Chinese Hamster Ovary Cells Contain ... - Journal of Virology

Vol. 68, No. 12

JOURNAL OF VIROLOGY, Dec. 1994, p. 7840-7849 0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Chinese Hamster Ovary Cells Contain Transcriptionally Active Full-Length Type C Proviruses YOLANDA S. LIE,1 ELICIA M. PENUEL,lt MARI-ANNE L. LOW,1 T. P. NGUYEN,2t JOCELYN 0. MANGAHAS,1 KEVIN P. ANDERSON,1§ AND CHRISTOS J. PETROPOULOSl* Molecular Virology Laboratory,1 and DNA Sequencing Laboratory,2 Genentech, Inc., South San Francisco, Califomia 94080 Received 22 April 1994/Accepted 7 September 1994 We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons.

Since their development in the 1950s, CHO cells have been used extensively for genetic studies (see reference 13). Recently, CHO cells have been used to manufacture recombinant protein therapeutics (7, 11, 18, 33). Several features make the CHO cell culture system a suitable choice for the manufacture of recombinant protein therapeutics (for discussions, see references 12, 24, and 40). Adherent CHO cell populations can be readily adapted to growth in suspension culture to facilitate scale-up and manufacturing. In general, complex proteins undergo more appropriate glycosylation and posttranslational modification when produced in CHO cells versus other eukaryotic (yeast or insect cells) or prokaryotic cell systems. An efficient system based on cell lines that are deficient in dihydrofolate reductase activity is available for generating cell clones that produce large amounts of exogenous protein (reviewed in reference 44; see reference 3 for methodology). In addition, CHO cells are nonpermissive for the replication of many human-pathogenic viruses (43). In contrast to many rodent cell lines, CHO cells do not produce demonstrable levels of infectious retrovirus (9, 17, 22). Most CHO cell lines are resistant to infection by all of the major classes of murine retroviruses, including amphotropic strains, which are known to infect cells from a wide variety of mammals (5, 14, 38). However, both intracytoplasmic type A and budding type C retrovirus-like particles have been detected in CHO cells by electron microscopy (16, 17, 22, 23, 39, 42). The small number of retrovirus-like particles produced by CHO cells and their lack of demonstrable infectivity suggest that they are the product of endogenous retrovirus sequences and not exogenous retrovirus infection. Previously, Anderson et al. (2) reported the molecular and biochemical character-

izations of the retrovirus-like particles concentrated from CHO cell culture fluids. Purified particles were shown to be immunologically related to mammalian type C retroviruses and to possess detectable levels of reverse transcriptase. It was also shown that the particles contained RNA sequences homologous to rodent retrovirus RNA. Related sequences were detected in CHO cell and Chinese hamster liver DNA preparations. To extend the investigation of CHO retrovirus-like particles, a recombinant X phage library was constructed from CHO genomic DNA and phage plaques containing retrovirus-related sequences were identified by using a cDNA probe that was generated from purified CHO retrovirus-like particle RNA. In this report, we present the first complete nucleotide sequence of a full-length type C provirus isolated from CHO cells. We show that it is a member of a family of full-length type C proviruses that are found both in CHO cells and in Chinese hamster liver. Some of these elements are actively transcribed and produce both full-length genomic RNA and subgenomic envelope mRNA. The observations we report are consistent with an endogenous origin of type C retrovirus-like particles in CHO cells.

MATERUILS AND METHODS CHO genomic DNA library. A genomic DNA library was constructed from a Sau3AI partial digestion of CHO cell DNA with the Lambda Fix vector system used in accordance with the manufacturer's (Stratagene, LaJolla, Calif.) instructions. X phage plaques containing type C retrovirus-related inserts were identified by conventional filter hybridization methods by using 32P-radiolabeled ML10 sequences (3). ML10 is a cDNA sequence that was generated from the RNA of CHO cell retrovirus-like particles and is homologous to the endonuclease region of rodent type C retroviruses (2). DNA sequence analysis. XML2G sequences were subcloned into the pUC219 vector by using standard molecular cloning protocols (3). DNA sequencing was performed by the dideoxychain termination method with single-stranded DNA templates (1-3). Internal primers were synthesized on the basis of derived DNA sequence information. Computer analyses of

* Corresponding author. Mailing address: Genentech, Inc., 460 Pt. San Bruno Blvd., South San Francisco, CA 94080. Phone: (415) 225-1328. Fax: (415) 225-2866. Electronic mail address: petropoulos.christos

@gene.com. t Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94702. t Present address: Applied Biosystems, Foster City, CA 94404. § Present address: Department of Molecular and Cell Biology, ISIS Pharmaceuticals, Carlsbad, CA 92008. 7840

VOL. 68, 1994

ENDOGENOUS TYPE C RETROVIRUS EXPRESSION IN CHO CELLS

DNA sequences (GenBank homology search, homology plots) were performed with the sequence analysis package (third edition) generated by the Scientific Computing Group of Genentech. DNA and RNA analysis. Genomic DNA was prepared from CHO cells by using proteinase K digestion and phenol-chloroform extraction (3). The products of restriction endonuclease digestion were separated by electrophoresis and analyzed by conventional Southern transfer hybridization (1-3). Total cellular RNA was prepared from cell cultures with RNAzol (Tel-Test "B", Inc., Friendswood, Tex.), and the poly(A)+ RNA fraction was isolated by oligo(dT) cellulose affinity chromatography (Pharmacia Biotech, Inc., Piscataway, N.J.). The poly(A)+ RNA fractions were analyzed by Northern (RNA) transfer hybridization methods (1). Hybridization probes were generated by preparing purified long terminal repeat (LTR), gag, pol, and env sequences as depicted in Fig. 1. Radiolabeled probes were prepared by nick translation (Gibco/BRL, Bethesda, Md.). Filter hybridizations were performed overnight at 42°C in hybridization buffer (50% formamide, 50 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.0], lx Denhardt's solution, 3x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 150 ,g of denatured salmon sperm DNA per ml). Filters were washed once for 15 min at room temperature in 2x SSC and twice for 60 min each time at 55°C in 0.1X SSC-0.1% sodium dodecyl sulfate. Radioanalytic quantitation of hybridization signal intensities was performed by using an AMBIS 100 Detector (Ambis, San Diego, Calif.). PCR. B52-related LTR sequences were amplified by PCR (3) from CHO cell genomic DNA by using the following oligonucleotide primers: 5'-CGC-GTC-GAC-TGA-AAGACC-CCT-GTC-C`-l-TAG-CCC-3' (forward) and 5'-GGGTCT-AGA-ATC-CCG-GAC-GAG-CCC-CCA-3' (reverse). The underlined sequences represent Sall and XbaI recognition sites that were incorporated into the forward and reverse primers, respectively. The PCR product was characterized by sequence analysis. CAT assays. The ML2G LTR region was inserted into the pCAT vector series (Promega Corporation, Madison, Wis.) between the Sall and XbaI sites by using a Sall site at position 1 and an XbaI site at position 1276 of the ML2G sequence. CHO Kl cells were transfected with LTR-chloramphenicol acetyltransferase (CAT) plasmids by the calcium phosphate precipitation protocol (3), and cell lysates were prepared 48 h after transfection. CHO cell lysates were adjusted to an equivalent protein concentration by using a modified Bradford assay (Bio-Rad Laboratories, Hercules, Calif.), and CAT activity was measured by conventional methods (3). Levels of CAT activity were quantitated by radioanalytic imaging (Am-

bis).

Nucleotide sequence accession number. The nucleotide sequence reported here (see Fig. 2) has been submitted to GenBank and assigned accession number U09104. RESULTS Isolation of endogenous retrovirus sequences from CHO cells. To isolate endogenous retrovirus sequences from CHO cells, a genomic DNA library was constructed in AFIX (see Materials and Methods for details). Approximately 5 x 104 recombinant phage, which we estimate represented 10 to 20% of the CHO cell genome, were screened by nucleic acid hybridization. The hybridization probe consisted of a cDNA sequence (named ML10) which is homologous to the endonuclease region of murine type C retroviruses. ML10 was isolated

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genomic RNA env mRNA FIG. 1. CHO cell endogenous provirus map. (A) Recombinant X phage ML2G was isolated from a genomic library of CHO cell DNA. XML2G contains 14.6 kb of CHO cell DNA (thick bar) that can be excised from the A vector arms (thin bars) by digestion with restriction endonuclease SalI (S). The 5' and 3' boundaries of the mammalian type C retrovirus sequences within the ML2G insert are defined by the provirus LTRs (solid boxes). An EcoRV (RV) recognition site is present in each LTR but not elsewhere in the provirus sequence. The provirus contains several unique restriction endonuclease recognition sites: ClaI (C), XhoI (X), SalI, (S) and ScaI (Sc). (B) Nucleotide sequence analysis has defined a complete type C provirus within ML2G. The positions of the gag, pol, and env genes (thin line) and flanking LTR sequences (solid boxes) are shown. Each region was subcloned separately to create nucleic acid hybridization probes (solid bars) specific for the LTR, gag,pol, and env regions. These probes were used to identify related sequences in CHO cell DNA and to define the structures of RNAs (solid lines) expressed in CHO cells. PstI, P; BamHI, B; XbaI, Xb; HindIII, H; SalI, S.

from a cDNA library generated from the RNA of CHO cell retrovirus-like particles. Its characterization has been reported elsewhere (2). Hybridization screening identified 19 recombinant A phage-containing ML10-related sequences. On the basis of preliminary restriction site mapping studies, clone XML2G, containing a 14.6-kb insert, was chosen for further

characterization (Fig. 1). AML2G contains a full-length retrovirus sequence. The nucleotide sequence of a continuous 9.6-kb stretch of the XML2G insert was determined by using conventional subcloning and dideoxy-chain termination sequence analysis protocols

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J. VIROL.

LIE ET AL.

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CGATA CC C ACCCAAGcma=} CATcOcAcc AWIG=A CATAGCAcr T1UAT= CiCWrC GOCAIttW, CCmrIc CroCIw Cri1it:r CrAct= CACCO ACCOCAAm CC GACIGAA AAACSIGPGM CIGAr AcGAG=AA GAGAETA GOIAAGAGA AGAACA[ AWI IA GGO'IAC GGCAAGACG GTIrOU= OCACI GC OR GAAXXMt CAC: ACG CGCC ATc TC.AGG CACOCCI CAGA.TICGC.C1TXk_ CI.MMACM GACCGAAP CACTCAA T GAS CACAACWIAC CAACCM AAOIGGAAAA ATAATA1A11UCIr11UICr GCACACt' r GCTGAI CC3 AAOOMG A.GAA.AAT MA GWGAAGAGAAA CAC1I'IM TACrGAG T1WCAGI = GGmAwIAaw GGCcAfccPAGCIGOtcAAG AAA=AGC GGrO1C CTI WfAGC CGAATGG TITTACrACr GAAC_flGTA GGACCCTCT V1UCU7IG3G77rIG CAGGAC ACCTACCAAT TACCGAACGr AATACPGG GC=GAAT COaXCrC T AGACAAAA GAATAr, AAUMATA= COUCCqAGAIC CAAAGAaU AGCAGO ACCACC MIVITA T rICA AGCCOCGGPC ATAAAA-CA AGcCCAAA GcIAAAAT TrACAAT ATACO'ICCA AGICITA AA=.PAGCAG AAnMUr C TATAAGAG GCIr AGAGAtC= CY=GGAA GAAAGAC AGAA &A R AGAACI'I7G AGAAAGGAA C GAAW =ACAG AGoGAATA AAGATGAGC AGOTATI ccAGrAGr GAGICAG AGACAGAATA GGCAGAGGA TGGCAGAAG GG0C0C

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GGCtAAG= COAOOCCAA GACIGT CTIOAAW TcAAaocr TwUGAAAG AAAAMAcA TiuGCAAAA GAATOWA AGM G ATC3GCAC cTIW.rAGCGATACAGSG AXI CGA AACAC'AGC vnNGAAG7TCAcAGAAMA GoccAGG C GTrAC'AAA CArATA= K=CGAAz CGIUC GA I MACCGAr GCAIGGI= AGAC'IC APGoaAAW CAanIT AT GACCAAATT oGcAI CAitcA crAnrTA_GCAT=T TI a AG CCCAGAT'I cccc TrAWrAGGA TIGACAI CCATAGAAGA LATACAGA AAG AA ATAcTI AG PGIUr AGCAAAGI ACAGOGCAG ACGAAI'CCrrArCCC AACAGGRA GC C CTcAAAAGGAA TrnAAcAm cAGGAAA=C TGATcAI GcTG A T MAG ACAG'AI AICrAAAr AAGOXAGA AGGAAIM CATcAAccAG GccAATA TAGrAAC AAAAGII CCTIAAA C AAGCAGAA CCAA7GAM ccAcATwm AAAGrrAC GcAAA TAAA c1rr CrCCAACCCT TATAATI 7GAGGAT GCOAAC TAoGoOAGTA cAGGATcIA oGAGGIA TAAAATA GAGACATAC A AT.IGG ATALMT AGAICTTAA GAAGATC'1:C CrATATTIG C=MUATI C.AGGAIGCXr CPACTIGGA CAGTAC 7CAAGG I AAGAAG OCUCTII MAAAGT TrAATAGG A MCAGG AccriAAT C A AAr C'ITAGr a GA.MA 'iU:Cltt'IX AGCCAAAAC AAGGAAGAAT GCAA MAAGG CACP.AG A I AGGATACAAG ATAaAGGA= CI CAZPB CCAArAAGC CCAGATAlU CAGAAACAGr C TC GAGGGGAIG GcAnGA G A GC GOCA1 ACAT ACCCACAAA ATaXXMCCA ACIGAGAGAG TMI CRCAG=C CA a cIanoc'r U oaMrAATGOCrnr GCrTFr%AA GGGAAGAGC CAAAAGAA rCC1 GOCt1ACCA ACAAAAA GAATCACrAG AOGACrA GCAAG= TIGAACI TATAGAMAG MAMA= I GCAC G AAGGP,C.CA GCATAL IC AAGAAAIT AGccT cAAAAAI GC ATGCGAAG. G ;GIGCAG AGAAAAGAT CM= CCATACA.W I lIlATG ACCAC ATCAGA I AGAMC C=AmCG AATACAT T1GACCA 1A IGG CAA-CGG GACTG GI GCilana GcAGCAW TGC'IttCTCr