Extrachromosomal DNA (Lentiviruses/Integration/Restriction Enzyme Analysis) JEFFREY D

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Proc. Natl. Acad. Sci. USA Vol. 81, pp. 7212-7215, November 1984 Microbiology Slow virus visna: Reproduction in vitro of virus from extrachromosomal DNA (lentiviruses/integration/restriction enzyme analysis) JEFFREY D. HARRIS*, HUBERT BLUM, JANE SCOTTt, BETTY TRAYNOR*, PETER VENTURA, AND ASHLEY HAASE§ Infectious Disease Section, V.A. Medical Center, San Francisco, CA 94121 Communicated by Howard M. Temin, July 27, 1984

ABSTRACT Under permissive conditions of growth in tis- Cloning of Visna DNA. Viral DNA for probes and for re- sue culture, the retrovirus visna multiplies over the course of a construction experiments was obtained by cloning. The rele- few days to high titer and kills the host cell. We show that in vant restriction enzyme sites in visna DNA (9) are shown in this lytic life cycle, viral DNA is tightly associated with, but not Fig. 1. DNA from the Hirt supernatant fraction from SCP covalently linked to, chromosomal DNA. This finding provides cells infected for 60 hr was digested with Sst I, and the frag- explanations for a number of the unusual properties of the len- ments were inserted into a X Charon 10 vector (13). Fifteen tivirus subfamily of retroviruses, and suggests potential mech- clones of recombinant bacteriophage were identified by hy- anisms for the block in virus gene expression in vivo responsi- bridization with a 32P-labeled probe transcribed from viral ble for the slow infection in nature. RNA with random primers (11). Four clones with the small (Vs) Sst I fragment and two clones with the large (VL) Sst I Visna virus is the prototype of the subfamily of retroviruses fragment were identified by restriction enzyme mapping. that cause slow infections in sheep characterized by patho- These clones were amplified and purified by banding in logical changes in the lungs and central nervous system (1, CsCl. DNA from purified bacteriophage was digested with 2). Both the persistence of virus in the face of the immune Sst I, and the small and large fragments were isolated after response mounted by the host and the slow evolution of dis- separation in agarose gels. ease can be explained by restriction in virus gene expression Hybridization Procedures. Restriction enzyme digestions, imposed at the transcriptional level (3, 4). Most of the cells electrophoretic separation of DNA in agarose, and transfer that harbor virus genomes have insufficient antigen to be de- to diazophenylthioether-paper followed published protocols tected and destroyed by immune surveillance, and limitation (11). Visna-specific probes were radiolabeled to 10 cpm/,g in synthesis of virus gene products allows the host cell to by nick-translation of cloned DNA. The number of copies of survive for the extended periods characteristic of slow infec- viral RNA in individual cells was evaluated by in situ hybrid- tions. ization (8). The mechanism of the block in transcription is unknown but has generally been assumed to be related to a lysogenic RESULTS state, since visna virus is a retrovirus, and viral DNA is associated with high molecular weight host DNA (5-7). Howev- Visna DNA Is Tightly Associated with High Molecular er, we recently found that transcription in vitro is governed Weight DNA. Most visna DNA extracted from cells is a lin- by the extent of early DNA synthesis and suggested that ex- ear duplex molecule of 9.5 kbp with a nick or gap near the trachromosomal DNA might be the template for viral RNA center of the molecule (11). This DNA is found in the nucle- (8). Moreover, Panganiban and Temin (9) have shown that us of infected cells within the first few hours of infection and production of spleen necrosis virus, an avian C-type retrovi- thereafter (8), and a signficant proportion (about 25%) parti- rus, can occur from unintegrated viral DNA. For these rea- tions into the Hirt pellet. The association with high molecu- sons, we reexamined the role of integration in the visna life lar weight cellular DNA is apparently quite stable, since the cycle in vitro and found that in the vast majority of cells un- same fraction of DNA is associated with high molecular integrated DNA must serve as the template for transcription weight DNA prepared by other procedures (data not shown), and for virus production. such as network formation (5) in alkali (14) or sedimentation of DNA through gradients after lysing the cells in detergent MATERIALS AND METHODS and 2 M NaCl (15). Infection of Cells and Isolation of DNA. Confluent cultures Restriction Enzyme Analysis. However, by restriction en- of sheep choroid plexus (SCP) cells were infected at a multi- zyme tests for integration (16, 17), visna DNA is not cova- plicity of 3 plaque-forming units per cell as described (8, 10). lently linked to host DNA. We first suspected that this was At 60-70 hr after infection, cells were removed by trypsini- the case when we found that we could no longer detect viral zation and either replated under agarose for infectious center DNA associated with host DNA 20 kpb or larger after elec- assay or separated into nuclear and cytoplasmic fractions trophoresis through low-gelling temperature agarose (Fig. (11). High molecular weight DNA was isolated by the Hirt 2C). Full-length viral DNA in the Hirt pellet (Fig. 2B, lane U) fractionation procedure (12). In some experiments, high mo- freed by this procedure comigrated with uncut viral DNA in lecular weight DNA was purified further by electrophoresis the Hirt supernatant (Fig. 2A), and identical species were in 0.5% low-gelling temperature agarose; the portion of the generated by digestion with BamHI, EcoRI, and Hindill gel containing DNA 20 kilobase pairs (kbp) or greater in size was excised and melted, and DNA was isolated by phen- Abbreviations: kbp, kilobase pair(s); SCP, sheep choroid plexus. ol/chloroform extraction. *Present address: Microgenics, Concord, CA 94520. tPresent address: Rockefeller University, New York, NY 10021. tPresent address: Academic Research Information System, Inc., The publication costs of this article were defrayed in part by page charge San Francisco, CA 94115. payment. This article must therefore be hereby marked "advertisement" §To whom correspondence should be addressed at: Department of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Microbiology, University of Minnesota, Minneapolis, MN 55455.

7212 Downloaded by guest on September 25, 2021 Microbiology: Harris et aL Proc. Natl. Acad. Sci USA 81 (1984) 7213

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Eco RI Eco RI Hind III Hind III Hind III Hind III FIG. 1. Map of the visna genome. Visna DNA is a linear duplex of 9.5 kbp comprised of an intact full-length minus strand, a plus strand interrupted by a nick or gap (I-4) in the center of the molecule, and two long repeat sequences (LTR; long terminal repeats) at the termini (11). Restriction enzyme sites in the DNA relevant to cloning and other experiments described in this article are indicated; the clone of visna DNA in X Charon 10 corresponding to the small Sst I fragment from 0.8-9.4 kbp is designated Vs; the clone corresponding to the large Sst I fragment from 0.8-9.4 kbp is designated VL- (Fig. 2 A and B, lanes B, RI, and HI11). Most important, we DNA cut with enzymes that do not cleave visna DNA, or could not detect in DNA 20 kbp or larger (Fig. 2C) fragments DNA cut with enzymes that do cleave visna DNA, we could of viral DNA internal to putative viral-host junctions that detect 0.25 copy of full-length virus DNA per cell, or even should have been released by these three restriction en- fragments of viral DNA (Fig. 3A). To further substantiate the zymes. sensitivity of the method, we also showed that we could de- Reconstruction Experiments, in Situ Hybridization, and In- tect a single copy of the sheep growth hormone gene on a fectious Center Assay. The sensitivity of the hybridization BamHI fragment of ovine DNA (Fig. 3B) and smaller frag- methods we used was more than sufficient to detect a single ments of that gene, using 5 pug of DNA and 0.7-kbp cloned copy of visna DNA integrated in high molecular weight probe (18). DNA. We established this by reconstruction experiments in At a time when DNA from infected cells lacks a single which we mixed the equivalent of 0.25 copy of viral DNA copy of integrated viral DNA, the cells are producing 50-100 per cell in 106 cells (3 pg of viral DNA) with high molecular plaque-forming units of virus per cell and several thousand weight DNA equivalent to that number of cells (5 Ag) from copies of viral RNA (8). We assured ourselves that this was uninfected cultures. After electrophoresis of uncut DNA, the case for the cultures from which DNA has been ana- lyzed, by showing by in situ hybridization that most cells had U B RI HIII U B RI HIII U B RI Hll viral RNA (Fig. 4) and by infectious center assay that 90% of Origin _ the cells were producing virus (data not shown). kbp DISCUSSION The production in vitro of visna virus from extrachromosomal DNA clearly distinguishes this representative of the subfamily of lentiviruses from retroviruses that transform cells and cause tumors and, by implication, highlights aspects of DNA structure important to integration. The vast majority of visna DNA molecules are linear duplexes comprised of a full-length minus strand and a plus strand interrupted by a nick or gap located at the center of the molecule (11). Only 0.1% of the molecules are covalently closed circles, in marked contrast to oncogenic retroviruses (11). If circular DNA is the topologically preferred form for integration, as a number of lines of evidence suggest (19-23), visna DNA predictably would not be expected to integrate. The insignificant role evidently played by integration in the visna life cycle in vitro holds the explanation we think for a number of peculiar features of the lentivirus subfamily,

FIG. 2. Restriction Enzyme Analysis of visna DNA Associated with high molecular weight Cell DNA. SCP cells infected for 66 hr were separated into nuclear and cytoplasmic fractions. DNA in the nucleus was partitioned by the Hirt procedure into supernatant (A) and pellet (B) fractions. (C) DNA in the Hirt pellet was electrophoresed in a gel of 0.5% low-gelling temperature agarose, and DNA 20 Z.J-~~~~~~~~ 'II.kX kbp or larger was identified from the position of markers in a parallel lane (bars at the left indicate the position and the size in kbp of X 2.0- DNA cut with HindIll), and excised and purified. Five micrograms of this DNA, equivalent to 1.2 x 106 SCP cells, was rerun in a gel of 1.2% agarose, either uncut (U) or after digestion with BamHI (B), EcoRI (RI), or HindIll (Hill). DNAs in the Hirt pellet and supernatant were treated similarly. After electrophoresis, the DNA was blot A B transferred to diazophenylthioether-paper and hybridized to a 32p_ IC labeled visna-specific probe (Vs + VL, 109 dpm/pg). The blots were Hirt Hirt Gel Purified exposed at -70'C to x-ray film in a cassette with intensifying screen Supernatant Pellet High Molecular for periods of hours to 9 days (C). The asterisks (*) in B indicate the pieces of viral DNA internal to the ends of the genome that should Weight DNA have been evident in C after release from putatively integrated viral from Hirt Pel let DNA. Downloaded by guest on September 25, 2021 7214 Microbiology: Harris et aL Proc. NatL. Acad. Sci. USA 81 (1984) 12 3 4 5 6 7 8 1 2 3 4 A kbp ;Ls i

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2.3 - BQ: 2.0- A FIG. 4. Detection of Visna RNA by in situ Hybridization. (A) An aliquot of the SCP cells infected for 66 hr described in the legend to FIG. 3. Reconstruction experiment to estimate the sensitivity of Fig. 2 was deposited by cytocentrifugation on coated glass slides. hybridization. (A) Five micrograms of DNA from uninfected SCP The cells were fixed, treated to enhance diffusion of the probe, and hybridized in situ (8) with a representative visna-specific probe re- cells, equivalent to 1.2 x 106 cells, was mixed with 3 pg of cloned verse to a visna DNA (VL) equivalent to 0.25 copy of viral DNA per cell in 106 transcribed from visna RNA with [3H]TTP specific activi- ty of 1.5 x 108 After with emulsion cells. DNA was electrophoresed, blot transferred, and hybridized as dpm/,g. coating NTB-2 and exposure at 40C for 3 the slides were and stained. described in the legend to Fig. 2; exposure time was 5 days. Lane 1, hr, developed uncut DNA; lane 2, DNA digested with BamHI; lane 3, DNA digest- (Photomicrograph taken with transillumination and incident polariz- ed with Bgl II; lane 4, DNA digested with EcoRI; lane 5, DNA di- ing light; original magnification, x500.) (B) Uninfected SCP cells; gested with HindflI; lanes 6-8, DNA digested with enzymes that do hybridization and exposure identical to that of A. not cleave visna DNA-Sal I, Sst II, and Xho I. Bars at the left indicate the position of X HindIll markers in kbp. (B) Five micrograms initially proposed by Battula and Temin (29) to account for of DNA from uninfected cells was digested with BamHI (lane 1), Bgl the cytopathic effects of spleen nicrosis virus. II (lane 2), EcoRI (lane 3), or HindIII (lane 4), electrophoresed, blot The persistence of visna virus and the slow pace of repli- transferred, and hybridized with a 700-nucleotide bovine growth cation in nature can be considered derivatives of restricted hormone insert in pBR322 labeled with 32p to 109 dpm/tzg. Exposure gene expression in vivo (1-4, 8). We have proposed two time was 5 days. mechanisms to account for this restriction, one equivalent to lysogeny on a grand scale in an animal, and the other a gene such as the remarkable resistance of lentiviruses to interfer- dosage model, in which transcription is regulated by the ex- on (24) and the lack of endogenous viral sequences (5). If tent of early viral DNA synthesis (8). among its other actions, interferon coordinately inhibits for- The findings reported here are consistent with both mech- mation of circular DNA, integration, and growth of oncogen- anisms of gene regulation. Under natural conditions of infec- ic retroviruses (25), its effects on visna predictably would be tion with small inocula, too few particles may enter cells to minimal, and the exogenous life cycle of visna in vitro pro- support the extensive early synthesis of viral DNA required vides a ready explanation for our failure to detect homology to achieve the high levels of transcription characteristic of between lentivirus DNA and cellular genes (5). the in vitro life cycles (8). Alternatively, virus DNA might be Visna virus in tissue culture fuses cells from without and, integrated in vivo at loci in host DNA where transcription is in the course of its growth cycle, causes giant cell formation repressed. with subsequent degeneration of the cells (26). We have at- tributed the cytopathic effects of visna virus to this demon- We thank Yvonne Guptill and Rosaria Cardella for preparation of strably damaging effect of the virus on the cell membrane this manuscript and Walter Miller for bovine growth hormone DNA. (1), although, by analogy with other cytopathic retroviruses This work was supported by grants from the American Cancer Soci- ety and the National Institute of Neurological and Communicative (27, 28), the large amount of unintegrated viral DNA in the Disorders and Stroke, by basic institutional support from the V.A., cell also could contribute to cell damage. What we can ex- and by a Heisenberg fellowship from the German government clude now in visna as the mechanism of cytotoxicity is multi- (H.B.). In the course of these studies, A.H. was a medical investiga- ple insertions of viral DNA into host DNA, a mechanism tor of the Veterans Administration. Downloaded by guest on September 25, 2021 Microbiology: Harris et aL Proc. Natl. Acad Sci. USA 81 (1984) 7215

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