introduce by transfection a murine leukemia virus infectious clone, or to infect them otherwise with such viruses. It is not known whether the DG-75 virus can infect primary human B-lymphocytes. In view of current efforts to design better retroviral vectors, especially those based on murine leukemia virus, further characterization of the cell tropism of the DG-75 retroviruses may be valuable.

Materials and methods Cell lines The EBV-negative human B-lymphoblastoid cell lines (and their sources) included the three DG-75 cell sublines, designated UW (B. Sugden, University of Wisconsin, Madison WI), KAR (G. Klein, Karolinska Institute, Stockholm Sweden), and HAD (H. Ben-Bassat, Hadassah Medical Center, Jerusalem Israel). The EBV-positive marmoset Bcell B95-8 cell line was provided by G. Miller (Yale University, New Haven, CT). These cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamine, and antibiotics. The human foreskin cell strain FS-7 (B. Brown, Children’s Hospital, Milwaukee, WI) and NIH/3T3 line (CRL1658, ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, as were the SC-1 mouse fibroblasts, the Mv1.Lu mink lung cells, and XC rat sarcoma cells (all from R. Risser, University of Wisconsin). Viral interference assay utilized human rhabdomyosarcoma TE671 cells (CRL8805) and murine NIH/3T3 cells and their derivatives (Takeuchi et al., 1994), which were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics. TE/ MLV-A and TE/MLV-X are TE671 cells chronically infected with MLV-A 1504 and MLV-X NZB virus strains, respectively. TELCeB6/AF-7 cells are packaging cells pro-

ducing helper-free LacZ(MLV-A) pseudotypes. TEL and 3T3L cells are TE671 and NIH/3T3 cells, respectively, harboring the MFGnlslacZ retroviral vector. TEL/MLV-X are TEL cells infected with MLV-X NZB, which thereby produce LacZ (MLV-X) pseudotypes as well as replicationcompetent MLV-X. Isolation of retroviral RNA Virus isolation and purification of the DG-75 (UW) retrovirus has been described previously (Raisch et al., 1998). Briefly, the filtered supernatant was precipitated with polyethylene glycol, and the virus was isolated in a sucrose step-gradient and then further purified through a continuous sucrose gradient. Reverse transcriptase-positive fractions were collected, and the virus was concentrated by ultracentrifugation. The virus pellet was suspended in 100 ␮l TEN (0.01 M Tris–HCl, pH 7.3; 0.001 M EDTA, 0.050 M NaCl) buffer, denatured in 4 M guanidine thiocyanate (500 ␮l) for 5 min at room temperature (RT), and then mixed with an equal volume of phenol:chloroform:isoamyl alcohol (25:24: 1). After 10 min at RT, the phases were separated at 15,000 g for 15 min. The aqueous phase was collected, and the organic phase was back-extracted with DEPC-treated H2O. The two aqueous phases were pooled and extracted once with chloroform:isoamyl alcohol (24:1). The aqueous phase was collected and precipitated with 20 ␮g glycogen, 0.1 vol of 3 M sodium acetate, pH 5.0, and 2.5 vol of 100% ethanol for 18 h at ⫺20°C. The RNA was pelleted at 15,000 g for 15 min at 4°C, washed once with ice-cold 70% ethanol, and suspended in DEPC–H2O. The RNA was quantitated spectrophotometrically at 260 nm. Cloning the R-U5 region The R-U5 cloning protocol of Lo¨ wer et al., (1993) was used with minor modifications (see Fig. 1A). In a 10-␮l reaction mixture, 80 ng of purified viral RNA was mixed with 1⫻ RT buffer (Gibco BRL, Gaithersburg, MD); 1 mM DTT; 10 ng RNase-free BSA (Amersham Pharmacia Biotech, Piscataway, NJ); 1 mM each dTTP, dATP, dGTP; 1 U RNase inhibitor (Ambion Inc., Austin, TX); 2 U M-MLV RT (Gibco BRL); 30 ␮Ci [32P]dCTP (3000 Ci/mmol, NEN, Boston, MA); and 300 ng PBS primer containing an EcoRI site restriction enzyme on the 5⬘ end. Twelve PBS primers were used and are presented in the 5⬘ to 3⬘ direction: arg1, CGT AGA ATT CCT CCT GGC TGG CTC ACC A; arg2, CGT AGA ATT CGT CCC TTC GTG GTC GCC A; gly, CGT AGA ATT CTC CCC GGC CAA TGC ACC A; gln, CGT AGA ATT CAT CTC AGT GGA ACC TCC A; his, CGT AGA ATT CAT CTG AGT CAC AGC ACC A; ile, CGT AGA ATT CTC CCT GTA TGG GCC ACC A; phe, CGT AGA ATT CTC CCG GGT TTC GGC ACC A; glu, CGT AGA ATT CTT CCT GGC CAG GGA ACC A; pro, CGT AGA ATT CAT CCC GGA CGA GCC CCC A; trp, CGT AGA ATT CAT CAC GTC GGG GTC ACC A; lys1,2,


CGT AGA ATT CGC CCC ACG TTG GGC GCC A; and lys3, CGT AGA ATT CTC CCT GTT CGG GCG CCA. The reaction mixture was incubated at 37°C for 40 min, supplemented with 1 ␮l dCTP (10 mM), and incubated for an additional 20 min. The reaction was stopped by adding 5 ␮l of stop solution (98% formamide, 10 mM EDTA, 0.025% bromphenol blue) and heated to 95°C for 3 min. The reaction products were separated in an 8 M urea:8% polyacrylamide sequencing gel. The wet gel was wrapped with plastic wrap and exposed to X-ray film to identify the single-stranded cDNA bands. The cDNA bands were excised from the gel and extracted in extraction buffer (0.5 M ammonium acetate; 10 mM magnesium acetate; 1 mM EDTA, pH 8.0; 0.1% SDS at 37°C for 4 – 6 h on a rotating wheel (Sambrook et al., 1989)). Any insoluble material was removed by centrifugation at 15,000 g for 15 min. The soluble phase was collected, and extraction buffer was added to the insoluble pellet for an additional 2 h at 37°C. The soluble phase was separated from the insoluble material and pooled with the first soluble phase. The cDNA was precipitated with 20 ␮g glycogen; 0.1 vol of 3 M sodium acetate, pH 5.2; and 2.2 vol of 100% ethanol for 18 h at ⫺20°C. The single-stranded cDNA was homopolymer-tailed with dCTP following the manufacturer’s protocol from the DNA Tailing kit (Boehringer Mannheim, Indianapolis, IN). The poly(dC)-tailed cDNA was amplified by PCR using 50 pmol of each primer, a poly(dG) primer containing either a BamHI (5⬘-TCT AGA GGA TCC (G)12-3⬘) or a HindIII (5⬘-GAA TAC AAG CTT (G)12-3⬘) restriction enzyme site, and the corresponding tRNA PBS primer containing an EcoRI restriction enzyme site. After amplification for 35 cycles at 94°C (30 s), 55°C (1 min), 72°C and (1 min), the purity of the PCR product was verified by agarose gel electrophoresis and then concentrated by ethanol precipitation (Sambrook et al., 1989). The amplified cDNA was suspended in H2O and digested with the appropriate combination of restriction enzymes. The restriction enzyme-cut PCR products were separated in low-melting-temperature (LMT) agarose gel (Gibco BRL) and isolated by gel extraction (Bio101, Vista, CA). The amplified cDNA was quantitated spectrophotometrically and then ligated into restriction enzyme-cut pGEM3Z (Promega, Madison, WI). Competent JM109 cells (Promega) were transformed according to the manufacturer’s protocol. Amplification of the conserved pol region Purified DG-75 viral RNA (1 ␮g) was reverse transcribed with random hexamer primers (Sambrook et al., 1989). The cDNA was amplified by PCR using degenerate pol primers as described by Shih et al., 1989 (see Fig. 1B). Briefly, the degenerate PCR primers containing an EcoRI restriction enzyme site presented in the 5⬘–3⬘ direction, gs, CGC GGA TCC TGG AAA GTG (C/T)T(A/G) CC(A/C) CA(A/G) GG; and gen, CGC CCA TCC GG(A/C) GGC

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CAG CAG (C/G)A(G/T) GTC ATC CA(C/T) CTA, were used to amplify the cDNA by standard PCR techniques of three cycles at 94°C (30 s), 37°C (30 s), and 72°C 1 min followed by 35 cycles at 94°C (30 s), 55°C (30 s), and 72°C 1 min. The 135-bp pol product was agarose gel-purified and ligated into the pGEM-T vector (Promega), which was used to transform competent JM109 cells. Amplification and cloning of the 5⬘ region An internal primer from the 135-bp pol region was synthesized to prime toward the 5⬘ end, DG-75POLr (5⬘-GTG GGA CTG TTT TTG AAA CCC TGT-3⬘). A second primer was synthesized from the putative R region of the R-U5 clone to prime toward the 3⬘ end, DG-75LTRf (5⬘-CCG TGT CCC AAT AAA GCC TC-3⬘). Amplification of the 5⬘ region using the expanded long template PCR kit (Boehringer Mannheim) was performed on oligo (dT)-primed viral RNA (see Fig. 1C). Briefly, DG-75 viral RNA (1 ␮g) was reverse-transcribed with oligo (dT)-primers using SuperScript RT (Gibco BRI). A 50-␮l PCR mixture consisted of 1⫻ PCR buffer containing 1.75 mM MgCl2, 350 ␮M each dNTP, 360 pM DG-750POLr primer, 360 pM DG-75LTRf primer, 0.75 ␮l of enzyme, and 100 ng of viral cDNA. The reaction mixture was denatured for 2 min at 90°C followed by 30 cycles of 94 °C for 15 s, 60°C for 30 s, and 68°C for 6 min, with 20 s added to each elongation step for the final 20 cycles. The PCR-generated fragment was agarose gelpurified, tailed with dATP using Taq DNA polymerase, and ligated into pGEM-T vector to transform competent DH-5 alpha cells (Gibco BRL). Amplification and cloning the 3⬘ end An internal primer from the 135-bp pol region was synthesized to prime toward the 3⬘ end, DG-75POLf (5⬘-ACA GGG TTT CAA AAA CAG TCC CAC-3⬘). A second primer was synthesized from the putative R region of the R-U5 clone to prime toward the 5⬘ end, DG-75LTRr (5⬘ATC AGC AAG AGG CTT TAT TGG GAA C-3⬘). Amplification and cloning of the 3⬘ region was performed on oligo (dT)-primed viral RNA as described for the amplification of the 5⬘ region (see Fig. 1D). The PCR-generated fragment was agarose gel-purified, tailed with dATP using Taq DNA polymerase, and then ligated into pGEM-T vector followed by transformation of competent DH-5 alpha cells. A 3⬘ RACE kit (Boehringer Mannheim) was used to clone the 3⬘ end of the viral RNA. Oligo(dT)-primed viral RNA was reverse-transcribed as described above and amplified by PCR with the oligo(dT)-abridged universal adaptor primer and a primer from the putative U3 region, 4793F (5⬘-CAG TTA CAA ATC AAG GCC G-3⬘). After 35 cycles at 94°C (30 s), 55°C (1 min), 72°C and (1 min), the PCR fragment was separated by agarose gel electrophoresis, purified, and ligated into pGEM-T vector followed by transformation of competent DH-5␣ cells.

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Sequencing DG-75 and analysis of retroviral clones Plasmid DNA was isolated from positive transformants using the WIZARD plasmid DNA isolation kit (Promega) and sequenced in both directions using the Pharmacia ALF system. Initially, T7, SP6, M13 forward, or M13 reverse fluorescently labeled primers were used to generate sequence data. Fluorescently labeled sequencing primers (Operon, Alameda, CA) designed from the sequence data were used to obtain overlapping sequence data in both directions for each clone. The data were analyzed and a consensus sequence identified using the GCG program (Madison, WI). Isolation and analysis of cellular mRNA Polyadenylated RNA from the DG-75-UW, ⫺KAR, and ⫺HAD cell sublines was isolated using oligo(dT)– cellulose columns according to the manufacturer’s protocol for the poly(A) Pure Kit (Ambion, Inc.). Five micrograms of polyadenylated RNA was separated using a standard 1% agarose gel with 0.24 M formaldehyde (Sambrook et al., 1989). The RNA was transferred to a nylon membrane (Bio-Rad) according to Beckers et al. (1994). Briefly, the gel was soaked two times for 5 min in DEPC-treated H2O. The RNA was denatured in the gel for 30 min using 0.05 N NaOH and then equilibrated for 30 min in 20⫻ SSC buffer. The RNA was transferred to a nylon membrane by capillary elution in 20⫻ SSC buffer for 24 h. The RNA was fixed to the membrane in 0.05 N NaOH for 5 min and washed in 2⫻ SSC for 2 min. The blot was blocked in prehybridization buffer (5⫻ SSPE, 5⫻ Denhardt’s, 0.5% SDS, 10 ␮g/ml denatured salmon sperm DNA) for 2 h. The R-U5 clone (Fig. 1A) was used to synthesize 32Plabeled riboprobes using MAXIscript SP6/T7 Kit (Ambion) (Fig. 2). Hybridization was performed at 60°C for 20 h in prehybridization buffer. Blots were washed under stringent conditions: 2⫻ SSC, 0.1% SDS for 15 min at 37°C; 2⫻ SSC, 0.1% SDS twice for 15 min each at 65°C, 1⫻ SSC, 0.1% SDS twice for 15 min each at 65°C; 0.1⫻ SSC, 0.1% SDS for 15 min at 65°C. The blots were analyzed by autoradiography. Radiolabeled DNA probes were generated by PCR for the gag (nts 1082–1520), pol (nts 3984 – 4689), and env (nts 6150 – 6686) regions (Fig. 2). The probes were separated by LMT agarose gel electrophoresis, extracted, and then melted at 65°C in hybridization buffer (500 mM NaHPO4, pH 7.2, 7% SDS). Hybridization to DG-75 (UW) mRNA was carried out at 65°C for 18 h followed by two washes in 40 mM NaHPO4, pH 7.2, 5% SDS, and two washes in 40 mM NaHPO4, pH 7.2, 1% SDS, all at 65°C for 30 min. The blots were analyzed by autoradiography.

were harvested in DMEM–10% FBS, filtered through a 0.45-␮m filter, and used immediately for LacZ infection assays. Harvested virus was diluted with DMEM–10% FBS containing 8 ␮g/ml of polybrene and plated on the assay cells in 24-well plates. After 4 h of infection, virus was removed and the cells were cultivated in growth medium. Two days after infection, the cells were stained with 5-bromo-4-chloro-3-indolyl-␤-D galactopyranoside (X-Gal) in situ. LacZ-positive colonies were counted as previously described (Cosset et al., 1995; Tailor et al., 1993; Takeuchi et al., 1994). Infectivity experiments Virus-containing DG-75 (UW) cell-culture supernatant, clarified by low-speed centrifugation and passed through a 0.2-␮m filter, was added with 2 ␮g/ml polybrene directly to 70% confluent cell monolayers or to 1⫻ 106 cells/ml in suspension. Suspension cultures were incubated at 36°C for 2 h and then diluted 1:2 with culture medium and incubated at 36°C for an additional 16 h. The medium of suspension and monolayer cultures was replaced after 18 h at 36°C with fresh medium and again subsequently as indicated. At 10, 21, and 30 days after infection, supernatant fluids were assayed for reverse transcriptase as previously described (Raisch et al., 1998), and genomic DNA from cell pellets was analyzed for integrated virus by nested PCR of the env region. Nucleotide sequence accession number The complete nucleotide sequence of the DG-75 MLV variant has been entered into the GenBank nucleotide sequence database (Accession No. AF221065). Acknowledgments We gratefully acknowledge the donation of: (i) sublines of DG-75 cells by Hannah Ben-Bassat (Hadassah Medical Center, Jerusalem, Israel), Bill Sugden (University of Wisconsin, Madison, WI), and George Klein (Karolinska Institute, Stockholm, Sweden); and (ii) Mv1.Lu mink lung, murine SC-1, and rat sarcoma XC cells by Rex Risser (University of Wisconsin, Madison, WI). We thank Monika Casey and Dean Pasko for excellent technical assistance. We are grateful to Robin A. Weiss for stimulating discussions and review of the manuscript. This research was supported in part by grants to S.E.G. from the National Institutes of Health (RO1-AI32710) and the Council for Tobacco Research, USA (No. 2970). Y.T. was supported by the Medical Research Council, UK. and M.P. by EU TMR Grant FMBICT 961804.

Interference assay

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