Cells inhibited by n-butyrate support adenovirus replication

VIROLOGY

lo?‘,

.514-519

(1980)

Cells Inhibited

by n-Butyrate

Support

ELLEN Department

of Molecular

Biology

and Virus

Replication

DANIELL

Laboratory,

Accepted

Adenovirus

University

August

of California,

Berkeley,

California

947.20

19, 1980

The ability of adenoviruses to replicate viral DNA and produce infectious virus particles in tissue culture cells blocked with n-butyrate has been examined. Cells are treated with millimolar amounts ofn-butyrate, gradually stopping cell division and DNA synthesis; the block is reversible when n-butyrate is removed. When HeLa cells which have been thus inhibited are infected with human adenovirus type 5 (Ad5) in the continued presence of inhibitor, the virus adsorbed and penetrated the cells normally and viral DNA synthesis ensued. A normal rate and time course of DNA synthesis was observed, and butyrate-treated cells yielded the same amount of infectious virus as uninfected cells. Adenoviral DNA synthesis occurred without detectable stimulation of cellular DNA replication or of histone synthesis. The mechanism of butvrate inhibition of cellular DNA replication and unique features of adenovirus replication are discussed.

Treatment of cultured mammalian cells with low concentrations of n-butyrate has a variety of effects, including inhibition of cell DNA synthesis and of cell division (20, 28). The most immediate and best understood effect of butyrate treatment is the inhibition of histone deacetylase (6, 9, 10, X2), resulting in hyperacetylation of histones (36). Many of these changes are completely reversible; cells resume normal rates of growth, DNA synthesis, and histone deacetylation when restored to butyratefree medium. The relationships among the varied effects of butyrate treatment are not clear. Hagopian et al. (20) have shown that in HeLa cells the extent of acetylation of core histone H4 increases in parallel with the decrease in DNA synthesis. In view of the close association of histones and DNA in chromatin (16) and the fact that acetylated histones are altered in their association with DNA (33), a link between histone hyperacetylation and DNA synthesis seemed likely. A role for reversible acetylation and deacetylation in chromatin replication has been previously suggested (30). In the study reported here, we have investigated the effect of n-butyrate treatment on replication of adenovirus DNA in order to elucidate some of the features of 0042.6822/80/160514-06$02.00/O Copyright All rights

C 1980 by Academic Press. Inc. of reproduction in any form reserved

514

the observed block to cell DNA synthesis. Adenovirus DNA replication is carried out by cellular DNA polymerases (1, 8, 17, 23, 26), though in many respects viral replication differs from cellular DNA replication (reviewed in (37)). For example, there is evidence that there are no histones associated with adenoviral DNA replication complexes (12, 24). Adenovirions do not contain histones; two virus-coded basic polypeptides, VII and V, are associated with DNA in the virus particle core (13, 31). The major core polypeptide, VII, is synthesized as a precursor, pVI1 (4) which binds to viral DNA before proteolytic processing to form VII (2). Unlike cellular histones, viral core proteins do not undergo reversible acetylation subject to enhancement by butyrate (15). We have undertaken a comparison of the effects of n-butyrate on cellular and on adenoviral DNA synthesis to elucidate the mechanism of butyrate inhibition as well as the dependence of adenovirus on host replicative functions. The results indicate that the adenovirus genome is completely independent of the block to cellular DNA replication imposed by butyrate. The concentration of n-butyrate required to efficiently inhibit DNA replication and cell division was determined for a variety of

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cell types. Cell cultures showed minimal levels of DNA synthesis in roughly one cell doubling time. Efficient shutoff of DNA synthesis, which we operationally defined as a level of thymidine incorporation less than 10% of normal, required a concentration of butyrate characteristic of cell type. The distinct changes in cell morphology accompanying butyrate treatment are illustrated in Figs. la and b. Overall rates of protein synthesis, as measured by incorporation of radioactive amino acids into TCA precipitable material, are not significantly altered in butyrate-treated cells. However, the amount of incorporation into acid-extractable nuclear proteins is less than 10% of normal in cells treated with butyrate for 24 hr. Gel analysis confirms that histone synthesis specifically has ceased (data not shown). This is not surprising, as histone synthesis and DNA synthesis are usually tightly coupled temporally (7). The effects of butyrate treatment at these concentrations are reversible. In all cases, cells were viable after 60 hr of treatment with concentrations of n-butyrate used to block DNA synthesis. Cells began dividing, resumed their “normal” morphologies and their ability to incorporate [3H]thymidine at normal rates within 24 hr after refeeding with butyrate-free medium. Furthermore, when butyratetreated cells are trypsinized to remove them from culture dishes, more than 90% of the cells attach to fresh dishes in the presence or in the absence of butyrate. The ability of adenoviruses to replicate in butyrate-treated cells was investigated. When HeLa cells blocked with n-butyrate were infected with human adenovirus type 5 (Ad5) in the continued presence of butyrate, the levels of DNA synthesis reached late in infection were as great as those in untreated infected cells (Fig. 2). DNA synthesis in uninfected cells remained inhibited throughout the experiment. The morphological characteristics of butyratetreated cells disappeared with infection; by late times after infection, they assumed the rounded appearance typical of adenovirus-infected cells (Figs. lc and d). The DNA synthesized in both but.yrate-

515

treated and untreated infected HeLa cells from 19 to 24 hr after infection consists almost completely of viral sequences as measured by filter hybridization to cellular and viral DNA (Table 1). As indicated in Fig. 2, in butyrate-inhibited cells, this viral synthesis occurs in the absence of cellular DNA synthesis. No incorporation of label into cellular DNA in the butyrate-treated cells is detected at any time after adenovirus infection. Experiments were also performed with simian adenovirus 7 (SA7) infection of butyrate-blocked BSC-1 cells, expanding the investigation to cells of a different species origin. Similar results were obtained. When n-butyrate was added to cells immediately following virus adsorption, rather than 24 hr before infection, the rate of DNA synthesis decreased rapidly in the first 3 hr, as occurs after treatment of uninfected cells (20). The timing of the onset of viral DNA synthesis was unaffected by butyrate treatment, viral DNA sequences being detectable 6-10 hr after infection. When butyrate was added to infected cells 24 hr after infection, no decrease in [“HIthymidine incorporation was observed. We have previously shown (25) that no histone synthesis is detectable 24 hr after Ad5 infection of HeLa cells. Amino acid label incorporated into acid-soluble nuclear material is in viral polypeptides pVI1, VII, and V. This is also true in butyrate-treated cells, as shown by gel electrophoresis of labeled proteins (data not shown). When butyrate-treated HeLa cells infected with Ad5 are labeled with [14C]arginine (1 pCi/ ml; 298 Ci/mmol) early in infection (1-8 hr postinfection), there is no incorporation of label into nuclear acid-soluble material above the background of uninfected cells. The amount of infectious Ad5 produced in butyrate-treated HeLa cells and of SA7 produced in butyrate-treated BSC cells was measured by plaque assay 48 hr after infection. The number of plaque-forming units produced per infected cell was the same in butyrate-treated and in untreated cells, at multiplicities of infection ranging from 0.5 to 100 PFU/cell. To ascertain whether adsorption and infection of butyrate-treated cells by in-

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FIG. 1. Morphology of HeLa cells treated with wbutyratr. (‘rll~ \vere plated ~parsrly onto W-mm Falcon tissue culture dishes in the presence or absence of hutyratr (7.5 n&V. Twenty-four hours late1 some plates were infected with adenovirw type 3. and others \vere mock infected as described. Photographs were taken with a Zeiss microscope and camera with Polaroid sheet film (type 35) 24 hr after infection. (a) -Rutgrate. uninfected, (b) +butyrate, uninfected, (c) -butyrate, Ad5 infected, cd) + butyrate. Ad5 infected.

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2.5 r--

n "0

I

I-.-I5

10

15 Hours

20 after

25 infection

30

35

40

FIG. 2. DNA synthesis in HeLa cells infected with Ad5 in the presence of n-butyrate. Cells were plated at densities so that cell numbers per 60-mm dish would be between 5 x loj and 1 x 10” at the time of labeling. In experiments where cells were infected 24 hr after treatment with butyrate, half as many cells were seeded on hutyrate-minus plates as on butyrate-plus plates, so that cell numbers were equivalent at the time of infection. At times indicated, cells were labeled with 1 &i/ml [“Hlthymidine for 1 hr, then chilled and removed from dishes with 2 ml oftrypsin. Incorporated thymidine was measured by a modification of the method of Hagopian et a/. (20). One milliliter was centrifuged, washed three times with phosphatebuffered saline by repeated centrifugation, lysed with 1 ml of 1 N NaOH at 60” for 5 min and precipitated with cold TCA (10%). The remainder of the cells in trypsin were used to count the cells in a Neuhauer counter; a cell count was obtained for each dish independently. TCA precipitates were collected by filtration onto glass fiber filters and counted in toluene with Liquifluour (New England Nuclear). Incorporation was normalized to cpm per lo5 cells. The four curves represent: (a) (0) hutyrate-treated cells, uninfected: (b) (A) butyrate-treated cells, Ad5 infected; (c) (0) untreated cells, uninfected; and (d) (A) untreated cells. Ad5 infected.

dividual virus particles was normal, plaque assays of Ad5 stocks were performed on butyrate-treated monolayers of HeLa cells. The efficiency of plaque formation by Ad5 was unaffected by prior treatment of cells with n-butyrate; apparently there is no enhancement or repression of the ability of virus particles to adsorb, penetrate or replicate. Interestingly, plaques could be counted 1 or 2 days earlier on butyratetreated than on untreated monolayer-s. This may be due to butyrate suppression of cell overgrowth of plaques (Daniel1 and Groff, manuscript in preparation). Adsorption and

517

penetration of virus to butyrate-treated cells were also monitored by infecting with [“Hlthymidine labeled virus particles and measuring the amount of label entering the cells and reaching the nucleus (11). In both treated and untreated HeLa cells, infected at a multiplicity of 10 PFUicell, about 30% of viral input DNA was in the nuclear fraction 4 hr after infection. Adenovirus replication is independent of the block to DNA synthesis imposed by sodium butyrate in HeLa cells and in BSC-1 cells. Viral DNA replication follows a normal time course, and proceeds in the absence of any detectable cellular DNA synthesis or histone synthesis. Neither the yield of complete virus particles nor their infectivity is altered by butyrate treatment of cells. The correlation between cell doubling time and the rate of shutoff of DNA synthesis by butyrate suggests that there is a block at some stage of the cell cycle. The fact that adenovirus efficiently infects butyrate-treated cells is thus consistent with a previous report that adenovirus growth is not dependent on the stage of the cell cycle at which cells are infected (21). Adenovirus DNA synthesis differs from cellular DNA synthesis in a number of ways. Adenovirus DNA synthesis is resistant to treatment with cycloheximide if the drug is administered after initiation of replication (22). In contrast, either cellular or papovavirus DNA synthesis is blocked when protein synthesis is inhibited (14,SS). Histone synthesis in uninfected cells is coupled to DNA replication ( 7); synthesis of adenovirus histone-like proteins continues in the absence of DNA replication in infected cells (25). These histone-like proteins are the viral substitute for histones in the virion (13, 31) and perhaps in viral replication complexes (24 >. We have found that butyrate has no effect on the acetylation of adenovirus histone-like proteins (15), which may well form the protein component of replicating viral chromatin. However, we cannot infer from this that the inhibition of cellular DNA synthesis by butyrate is due to the hyperacetylation of histones. To the contrary, we have shown that SV40 induces cellular DNA

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1

HYBRIDIZATION OF LABELED DNA FROM INFECTED BUTYRATE-TREATED OR UNTREATED CELLS Ad5 AND TO HeLa DNA”

Probe (cells/virus) HeLa:AdB HeLa:Ad5 HeLa:Ad5 HeLa:Ad5 HeLa:Mock HeLa:Mock

Butyrate

Specific activity ( x 10” CPmi~cLg)

Total cpm (X10’)

+ + + -

6 6 4 4 1” 7

2.5 5.0 2.5 ‘0 ;:5 2.5

cpm hybridized to filter HeLa Ad5 300 500 600 1000 2300 2400

7,000 13,500 5,900 7,600 40 110

” Hybridizations were performed by the method of Denhardt (19). Filters contained 50 /*g of HeLa DNA. Each probe was denatured, sonicated, and incubated with three one each of the Ad5 and HeLa DNA-containing filters. * Specific activity of the uninfected butyrate-treated cell samples was only seven-fold treated cells because twice the specific activity of thymidine was used to label this to get a useable probe.

and histone synthesis in butyrate-treated monkey cells, while hyperacetylation of histones persists (Daniell, Fedor, and Burg, manuscript in preparation). This suggests that the abnormal histone modification does not directly block DNA synthesis. Using a different experimental approach, Rubenstein et al. (29) have also concluded that the butyrate-induced block to DNA replication is not a direct result of histone hyperacetylation. Heretofore, it was established that cellular DNA synthesis and histone synthesis are shut off late in adenovirus infection, but little could be inferred about the necessity (or lack thereof) for these cellular processes early in infection. Inhibitors of DNA replication administered before the onset of adenoviral DNA synthesis restrict infection to its early phase (5, 27). However, these experiments shed no light on requirements for synthesis specifically of cellular DNA. Cellular DNA polymerases (Y and y are both involved in adenovirus DNA replication (1, 8,23,26,35) and the inhibitors used directly block viral as well as cellular DNA synthesis by these enzymes. Using butyrate to block cell DNA synthesis indirectly we have shown that adenovirus can complete its infectious cycle without replication of cellular DNA.

HeLafAd5 (cpm)

0.04 0.03 0.10 0.14 -

10 c(g of Ad5 DNA or filters, one blank and lower in the butyratepreparation in order

It is possible that adenovirus DNA, which enters the nucleus with its own complement of histone-like proteins (3) has no requirement for interaction with histones in the course of infection. It should be noted that deproteinized adenoviral DNA is infectious, so there is no absolute prerequisite for viral core proteins to initiate infection (19). The fact that Ad5 and SA7 are unaffected by butyrate-induced inhibition of histone synthesis supports the model that permissive infection by adenovirus can proceed without association of viral DNA with histones. However, these data also permit the interpretation that such association is essential and that a small pool of histone is sufficient to complex with infecting DNA. The ability to block cellular DNA synthesis without affecting the permissive infection of adenovirus suggests an investigation of nonpermissive infection using butyrate. Adenovirus stimulates cellular DNA synthesis in serum-arrested BHK-21 cells (34). These cells are nonpermissive for adenoviruses of groups A and B, and semipermissive for group C adenoviruses (including Ad5). Whether this stimulation of cellular DNA synthesis will occur in the presence of butyrate, and how butyrate may affect abortive infection and/or transformation of rodent cells by adenovirus

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are areas of investigation which may increase our understanding of the mechanisms of viral transformation and hostrange restriction. ACKNOWLEDGMENTS Most of the materials incorporated in this work were developed with the financial support of the National Science Foundation Grant PCM78-21098. The investigation was also supported by NIH Research Grant CA 19058 from the National Cancer Institute, and by BRSG Grant RR-7006 from the Biomedical Research Support Program, Division of Research Resources, NIH. The author thanks M. Fedor and L. Burg for patient revision of the manuscript and Barbara Kellogg for her excellent typing.

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