Virus Research, 14 (1989) 339-346 Elsevier

339

VIRUS 00541

Restricted replication of human adenovirus type 5 in mouse cell lines

G. Eric Blair ‘, Sara C. Dixon I**, Susan A. Griffiths L** and Maria E. Blair Zajdel *

’ Department of Biochemistv, University of Lee&, Lee&, U.K. and ’ Department of Biomedical Sciences, City of Sheffield Polytechnic, Sheffield U.K.

(Accepted 5 September 1989)

Infection of mouse BALB/c 3T3 cells by adenovirus 5 resulted in at least lOOO-fold lowered yields of virus compared to human cells. The molecular basis of this restriction was analysed at the level of viral gene expression. Steady-state levels of viral DNA and RNA were greatly reduced in infected mouse, compared to human cells. Both early region 1A (ElA) and ElB mRNAs were decreased in mouse cells and their protein products were barely detectable by metabolic labelling of infected cells. The E2A-72 kDa protein and the hexon protein were detected by metabolic labelling, and immunocytochemical analysis showed that they were correctly located in nuclei of infected mouse cells. Only a minor proportion of infected mouse 3T3 cells expressed the E2A-72 kDa or hexon proteins. Low yields of virus were obtained by infection of SV40 transformed BALB/c 3T3 cells showing that SV40 does not provide a helper function for adenovirus 5 growth in this cell system

Adenovirus 5; Mouse cell; Semi-permissive replication; Viral gene expression

The level of human adenovirus (Ad) replication varies depending on the origin of the host cell and may lead to permissive, semi-permissive or non-permissive infec-

Correspondence to: Dr. G.E. Blair, Department of Biochemistry, University of Leeds, Leeds LS2 9JT,

U.K.

Present addresses: * Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.; * * MRC Toxicology Unit, Carshalton, Surrey, U.K.

0168-1702/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

340

tion. In contrast to the fully permissive infection of human cells, interaction of group C adenoviruses with many cell lines of non-human origin results in restricted virus growth. Infection of primary African green monkey kidney cells with Ad2

provides one of the best investigated semi-permissive systems. The block to viral replication in monkey cells occurs at the level of processing and translation of

mRNA encoding late proteins (Klessig, 1984). Large T antigen of SV40 specifies helper function necessary for unrestricted growth of Ad2 in monkey cells (Kimura, 1974; Cole et al., 1979). Although Ad2 and Ad5 can replicate efficiently in certain

hamster cells (Williams, 1973) infection of rodent cells is usually characterised by much lower virus yields than in fully permissive host cells. The molecular basis of the restricted growth of Ad2 or Ad5 in rodent cells has not yet been clearly elucidated. In this report we have examined the expression of Ad5 genes in infected BALB/c 3T3 cells and analyze the possible helper function of SV40 large T antigen in Ad5 infection of rodent cells.

A number of cell lines were infected with Ad5 at either 10 or 100 fluorescent focus units (FFU)/cell. After 48 h of infection at 37” C, cells were collected,

freeze-thawed several times and virus was titrated by a fluorescent focus assay on monolayers of Hep2 cells (Philipson, 1961). Infection of Hep2 or MRCS fibroblast cells by Ad5 yielded at least 100 times more infectious virus than any mouse cell tested (Table 1). Both BALB/c 3T3 and 3T6 cells showed marked restriction in Ad5 virus growth, and this restriction was not significantly relieved either by increasing the multiplicity of infection or by SV40 large T antigen, present at high levels in

wild-type form in SV3T3 cells (Milner et al., 1989) or by Ad5 ElA proteins (in 3T6/ElA cells). Thus, unlike the Ad2/monkey kidney cell system, SV40 large T antigen did not provide a helper function for Ad5 growth in mouse fibroblasts. Although Ad5 ElA proteins have been shown to trans-activate viral and cellular genes (Berk, 1986) the presence of an endogenous ElA gene in 3T6 cells did not

result in increased virus yield. Experiments were then conducted to determine the steady-state levels of viral

DNA and RNA in Ad5 infected BALB/c 3T3 cells. Total cell DNA was isolated 48 h post-infection using standard procedures, involving proteinase K and ribonuclease A treatment followed by phenol extraction (Maniatis et al., 1982). Steady-state

levels of viral DNA in infected cells were analysed by dot hybridization of serial dilutions of each DNA sample, using as a probe, 32P-adenovirus 5 DNA labelled by nick-translation (Rigby et al., 1977). This analysis showed that the steady-state level of Ad5 DNA in infected 3T3 cells was reduced approximately lOO-fold compared to that in infected Hep2 cells (Fig. 1A). A similar dot hybridization experiment was

performed to estimate the steady state level of AdS-specific cytoplasmic RNA. Total cytoplasmic RNA was isolated by phenol extraction of infected or mock-infected cells, serial dilutions immobilized as dots on nitrocellulose and hybridized with nick-translated 32P-labelled Ad5 DNA. The steady-state level of AdS-specific RNA was reduced approximately 30-fold compared to that of infected Hep2 cells (Fig.

1B). In order to study specific viral mRNAs, it was decided to select early region 1

mRNAs since they are synthesized soon after infection of human cells (Nevins et al.,

341

A

Ad5/3T3

AdS/Hep 2

28

18

EIA

13s

12s

EIB

5678

/I? - actin

Fig. 1. Analysis of AdS-specific nucleic acids in infected mouse and human cells. (A) Serial two-fold dilutions of total cell DNA from 3T3 or Hep2 cells infected with Ad5 were analysed by dot hybridiza- tion, beginning with 5 pg at the left. Hybridization with 32 P-labelled Ad5 DNA and washing were exactly as previously described (A&ill and Blair, 1988). (B) Serial two-fold dilutions of total cytoplasmic RNA were analyzed by dot hybridization using 32P-labelled Ad5 DNA, beginning with 5 pg on the left. (C) Northern blotting of polyadenylated RNA (5 cg) isolated from Hep2 (lanes 1, 8, 9), SV3T3 (lanes 2, 6, 10) or 3T3 cells (lanes 3, 7, 11) infected with Ad5 or uninfected 293 cells (lanes 4, 5, 12) using conditions previously described (A&ill and Blair, 1988) and 32P-labelled cloned DNA fragments for the Ad5 ElA

or ElB genes or mouse /3-actin cDNA.

1979) and the ElA products are necessary for trans-activation of other early adenovirus genes (Berk, 1986). Northern blotting of polyadenylated RNA isolated from Hep2, 3T3 or SV3T3 cells infected by Ad5 was performed using nick-trans- lated ElA or ElB DNA probes (Fig. 1C). Polyadenylated RNA was isolated from uninfected AdS-transformed human cells (293 cells) as control. Steady-state levels of

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TABLE I

Yields of adenovirus 5 grown in mouse and human cell lines

Cell line Virus yield (FFU cell -- ’ )

Infected at Infected at 10 FFU cell - ’ 100 FFU cell-’

3T3 0.046 0.280 SV40-transformed 3T3 0.180 0.470 (SV3T3) 3T6 0.125 n.d. 3T6,‘Ad5ElA 0.240 n.d. Hep2 500 420 MRC 5 455 n.d.

Virus yields were determined by fluorescent focus assay on Hep2 monolayers using a rabbit monospecific antibody against the Ad5 hexon protein. Numbers given are the average of three independent experi- ments.

the 13s and 12s ElA mRNAs in Ad5 infected 3T3 and SV3T3 cells (Fig. lC, tracks 2, 3) were only slightly lower than those in uninfected 293 cells (Fig. lC, track 4) although considerably lower than in infected Hep2 cells (Fig. lC, track 1). When ElB mRNAs were examined, the 22s ElB mRNA was barely detectable in Ad5infected SV3T3 and undetectable in infected 3T3 cells (Fig. lC, tracks 6, 7) although this 22s mRNA was clearly evident in both AD5 infected Hep2 cells and uninfected 293 cells (Fig. lC, tracks 5, 8). The ElB 13s mRNA was also present in Ad5 infected Hep2 cells (Fig. lC, track 8); the mRNA species migrating more slowly than the 13s mRNA probably represent the minor 14s and 14.5s species found late in infection in human cells (reviewed by Stillman, 1986). The 13s mRNA was found at very low level in uninfected 293 cells (Fig. lC, track 5) as previously described (Spector et al., 1980), and was undetectable in either SV3T3 (Fig. lC, track 6) or 3T3 (Fig. lC, track 7) cells infected with Ad5. Analysis of the same RNA samples with a nick-translated p-actin cDNA insert showed that equal masses of RNA had been analyzed (Fig. lC, tracks 9-12). Thus both total Ad5 virus-specific RNA and individual mRNAs from the ElA and ElB ~~sc~ptional units are reduced in level in infected mouse, compared to infected human cells. In particular the steady-state level of the ElB 22s mRNA was more severely reduced than the ElA mRNAs. ElA mRNAs have previously been detected in Swiss 3T3 cells infected by Ad2 (Iseki and Baserga, 1983) and it is apparent from the results obtained in that study that steady-state levels of ElA and E2 mRNAs were reduced compared to AdZinfected HeLa cells.

The biosynthesis of certain Ad5 proteins was investigated by metabolic labelling of SV3T3 and 3T3 cells infected by Ad5, followed by immunoprecipitation with either monoclonal or monospecific antibodies for either the hexon protein, the ElA proteins, the E2A-72 kDa single-stranded DNA binding protein or the ElB-58 kDa protein (Fig. 2A). In both 3T3 and SV3T3 cells synthesis of the E2A-72 kDA protein was evident (Fig. 2A, tracks 4, 8) but substantial degradation of this protein

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E2a

ElB

E2a

iexc

-72 K

-50 K

El A

-45 NK

m 1 2

zoo

90

69

46

30

14

Fig. 2. Synthesis of Ad5 proteins in infected mouse and human cells. Infected SV3T3 (A, lanes l-5), 3T3 (A, lanes 6-10) and Hep2 cells (B, lanes 1,2) were labelled with [35S]methionine and immunoprecipitated as previously described (Zajdel Blair et al., 1985). Equal incorporated c.p.m. were used for each cell line. Immunoprecipitates were analyxed on 15% SDS gels using (A) tracks 1 and 6, normal mouse serum; 2 and 7, anti-ElA antibody M73 (HarIow et al., 1985); 3 and 10, hamster anti-ElB-58 kDa; 4 and 8, rabbit anti-E2A-72 kDa; 5 and 10, rabbit anti-hexon. (B) 1, normal rabbit serum; 2, rabbit anti-E2A-72 kDa.

Molecular weight markers (m) are shown in kDa. Exposure to X-ray film was 14d in A., 3d in B.

had taken place, yielding the previously-identified 45 kDa derivative (Linne and Philipson, 1980). The conditions used for i~unopr~ipitation were unlikely to be responsible for this degradation since analysis of lysates of AdS-infected Hep2 cells showed that a relatively minor fraction of the immunoprecipitate was degraded to the 45 kDa species (Fig. 2B, track 2). The ElA proteins (Fig. 2A, tracks 2, 6) and the ElB-58 kDa protein (Fig. 2A, tracks 3, 7) were immunoprecipitated at low level from SV3T3 lysates but were not detectable in lysates from 3T3 cells. Biosynthesis of the 120 kDa hexon protein was evident in both SV3T3 (Fig. 2A, track 5) and 3T3 (Fig. 2A, track 9), although the intensity of the band in SV3T3 cells appeared greater.

In order to determine whether adenovirus proteins were correctly transported within infected cells, indirect immunofluorescent antibody staining was performed on fixed 3T3 or Hep2 cells infected by Ad5 (Fig. 3). The two viral proteins shown to be synthesized most readily in infected 3T3 cells by the i~unopr~pitation analysis (Fig. 2A) were the E2A-72 kDa protein and the hexon protein. They also represent, respectively, an early and a late protein. Uninfected cells did not react with antibodies to these proteins in immunofluorescence (results not shown). Cells were grown on glass coverslips, infected for 24 h (Hep2) or 48 h (3T3), fixed in

Fig. 3. Intracellular location of Ad5 proteins in infected mouse and human cells. Infected cells were fixed and analysed by indirect immunofluorescence using: (a-d), rabbit anti-E2A-72 kDa; e-h, rabbit

anti-hexon. Cells were viewed under phase contrast (a, c, e, g) or UV (b, d, f, h) optics. Bar = 10 pm.

345

methanol, washed in PBS and reacted with monosp~cific rabbit antibodies against either the Ad5 72 kDa protein or hexon protein for 1 h at 37 o C, washed in PBS and incubated for a further hour at 37 o C with a 1: 50 dilution of FITC-conjugated goat anti-rabbit IgG (Sigma), washed in PBS, mounted in gelvatol and photographed under UV epifluorescence. The results obtained (Fig. 3) showed that the Ad5 E2A-72 kDa protein was present in the nucleus as aggregates of varying size in both infected 3T3 (Fig. 3, panels a and b) and Hep2 cells (Fig. 3, panels c and d). Neither the ElA nor the ElB proteins could be detected by immunofluorescent staining in infected 3T3 cells (results not shown). The hexon protein was located in the nucleus of infected 3T3 cells (Fig. 3, panels e and f) and Hep2 cells (Fig. 3, panels g and h). However, it was noted that only a minority (between 1 and 10%) of 3T3 cells in the culture reacted with antibodies to either E2A-72 kDa or hexon whereas, at the same multiplicity of infection, all Hep2 cells were positive for both hexon and 72 kDa protein. Extension of the time of infection of 3T3 cells to 72 h did not increase the proportion of hexon-positive cells (results not shown).

This work has shown that both BALB/c 3T3 and SV40-transformed BALB/c 3T3 cells are severely restricted in their capacity to support Ad5 growth. The failure of SV40 large T antigen to provide a helper function distinguish~ the mouse cell from the monkey cell system and suggests that the large T helper function may require primate-specific cellular factor(s) for support of Ad5 growth. Previous studies have also shown restricted growth of Ad5 in other mouse cells, although the nature of the defect in Ad5 replication may differ depending on the host mouse cell (Younghusband et al., 1979; Cheng and Praszkier, 1982; Zucker and Flint, 1985; Eggerding and Pierce, 1986). In the undifferentiated mouse terat~~cinoma cell line F9, the defect in virus replication was suggested to be at the level of transcription and translation of late viral mRNAs (Cheng and Praszkier, 1982). Infection of primary mouse embryo or baby mouse kidney cells with Ad5 resulted in replication of viral DNA and synthesis of late structural proteins, indicating that defective virus assembly or maturation may be responsible for restricted virus growth in primary cells (Zucker and Flint, 1985). An extensive study of Ad5 replication in established rat and mouse cell lines revealed that in general, the level of late structural proteins was reduced and that in Swiss 3T3 cells, late structural proteins were undetectable (Eggerding and Pierce, 1986). In this paper we have shown that synthesis of viral DNA, RNA and proteins is greatly reduced in AdS-infected BALB/c 3T3 cells. In addition i~uno~uores~n~ studies detected viral proteins in a minority of cells, indicating that very early events in adenovirus infection, such as adsorption, penetration and uncoating of virions may also be affected in BALB/c 3T3 cells.

We thank our colleagues for generous gifts of cells and antisera and the Yorkshire Cancer Research Campaign for support.

346

References

Ackrill, A.M. and Blair, G.E. (1988) Regulation of major histocompatibility class I gene expression at the level of transcription in highly oncogenic adenovirus transformed cells. Oncogene 3, 483-487.

Berk, A. (1986) Adenovirus promoters and ElA transactivation. Ann. Rev. Genetics 20, 45-79. Cheng, C. and Praszkier, J. (1982) Regulation of type 5 adenovirus replication in murine teratocarcinoma

cell lines. Virology 123, 45-59. Cole, C.N., Crawford, L.V. and Berg, P. (1979) Simian virus 40 mutants with deletions at the 3’ end of

the early region are defective in adenovirus helper function. J. Virol. 30, 683-691. Eggerding, F.A. and Pierce, W.C. (1986) Molecular biology of adenovirus type 2 semi-permissive

infections. I. Viral growth and expression of viral replicative functions during restricted adenovirus infection. Virology 148, 97-113.

Harlow, E., Franza, B.R. Jr. and Schley, C. (1985) Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products. J. Virol. 55, 533-546.

Iseki, S. and Baserga, R. (1983) Effect of butyrate on adenovirus infection in set-pe~ssive cells. Virology 124, 188-191.

Kimura, G. (1974) Genetic evidence for SV40 gene function in enhancement of replication of human adenovirus in simian cells. Nature (London) 248, 590-591.

Klessig, D.F. (1984) Adenovirus-simian virus 40 interactions. In: H.S. Ginsberg (Ed.), The Adenoviruses, 399-449. Plenum Press, New York.

Lint& T. and Philipson, L. (1980) The nature of ‘the phosphate moiety of the adenovirus 2 DNA-binding protein. Eur. J. B&hem. 103, 259-268.

Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual, pp. 288-291, Cold Spring Harbor Laboratory, NY.

Milner, J., Gamble, J. and Cook, A. (1989) p53 is associated with a 35 kD protein in cells transformed by simian virus 40. Oncogene 4, 665-668.

Nevins, J.R., Ginsberg, H.S., Blanchard, J.M., Wilson, M.C. and Darnell, J.E. (1979) Regulation of the primary expression of the early adenovirus transcription units. .I. Virol. 32, 727-733.

Philipson, L. (1961) Adenovirus assay by a fluorescent cell-counting procedure. Virology 15, 263-268. Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977) Labelling deoxyribonucleic acid to high

specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237-251. Spector, D.J., Halbert,. D.N. and Raskas, H.J. (1980) Regulation of integrated adenovirus sequences

during adenovirus infection of transformed cells. J. Virol. 36, 860-871. Stillman, B. (1986) Functions of the adenovirus ElB tumour antigens. Cancer Surveys 5, 389-404. Williams, J.F. (1973) Oncogenic transformation of hamster embryo cells in vitro by adenovirus type 5.

Nature (London) 243, 162-163. Younghusband, H.B., Tyndall. C. and Bdlett, A.J.D. (1979) Replication and interaction of virus DNA

and cellular DNA in mouse cells infected by a human adenovirus. 3. Gen. Virol. 45, 455-467. Zajdel Blair, M.E., Barker, M.D., Dixon, S.C. and Blair, G.E. (1985) The use of monoclonal antibodies to

study the proteins specified by the transforming region of human adenoviruses. Biochem J. 225, 649-655.

Zueker, M.L. and Flint, S.J. (1985) Infection and transformation of mouse cells by human adenovirus type 2. Virology 147, 126-141.

(Received 6 June 1989; revision received 5 September 1989)