Vol. 68, No. 5
JOURNAL OF VIROLOGY, May 1994, p. 3443-3447
0022-538X/94/$04.00+0 Copyright C) 1994, American Society for Microbiology
Modification of the Cascade Model for Regulation of Vaccinia Virus Gene Expression: Purification of a Prereplicative, Late-Stage-Specific Transcription Factor GERALD R. KOVACS, RICARDO ROSALES,t JAMES G. KECK,4 AND BERNARD MOSS* Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892 Received 28 December 1993/Accepted 8 February 1994
In vivo and in vitro studies have provided evidence that vaccinia virus late gene transcription factors are intermediate gene products synthesized exclusively after DNA replication. Here, we describe an additional transcription factor (P3 factor) that stimulates late gene transcription between 10- and 40-fold but is made in the absence of viral DNA replication. P3 factor activity was not detected either in uninfected cells or in purified virions. A >1,500-fold purification of P3 factor was achieved by column chromatography of cytoplasmic extracts prepared from cells infected with vaccinia virus in the presence of a DNA replication inhibitor. P3 factor was stage specific, since it could not substitute for early or intermediate transcription factors. Evidence that late stage-specific transcription factors are made both before and after DNA replication necessitates a modification of the cascade model for vaccinia virus gene regulation. been fully characterized and exceptions to this model are possible. For example, the genetic approach used to identify the late transcription factors could not have revealed additional prereplicative, late-stage-specific transcription factors. Using biochemical methods, we now provide evidence for such a late transcription factor and propose a modification of the cascade model. An in vitro transcription assay was developed to monitor the purification of vaccinia virus late transcription factors (VLTFs [28]). Initial studies (29) demonstrated that an infected-cell cytoplasmic extract could be fractionated into three components by phosphocellulose chromatography: P1 (0.1 M NaCl flowthrough), P2 (0.3 M NaCl eluate), and P3 (1.0 M NaCl eluate). Subsequent analyses of the phosphocellulose fractions demonstrated that the three transactivators of 17, 26, and 30 kDa, encoded by intermediate genes, were all present in the P1 fraction (13, 27). The P2 fraction contained most of the RNA polymerase activity and an essential factor that sedimented from a glycerol gradient with an apparent mass of 32 kDa (29). The P3 fraction contained low amounts of RNA polymerase and many additional polypeptides (29). On the basis of the genetic and biochemical analyses, one might have predicted that only the P1 and P2 fractions would be required for in vitro transcription of late genes. However, the P3 fraction was necessary for high levels of transcription in our analyses. This result indicated to us that the VLTF(s) in the P3 fraction must be either (i) present in uninfected cells, (ii) carried in by virus particles, or (iii) synthesized in infected cells prior to viral DNA replication. Biochemical fractionation techniques were used in this study to characterize the VLTF present in the P3 fraction. To determine whether the P3 factor is a host protein, we assayed an uninfected HeLa cell extract for VLTF activity. Five liters of uninfected HeLa cells and ten liters of cells infected with 10 PFU of vaccinia virus per cell were incubated at 37°C for 18 h. Cells were harvested by centrifugation, and cytoplasmic extracts were prepared by Dounce homogenization followed by centrifugation at 10,000 rpm in a Beckman Ti6O for 15 min to remove nuclei. The supernatants were processed essentially as described by Manley and Gefter (16), and the final extracts were dialyzed against buffer A (50 mM
A combination of biochemical and genetic analyses has shown that the successive expression of vaccinia virus early-, intermediate-, and late-stage genes within the cytoplasm of infected cells is regulated principally at the transcriptional level (for reviews, see references 17 and 18). Transcription of early genes occurs immediately after infection, since the virusencoded enzymes and factors, including a DNA-dependent RNA polymerase with an associated specificity factor termed RAP94 (3) and an early promoter-binding protein, vaccinia virus early transcription factor (VETF) (9), are packaged within the virions. Consequently, transcription of early genes is resistant to inhibitors of either protein or DNA synthesis. In contrast, both viral gene expression and DNA replication must precede the transcription of intermediate and late genes (5, 25). Viral early proteins needed for transcription of intermediate genes, including RNA polymerase, capping enzyme, and an intermediate transcription factor, have been isolated from cells infected in the presence of an inhibitor of DNA replication (11, 23, 24). The inability of a transfected late promoterregulated reporter plasmid to be expressed in cells treated with a DNA replication inhibitor formed the basis for screening a library of cloned viral DNA fragments for genes encoding late transcription factors (12). In that transfection assay, the intermediate genes AlL, A2L, and G8R, encoding 17-, 26-, and 30-kDa proteins, were necessary and sufficient for late transcription. The virion-associated early transcription factors, RAP94 (3) and VETF (7, 10), are products of late genes. Thus, the data are consistent with a cascade cycle (early-> intermediate-Aate--early) in which stage-specific transcription factors are encoded by genes of the immediately preceding regulatory class. However, not all the transcription factors have * Corresponding author. Mailing address: Laboratory of Viral Diseases, National Institutes of Health, Building 4, Room 229, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-9869. Fax: (301) 480-1147. Electronic mail address:
[email protected].
niaid.nih.gov. t Present address: Instituto de Investigaciones Biomedicas, Ciudad Universitaria, 04510 Mexico, D.F., Mexico. t Present address: Tularik, Inc., 270 E. Grand Ave., San Francisco, CA 94080. 3443
3444
NOTES
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1 2 3 4 5 FIG. 1. Late transcription requires the P3 fraction of infected cells. Cytoplasmic extracts from vaccinia virus-infected and uninfected HeLa cells were fractionated by phosphocellulose chromatography. In vitro transcriptions were carried out with (+) or without ( - ) 5 pL. of P1 (18 mg of protein per ml), 5 .1l of P2 (13 mg of protein per ml), and 10 p.1 of P3 (3.3 mg of protein per ml) in an assay mixture containing a late promoter-regulated G-less cassette. RNA was radiolabelled with [Uc--32P]UTP and analyzed by electrophoresis through a 4%c polyacrylamide gel. Shown is an autoradiogram of the dried gel. Indicated on the left is the size (in nucleotides) of the predicted RNA product.
Tris-HCl [pH 8.0]-0.1 mM EDTA-O.01% Nonidet P-40-10% glycerol-2 mM dithiothreitol) containing 0.1 M NaCl. Both extracts were fractionated by phosphocellulose chromatography as previously described (29). Briefly, the dialyzed extracts were applied to phosphocellulose (P11) columns that had been equilibrated with buffer A-0.1 M NaCl, and unbound proteins (P1 fraction) were collected. Bound proteins were eluted with two consecutive washes of buffer A containing 0.3 M NaCl (P2 fraction) and 1.0 M NaCI (P3 fraction). The P2 and P3 fractions were dialyzed against buffer A-0.1 M NaCl for 4 h prior to analysis. An in vitro transcription assay with a plasmid (pCFW9 [29]) that contains the vaccinia virus 11K late promoter fused to a DNA segment that lacks G residues in the nontemplate strand (G-less cassette [22]) was used to assess the activities of the reconstituted fractions (Fig. 1). Standard specific transcription assays (29) were performed at 30°C for 30 min with the exception that the final volumes were 20 [1I. Very low activity was obtained by combining the infected-cell P1 and P2 fractions (Fig. 1, lane 1). In this assay, the P3 fraction stimulated transcription approximately 40-fold (Fig. 1, lane 2). Addition of more of the infected-cell P3 fraction to the transcription reaction did not result in greater stimulation. Of significance, uninfected-cell fractions did not substitute for the P1, P2, or P3 fractions of infected cells (Fig. 1, lanes 3, 4, and 5), nor did they stimulate late transcription when combined in the absence of infected cell fractions (data not shown). Protein concentrations in the corresponding uninfected- and infectedcell P1, P2, and P3 fractions were similar as determined by the method of Bradford (6). We also assayed an uninfected whole-cell extract and found no stimulatory activity (data not shown). Thus, neither the cytoplasmic nor the nuclear fractions of uninfected HeLa cells contained the stimulatory activity in the infected-cell P3 fraction. We also considered the possibility that the infecting virion may bring P3 factor into the cell for use during the late phase. It has been shown that vaccinia virus particles contain all the factors essential for early gene transcription. Moreover, a functional intermediate transcription system can be reconstituted by combining the appropriate vaccinia virus intermediate transcription factors (VITFs) with virion-derived RNA poly-
1 2 3 4 5 6 7 8 9 FIG. 2. Virion extracts do not contain the P3 factor. A virion extract (VE) was prepared (19) and dialyzed against buffer A-0.1 M NaCI. This extract (5 ,ul at 0.45 mg of protein per ml) was combined with 5 p.l of the indicated phosphocellulose fractions (described in Fig. 1) from an infected-cell extract. RNA synthesis was analyzed as described in Fig. 1.
merase and capping enzyme (21, 23, 24). Therefore, we wished to determine if a virion extract could substitute for any of the infected-cell phosphocellulose fractions (Fig. 2). The basal level of transcription supported by the combined P1 and P2 fractions from infected cells is shown in Fig. 2, lane 9, and the stimulation of transcription is shown by the P3 fraction in lane 8. The low signal in lane 5 (Fig. 2) indicated that virion extracts do not contain the stimulatory activity found in the P3 fraction. Similarly, the virion extract could not replace the P1 fraction containing the 17-, 26-, and 30-kDa proteins, suggesting that these VLTFs are also not packaged. In contrast, the virion extract could replace the P2 fraction (Fig. 2, lane 7), indicating that the virion-associated RNA polymerase can functionally interact with VLTFs. Since the stimulatory activity in the P3 fraction was not found in either uninfected-cell or virion extracts, we suspected that this activity is virus-encoded or virus-induced and appears prior to DNA replication. To investigate this possibility, we prepared an extract from infected HeLa cells treated with AraC (1-f3-D-arabinofuranosylcytosine), fractionated it by
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 FIG. 3. The P3 factor is present in the absence of viral DNA replication. Cytoplasmic extracts were prepared from cells that were treated (+AraC) or untreated ( - AraC) with AraC (44 p.g/ml) starting 2 h prior to infection. Indicated phosphocellulose fractions (5 ,ul) were combined for the transcription assays, and RNA synthesis was analyzed as described in Fig. 1.
VOL. 68, 1994
NOTES
A
3445
B Cytoplasmic extract (+AraC)
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