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Biological Activity Ofcloned Retroviral DNA in Microinjected Cells

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Biological Activity Ofcloned Retroviral DNA in Microinjected Cells Proc. Natl Acad. Sci. USA Vol. 78, No. 7, pp. 4383-4387, July 1981 Cell Biology Biological activity of cloned retroviral DNA in microinjected cells (long terminal repeat/transcription/promoter assay) JOHN J. KOPCHICK, GRACE Ju, A. M. SKALKA, AND DENNIS W. STACEY Roche Institute of Molecular Biology, Nutley, NewJersey 07110 Communicated by B. L. Horecker, April 1, 1981 ABSTRACT Avian retroviral DNA molecules that had been 2.2) (2). The LTR is believed to contain the promoter se- cloned from infected cells by using recombinant DNA techniques quence(s) for viral transcription (8, 9). were microinjected into either uninfected chicken embryo fibro- Because the enzyme Sal I used to clone the circular DNA blasts (CEF) or CEF transformed by the envelope glycoprotein- cleaves within the viral env gene (1, 2, 9), the cloned molecules deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Ret- contained a permuted gene order with portions of env gene at roviral DNA injected into RSV(-) cells directed transcription of each terminus of the virus-specific region within the linear A envelope mRNA, which was then able to complement the RSV(-) vector (Fig. IA) (2). Therefore, the production of env mRNA env deficiency and promote the production of infectious trans- in a recipient cell would require (i) removal of virus-specific forming virus. The retroviral DNA also directed the production DNA from the A vector, (ii) ligation of the ends of the cloned of fully infectious virus after injection into uninfected cells or and RSV(-) cells. Virus production began within 3-4 hr after microin- DNA to form a circle or linear concatemer (Fig. 1B), (iii) jections. When 100 DNA molecules per cell were injected, almost transcription of the injected DNA. Subsequent translation of all injected cells produced infectious virus. As the number of in- the mRNA would produce the envelope precursor protein, jected molecules per cell was decreased, a corresponding decrease which would be processed and incorporated into budding was observed in the number ofcells that produced infectious virus. virions. DNA injected into the cytoplasm was 1/50th to 1/10th as effective The result reported here suggests that DNA ligation and in virus production as DNA molecules injected into the nucleus. transcription occur rapidly and directly from injected DNA DNA molecules containing one or two tandem copies of the viral molecules. The data also indicate that our microinjection system long terminal repeat were equally effective in virus production. provides a sensitive assay in which to explore the relationship between eukaryotic promoter structure and function. DNA cloning, restriction endonuclease mapping, and nucleo- tide sequence determination experiments have revealed many MATERIALS AND METHODS details of the molecular structure of avian retroviruses (1-10). Cell Culture. CEF preparations and culture conditions have In order to correlate structural datawith information concerning been described (14-16). CEF preparations were negative in the function, we have studied the biological activity ofcloned retro- expression of endogenous group-specific antigens and endog- viral DNA molecules after microinjection into cultured chicken enous env activity (gs-, chf; from SPAFAS). RSV(-) refers to cells. CEF infected with the Bryan strain of RSV in the presence of In previous reports, viral RNA molecules were studied by ultraviolet-light-inactivated Sendai virus (14-16). RSV(-) vi- using the technique of microinjection with glass micropipettes rions produced by RSV(-)-transformed cells contain no helper (11-13). For these studies, injections were performed into virus (11). No virus infectious for CEF (C/E type, which are chicken embryo fibroblasts (CEF) transformed by the envelope resistant to infection with subgroup E virus) were released from glycoprotein-deficient Bryan strain of Rous sarcoma virus RSV(-)-transformed cells. [RSV(-) cells]. Envelope glycoprotein formed within the in- Microinjection. For each microinjection experiment, RSV(-) jected cells complemented the env deficiency ofthe Bryan RSV, cells from the same preparation were plated onto individual resulting in the production of focus-forming units (FFU) of in- glass coverslips. Prior to injection, the coverslip was placed in fectious RSV. With this sensitive assay env mRNA and its nu- a 35-mm plate and incubated with 2 ml of growth medium at clear precursor were detected in cells and virus particles. FFU 370C for 1 hr. Microinjection using glass pipettes with an out- production began approximately 3 hr after RNA injection and side diameter of 0.5 um was performed on 300 cells at a mag- continued for 48 hr. A direct relationship was observed between nification of X640 in approximately 30 min at room tempera- FFU production and the amount of env mRNA injected. ture. A glass syringe was used manually to produce and regulate In the studies presented in this paper, cloned retroviral pressure necessary for microinjections. We also have performed DNAs were injected into RSV(-) cells as well as normal un- microinjections with uninterrupted flow from the micropipette infected CEF. Transcription of the injected DNA was assayed and found no difference in results between cytoplasmic and by the production ofinfectious virus. The cloned DNA used in nuclear injections. This pressure was created only when the orifice of the micropipette was within the cell. Cytoplasmic in- these studies originated from transformation-defective uninte- jections were visualized by movements of granules away from grated circular molecules isolated from CEF infected by the site of the cytoplasm at the site ofinjection. Nuclear injec- Schmidt-Ruppin B RSV (td SR-RSV-B). This DNA, cloned in tions were apparent with an immediate change ofthe refractive the bacteriophage A vector Charon 21A, was shown previously index of the nucleoplasm upon microinjection (17). Spillage of to contain either one (ASRBtd-2.4) or a mixture ofone and two tandem copies ofthe viral long terminal repeat (LTR) (ASRBtd- Abbreviations: CEF, chicken embryo fibroblasts; RSV, Rous sarcoma virus; RSV(-) cells, CEF transformed by envelope glycoprotein-defi- The publication costs ofthis article were defrayed in part by page charge cient RSV; SR-RSV-B, Schmidt-Ruppin strain B of RSV; td, transfor- payment. This article must therefore be hereby marked "advertise- mation-defective; FFU, focus-forming units; LTR, long terminal ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. repeat. 4383 Downloaded by guest on October 3, 2021 4384 Cell Biology: Kopchick et al. Proc. Natl. Acad. Sci. USA 78 (1981) Sal I Sol I 4 LTR 4 A I C F a gog pozzzi Sal I Sal I Sal I *I LTR * LTR 4 B NZA c gag pol c/// gag pol FIG. 1. (A) Sal I restriction endonuclease map of the ASRBtd-2.4 recombinant clone. The env gene is indicated by hatching and the LTR by stippling. Sal I sites indicate the boundary between bacteriophage A DNA and virus-specific DNA. The relative order ofthe viral genes in the viral RNA genome is: (5')gag,pol, env, C(3'). The virus-specific sequence in this clone is permuted sa as to divide the env gene. (B) Diagram ofa concatemer of virus-specific DNA molecules. Virus-specific DNA (shown in A) was freed from bacteriophage A sequences by Sal I treatment prior to microin- jection. If two (or more) such molecules were ligated together after injection, the DNA molecule diagrammed above would result. This molecule contains a structure, noted by broken lines, analogous to proviral DNA integrated in the chromosome of an infected cell. It is conceivable that this structure would be transcribed as though it were a normal provirus. solutions into the cytoplasm during nuclear injections could not Recombinant DNA was handled in accordance with the be completely avoided. After injections, the coverslip was guidelines of the National Institutes of Health. placed in 2 ml of growth medium and incubated at 370C for various time intervals. After incubation, the entire 2 ml ofcul- RESULTS ture fluid was collected for virus assay. Assays for env mRNA Production: Time Course of RSV Infectious RSV production by injected RSV(-) cells was de- Release. Virus-specific DNA was freed from the A vector DNA termined in an infectivity assay for FFU as described (11-13). by cleavage with Sal I and purified by sucrose gradient sedi- Infectious, nontransforming virus (td SR-RSV-B) produced by mentation (see Materials and Methods). When this virus-spe- microinjected normal CEF were assayed indirectly by using the cific DNA was microinjected into the nuclei of RSV(-) trans- following protocol: Culture fluids, collected at various time in- formed cells, infectious RSV were first detected at 3 hr after tervals from microinjected CEF, were added to a culture dish injection, and the number increased continuously for 48 hr containing 2.0 X 105 CEF and 1 X 105 RSV(-)-transformed thereafter (Table 1A). This is similar to the time course ofvirus cells,,followed by incubation at 370C for 48 hr. Infectious RSV production observed after injections of env mRNA into the cy- produced by these infected cultures was determined as de- toplasm ofRSV(-) cells (11). When virus-specific DNA was in- scribed above (11-13). jected into the cytoplasm ofRSV(-) cells, virus production also The following values were used in the calculation ofthe num- began at 3 hr (Table 1). Thus, it appears that the transport of bers of DNA molecules microinjected per cell: (i) the final con- the injected molecules into the nucleus occurred rapidly. The centration of cloned DNA used in a given microinjection ex- results presented in Table 1 indicate that nuclear injections are periment (0.2-200 ,ug/ml); (ii) the total volume microinjected about 4- to 6-fold more efficient in the production ofinfectious per cell, which has been determined to be 5.0 x 10-11 ml for virus from RSV(-) cells relative to cytoplasmic injections.
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