Hyperexpression of baculovirus polyhedrin and p10 is inversely correlated with actin synthesis

VIROLOGY

191142-48

(1992)

Hyperexpression of Baculovirus Polyhedrin and pl0 Is Inversely Correlated with Actin Synthesis NING WEI* AND LOY E. VOLKMAN?’ *Department

of Biology, Yale University, New Haven, Connecticut 06511; and tDepartment University of California, Berkeley, California 94720

of Entomology,

Received June 5, 1992; accepted July 8, 1992 Polyhedrin and ~10, two proteins encoded by Autographa californica M nuclear polyhedrosis virus, are hyperexpressed very late during normal infections. In this study we found that cytochalasin D, a drug that leads to increased actin synthesis in infected and uninfected host cells, delayed the amplified expression of polyhedrin and p10 when added to infected cells before hyperexpression was already in progress. Restoration of polyhedrin and p10 hyperexpression could be achieved by removal of the drug, but required new protein synthesis. An inverse correlation was observed between polyhedrin/plO mRNA levels and actin mRNA levels at late and very late times during infection, regardless of whether cytochalasin D was added, removed, or never present. In comparison to mRNAs of polyhedrin and ~10, the mRNA levels of the early/late viral gene 39K were much less affected by cytochalasin D and responded to drug removal more slowly. The results of these studies revealed an apparent correlation between the shut down of host actin genes and the amplified expression of polyhedrin and p10 in the presence and absence of cytochalasin D. The possibility that newly synthesized actin itself, either directly or indirectly, plays a negative regulatory role in the accuo 1992 Academic PWSS. IIIC. mulation of polyhedrin and p10 mRNAs is discussed.

INTRODUCTION

(Possee and Howard, 1987; Rankin era/., 1988; Weyer and Possee, 1989; Ooi et al., 1989; Blissard and Rohrmann, 1990). Actin, one of the major cytoskeletal components of eukaryotic cells, exists in dynamic equilibrium between two forms in vivo: soluble globular monomers (g-actin) and insoluble filamentous polymers (f-actin). Actin microfilaments are known to be involved in various cellular cytoplasmic functions such as motility, morphogenesis, polar distribution of macromolecules, and cytokinesis (reviewed by Schliwa, 1986). Globular actin, on the other hand, has been reported to stimulate transcriptional activity of RNA polymerase II (Egly et a/., 1984; Scheer et a/., 1984). Like some other cellular components, actin is thought to be used by viruses for diverse functions (reviewed by Charlton and Volkman, 1992). These include budding, intracytoplasmic transport of structural proteins (Murti et al., 1985; Bohn et al., 1986; Stallcup et a/., 1983), cytoplasmic virus replication, transcription (De et al., 1991), and possibly nucleocapsid assembly (Volkman, 1988; Volkman et al., 1992). Cytochalasin D (CD) is a fungal metabolite that disables actin microfilaments and has been used widely to identify actin-based processes (Schiiwa, 1982; Yahara et al., 1982). Its primary mode of action is to bind to the fast-growing ends of the filaments and reduce their elongation rate (Cooper, 1987; Ohmori et a/., 1992). CD disrupts AcMNPV nucleocapsid morphogenesis, presumably by interfering with the virus-in-

Autographa californica M nuclear polyhedrosis virus (AcMNPV), the most studied member of baculoviridae, is a large double-stranded DNA virus of lepidopteran insects. Infection of host cells by AcMNPV triggers a series of temporally regulated changes in both viral and host gene expression. Viral genes are expressed in a cascade pattern and host gene expression is terminated in the late phase of infection (see Blissard and Rohrmann, 1990, for review). The a-amanitin-sensitive host RNA polymerase II is considered to be responsible for transcription of early viral genes (immediate early and delayed early), while an cu-amanitin-resistant form of RNA polymerase is thought to be involved in the expression of late and very late genes (Grula et al., 1981; Fuchs et al., 1983; Huh and Weaver, 1990). The switch from late to very late stage is characterized by the gradual shut down of late viral genes and the intensified expression of polyhedrin and ~10. This switch is particularly important as it underlies the transition from production of extracellular to occluded virus, the two morphological forms of the virus that drive the infection cycle in nature (Volkman and Keddie, 1990). The molecular mechanism(s) governing this switch is not understood. The minimal promoters required for hyperexpression of polyhedrin and p10 have been identified, but little is known about the regulation of these genes ’ To whom reprint requests should be addressed. 0042-6822192

$5.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction I” any form reserved.

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duced polymerization of actin within the nucleus during this process (Volkman, 1988; Volkman eta/., 1992). CD also stimulates actin synthesis in a variety of ver-tebrate mesenchymal cells, apparently by increasing the level of transcription of actin genes (Brett and Tannenbaum, 1985; Rebillard et a/., 1987; Sympson and Geoghegan, 1990). Although the molecular basis for this effect is not well understood, Lloyd et a/. (1992) recently demonstrated that the organization of the actin cytoskeleton is involved in a feedback-regulatory mechanism governing the synthesis of nonmuscle yactin in mice. A similar increase in actin synthesis occurs in CD-treated Spodoptera frugiperda cells (Talhouk and Volkman, 1991). In untreated cells, actin synthesis represents approximately 3.5% total protein synthesis per unit time, while in cells treated with CD for 8 hr or more, actin represents approximately 13.5% of the total protein synthesized per unit time. In infected cells, actin synthesis is similarly enhanced and virus-mediated shut off of this protein is delayed from 12 to 16 hr postinfection (hpi) to approximately 30 hpi. The effect of CD on the rates of synthesis of other host proteins appears to be nominal, although virus-mediated shut off of tubulin synthesis is delayed from approximately 12 hpi to 18 hpi (Talhouk and Volkman, 1991). CD also interferes with several aspects of virusprogrammed gene expression in AcMNPV-infected S. frugiperda cells. It causes a delay in the onset and/or shut down of at least five viral proteins. During the time of late and very late gene expression, from 18 to 30 hpi, one can observe a delay in the shut down of gp64, a 43K and a 34K protein, and proteins in the 37-38K region, and in addition observe at least an 8-hr delay in the amplification of polyhedrin. Other than these differences, however, synthesis of late viral proteins detectable by [35S]methionine labeling, such as the capsid protein, appears to be similar in CD-treated and untreated, infected cells during this period. In this study, we investigated further the effect of CD on the hyperexpression of AcMNPV very late genes, We paid close attention to the relationship between the expression of host actin genes and viral polyhedrin and pl0 genes. We found that the expression levels of host actin genes and viral polyhedrin and p10 genes were inversely correlated under all experimental conditions tested. We interpreted these results as evidence of the possible involvement of actin, either directly or indirectly, in the regulation of polyhedrin and pl0 expression. MATERIAL Cell, viral infection,

AND METHODS

and drug treatment

All experiments were conducted using Sf-9 cells (a clonal isolate of S. frugiperda IPLB-Sf21-AE cells)

POLYHEDRIN

43

grown at 27” in Grace’s medium with 10% fetal bovine serum. Cells were grown in suspension to early log phase then seeded into 6-well culture dishes (1.5 x lo6 cells/well) and incubated for 30 min to form subconfluent monolayers before virus inoculation. The virus used was a second passage E2 strain of AcMNPV extracellular virus as described (Talhouk and Volkman, 1991). Cells were routinely infected at an m.o.i. of 40. Time zero was defined as the time at which viral inoculation was removed following a 1.5 hr adsorption period. CD (Sigma) was dissolved in DMSO (dimethyl sulfoxide, Sigma) at 5 mg/ml for a stock solution and diluted to a final concentration of 5 pg/ml in culture medium before use (Talhouk and Volkman, 1991). For drug release experiments, the CD-containing medium was removed from the cell monolayers, and fresh medium was used to rinse the cells twice before further incubation. Cycloheximide (Sigma) was prepared in cell culture medium and was used at a final concentration of 0.2 mg/ml. Protein radiolabeling Cells were rinsed twice with leucine-deficient medium and starved in the same medium for 1 hr. [3H]Leucine (Amersham, 141 Ci/mmol) was then added to a final concentration of 30 &i/ml. After 1 hr of labeling, cells were rinsed with cold phosphate-buffered saline (PBS) and lysed in 2X SDS-PAGE sample buffer. Samples containing equal cpm (determined using a liquid scintillation counter) were loaded for analysis on 15% SDS-polyacrylamide gels. The [3H] signal was enhanced by soaking the gel in 1 AJsodium salicylate solution for 20 min. The gels were then dried on Whatman 3 MM paper and [3H]-labeled proteins were detected by autoradiography. RNA isolation

and analysis

Total RNA isolation from cultured cells was performed essentially as described by Chomczynski and Sacchi (1987) with minor modifications. Cells in 6-well culture plates were rinsed with cold PBS and lysed with 500 ~1 of guanidinium solution (4 M GuSCN, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.7% mercaptoethanol). Samples were then mixed with 50 ~1 of sodium acetate (3 &J, pH 4.0) and extracted with 500 ~1of water-saturated phenol and 100 ~1 of chloroform:isoamyl alcohol (24: 1). The mixture was chilled on ice for 5 min and centrifuged for 15 min at 4’ at full speed in a microfuge. The aqueous phase was mixed with 1 vol of isopropanol and incubated at -20” for 1 hr or over-

WEI AND VOLKMAN

44

(t-vi) 2024

30

36

72

CD

Polyhedrin )

PlO ) FIG. 1. Effect of CD on polyhedrin and p10 protein synthesis. AcMNPV-infected cells were labeled with t3H]leucine for 1 hr before harvest at 20, 24, 30, 36, and 72 hpi. CD was added to the cells at 0 hpi in lanes (+), and removed from the cells at 24 hpi in lanes (+/-). Cells in lanes (-) were not treated with the drug.

night. The RNA was pelleted after a 15 min centrifugation at 4’, completely dissolved in 600 ~1 of guanidinium buffer, and reprecipitated with 1 vol (600 ~1)of isopropanol at -20”. After 1 hr or overnight, the sample was centrifuged for 15 min, rinsed with cold 75% ethanol, and resuspended in DEPC-treated distilled water. The concentration of total RNA was then measured by adsorption at OD 260 nm. The RNA (5 pg/lane) was fractionated on 1% formaldehyde-denaturing gels (Sambrook et a/., 1989) and transferred by capillary action to Nytran membranes (Schleicher & Schuell) overnight in 10X SSC buffer. The membranes were rinsed briefly in 1X SSC, and the RNA was fixed to the membrane by uv irradiation with a uv cross-linker (Stratagene).

RESULTS Effect of CD on polyhedrin and PlO gene expression The effect of CD on polyhedrin and Pl 0 protein synthesis was evaluated by amino acid pulse labeling infected cells treated or untreated with the drug (Fig. 1). [3H]Leucine was used to label polyhedrin and ~10. As shown in Fig. 1, addition of CD at 0 hpi drastically delayed and slowed down the amplified expression of both genes. Typically, in the presence of CD, polyhedrin synthesis did not display a significant increase until around 30 hpi (6-l 0 hr later than normal). More severe retardation was observed in the amplified expression of ~10. The CD-induced effect was at least partially reversible, however. When CD was removed at 24 hpi, a considerable increase in polyhedrin and pl0 synthesis was observed for CD-released samples [lanes (+/-)I compared with samples subjected to continuous CD treatment [corresponding lanes (+) in Fig. 1.1. The increase was detectable within 6 hr (30 hpi) and obvious by 12 hr (36 hpi). Polyhedrin synthesis appeared to be more fully reversible than pl0 synthesis. The same pattern of response to CD was also detected at the mRNA level by Northern analyses of polyhedrin and p10 mRNAs (Fig. 2). This result indicates that the effect of CD on the expression of these two major very late genes is at the transcriptional or posttranscriptional level. Interestingly, although polyhedrin and pl0 are both hyperexpressed at terminal stages of infection and have been thought to be coordinately regulated (Leisy et al., 1986) their responses toward CD appeared to be similar but not identical. We were also interested in whether new protein synthesis was necessary for the process of restoration of

A

CD

Northern hybridization and probe preparation Hybridization probes were prepared using a randomprime DNA labeling kit from United State Biochemical (USB). Plasmids containing non-full-length cDNA clones specific for AcMNPV polyhedrin (pMA-VI) and pl0 (pMA-P[Q]P) were kindly provided by Paul Friesen (Friesen and Miller, 1985). The 39K-specific probe was made from a plasmid containing the AcMNPV Pstl K fragment, obtained from Linda Guarino, Texas A&M University (Guarino and Smith, 1990). The actin probe, kindly provided by Sara Tobin, University of Oklahoma, was made by polymerase chain reaction from the DNA of pD11 which contains a Drosophila melanogaster actin 5C cDNA (Vigoreaux and Tobin, 1987). Hybridization was carried out as described by Jones eta/. (1990).

(hpi)

Polyhedrin b

B

O-PO CD PlO,

FIG. 2. Effect of CD on polyhedrin and p10 mRNA levels. Total RNAs extracted at 20, 24, 30, 36, and 72 hpi were analyzed by Northern hybridization using a polyhedrin-specific probe (A) or a pl O-specific probe (B). Drug treatments included untreated controls (lanes -), continual treatment with CD from 0 hpi (lanes +), and removal of CD at 24 hpi (lanes +/-).

HYPEREXPRESSION

OF SACULOVIRUS

POLYHEDRIN

45

Polyheckln b

FIG. 3. Effect of CH on restoration of polyhedrin and ~10 mRNA levels upon removal of CD. Cells incubated in the absence of CD (lanes -), presence of CD (lanes +), or presence of CD until removal at 24 hpi (lanes +/-), were harvested at 24, 30, 36, and 48 hpi. CH was added to a duplicate set of cultures at 24 hpi as indicated [lanes 30(CH), 36(CH), and 48(CH)]. The RNAs were probed with a polyhedrin-specific probe (A) and a pl O-specific probe (El).

polyhedrin and pl0 amplification after the removal of CD. To address this question a protein synthesis inhibitor, cycloheximide (CH), was added at the time of CD removal (24 hpi). Total RNAs were extracted at various time points for Northern analysis after CD removal and CH addition or CD removal without CH addition (Fig. 3). It is clear that CH significantly reduced the increase of both polyhedrin and p10 mRNAs following drug release [compare lanes 30 with 30&H); lanes 36 with 36&H); and lanes 48 with 48(CH)]. The result demonstrated, therefore, that after withdrawal of CD, newly synthesized protein was required to resume amplified expression of polyhedrin and ~10.

FIG. 4. Effect of CD on polyhedrin and pl0 protein synthesis when added at different stages during infection. Cells were labeled with [3H]leucine for 1 hr before harvest at the designated time points shown at the top of each lane. CD treatments were started from 0 hpi in lanes O-l 5. O-l 9, O-23, and O-27; from 15 hpi in lanes 1519, 15-23, and 15-27; from 19 hpi in lanes 19-23 and 19-27; and from 23 hpi in lane 23-27. The drug treatments were terminated by sampling for gel analysis at 15, 19, 23, and 27 hpi.

time when polyhedrin and pl0 were already being hyperexpressed, such as at 23 hpi (Fig. 4) no effect on polyhedrin and ~10 was detected (compare lane 27 with lanes 23-27, 19-27, 15-27, and O-27). Inverse correlation between actin gene expression

polyhedrin/plO

and

During normal infection, amplification of polyhedrin and pl0 expression is preceded by the shut down of host genes. It is possible that the delayed hyperexpression of polyhedrin and pl0 in the presence of CD is

CD-induced inhibition of polyhedrin and p10 hyperexpression only occurs when CD is present prior to the onset of hyperexpression To define the CD-sensitive time range, CD was added at specified time points postinfection and the expression of polyhedrin and pl0 was followed at both the protein (Fig. 4) and mRNA (Fig. 5) levels. As shown in Fig. 4, CD added at 15 hpi or even 19 hpi still affected polyhedrin and pl0 protein synthesis although the effectiveness of the drug was diminished when added at later times (compare Fig. 4, lanes 0-, 15-, and 19-). Such diminished effects were apparent not only in a reduction in the degree of inhibition, but also in a shortening of the delay period. For example, when CD was added at 19 hpi, synthesis of polyhedrin and pl0 was slightly reduced at 23 hpi, but appeared almost normal by 27 hpi (Fig. 4) whereas when CD was added at 0 hpi, a similar level of recovery did not occur until after 36 hpi (Fig. 1). Eventually, if CD was added at a

Polyhedrin

PlO

actin

39K FIG. 5. Northern analysis of infected cells receiving CD treatment at different stages. Cells were incubated with CD for the periods indicated at the top of each of the right 7 lanes, harvested, and analyzed. The untreated control cells (left 5 lanes) were harvested at the time points indicated at the top of each lane. RNAs were extracted and probed for transcripts of polyhedrin, ~10, actin, and 39K as indicated on the left.

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(hpi)

-

CD treat o-12 7r

CD treat o-10 7

CD treat r O-20,

CD

12 16 20 24 12 16 20 24 16 20 24 20 24 O-24 Polyhedrln

removal within 4 hr, whereas 39K showed little change until about 8 hr after removal. Such a tight inverse correlation of polyhedrin and pl0 mRNA levels relative to actin mRNA levels suggests the possibility that delayed shutdown of actin synthesis may contribute to the delayed hyperexpression of polyhedrin and ~10.

actin

DISCUSSION 39K FIG. 6. Northern analysis of infected cells responding to the removal of CD at various time points. Durations of the CD treatments are indicated at the very top of the figure. After removal of the drug, cells were sampled at the indicated times shown directly above each lane. The untreated control cells (lef-l 4 lanes) were also sampled at the time points shown at the top of the lanes. RNA was extracted and probed for transcripts of polyhedrin, actin, and 39K as labeled on the left.

caused by the delayed shut off of one or more host gene products. In a previous study, Talhouk and Volkman (1991) showed that actin gene expression was greatly stimulated by CD in both uninfected and infected cells, while the synthesis of other host proteins was not similarly affected. Further, they showed that addition of CD to infected cells after host protein synthesis had ceased (at 16 hpi) resulted in an induction of actin synthesis. We decided to examine actin gene expression more carefully, therefore, in relation to that of polyhedrin and ~10. We also followed the expression of the viral gene 39K as an internal control. The 39K gene product is expressed at delayed early and late stages of infection (Guarino and Smith, 1990) and has been found associated with nuclear matrices (Wilson and Price, 1988). To investigate the expression patterns of these genes in response to CD treatment, we either added (Fig. 5) or removed (Fig. 6) the drug at different times during infection and followed the mRNA levels of polyhedrin, p10 (Fig. 5 only), actin, and 39K. The results showed that an increase in the level of polyhedrin and pl0 mRNAs correlated with a decrease in the level of actin mRNAs. The pattern of mRNA levels of the 39K gene showed a slight tendency to reflect that of actin mRNA, but the degree of change was far less extreme. The inverse relationship between polyhedrin and actin mRNA levels is especially clear in the CD removal experiment shown in Fig. 6. When CD was removed at 20 hpi, a dramatic increase in polyhedrin message and decrease in actin message was evident by 24 hpi, whereas little change was detected for 39K mRNA. Moreover, both polyhedrin and actin responded to CD

Actin microfilaments in AcMNPV-infected cells undergo a series of changes that correlate temporally with the sequential expression of virus gene classes (Charlton and Volkman, 1991). At the time of nucleocapsid morphogenesis (12-24 hpi), actin polymerizes within the nucleus and co-localizes with the major capsid protein. However, both nucleocapsid morphogenesis and nuclear co-localization of f-actin with the capsid protein can be disrupted reversibly by treating infected cells with CD (Volkman et a/., 1987; Volkman, 1988; Volkman et al., 1992, in press). CD also causes a delay in the onset and/or shut down of at least five viral proteins and inhibits the amplification of polyhedrin (Talhouk and Volkman, 1991). In this study we show that shut down of host actin synthesis and amplified production of polyhedrin and pl0 are both delayed when CD is added to AcMNPVinfected cells before the very late genes are hyperexpressed. Furthermore, the expression of actin and polyhedrin/pl 0 appears to be inversely correlated at both the mRNA and protein levels under all treatment regimens. This correlation, together with the observations that the synthesis of other host proteins is not so dramatically affected by CD as is actin, and that in the presence of CD, the virus appears to shut down the synthesis of other host proteins hours before it shuts down the synthesis of actin (Talhouk and Volkman, 1991), suggest the possibility that newly synthesized actin itself, either directly or indirectly, plays a negative regulatory role in the accumulation of polyhedrin and p10 mRNAs. Recovery of polyhedrin/pl 0 hyperexpression from the CD-induced delay is dependent upon new protein synthesis. This result suggests that a protein factor(s), probably a late viral gene product (not required for late gene expression), is required for the hyperexpression of p10 and polyhedrin. It is possible that in the presence of CD the factor is not synthesized or appropriately modified. This is unlikely, however, since the CDinduced delay in hyperexpression cannot be extended beyond the normal delay period by exchanging originally added CD with fresh CD at 24 hpi (unpublished observations). Alternatively, the putative factor could be adsorbed by or effectively competed out by newly synthesized actin. If so, synthesis of this protein would

HYPEREXPRESSION

OF BACULOVIRUS

be necessary after actin synthesis is completely shut down to allow high level expression of polyhedrin and ~10. This possibility is consistent with the observation that exchanging CD at 24 hpi does not prolong actin synthesis any more than the continuous presence of CD added at the onset of infection (unpublished observations). These results also indicate that neither the eventual shut down of host actin synthesis nor the recovered hyperexpression of pl0 and polyhedrin in CDtreated infected cells are due to the deterioration of CD. The possibility that globular actin may have a regulatory effect on gene expression is not completely without precedent. Although actin is known mostly for its cytoplasmic functions, it occurs as a major non-histone protein in nuclei isolated from a number of different organisms (Clark and Merriam, 1977; Clark and Rosenbaum, 1979; Krohne and Franke, 1980; Gounon and Karsenti, 1981). Globular actin has been reported to stimulate transcription of RNA polymerase II by its involvement in preinitiation complexes of both cellular and viral genes (Egly et a/., 1984). The stimulatory affect is specific since total RNA synthesis on a nonspecific template (calf thymus DNA) is not altered. In addition, Scheer et a/. (1984) reported that transcription of lampbrush chromosomes can be inhibited by injection of anti-actin antibodies or certain actin-binding proteins into the nuclei of amphibian oocytes. Further, they observed polymerized actin around the lampbrush chromosomes when transcription was arrested with cu-amanitin. Polymerized actin in the nucleus is first detected in AcMNPV-infected cells at 12 hpi and becomes increasingly prominent as infection progresses through 24 hpi (Charlton and Volkman, 1991). Host protein synthesis is normally shut off 12-l 8 hpi and polyhedrin and pl0 are normally hyperexpressed at 20-24 hpi (Ooi and Miller, 1988; Talhouk and Volkman, 1991). These observations place actin within the nucleus at the time of the transition from late to very late gene hyperexpression, and host gene shut-off. It is possible that the transition of nuclear actin from a soluble to insoluble form might have an effect on both host and viral gene regulation. Experiments are in progress to test this possibility further.

ACKNOWLEDGMENTS The authors are indebted to Salma Talhouk and Carol Charlton for showing the way, and to Sally Tobin for her helpful comments regarding the manuscript. This researchwas supported financially by USDA Competitive Research Grants 90-37153-5556, 91-373025874, and by federal Hatch funds.

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