key: cord-0004948-49uq4wiq
authors: Gomes, S. A.; Niel, C.; D'Halluin, J. C.
title: Growth of fastidious adenovirus serotype 40 in HRT 18 cells: Interactions with E 1 A and E 1 B deletion mutants of subgenus C adenoviruses
date: 1992
journal: Arch Virol
DOI: 10.1007/bf01314624
sha: 8d1416fec6111d311eb3e4d22326c37a6f56b945
doc_id: 4948
cord_uid: 49uq4wiq

Growth of fastidious adenovirus serotype 40 (Ad 40) in several cell lines was investigated. Ad 40 was able to readily propagate in human intestinal cell line, HRT 18. Coinfection assays were made in non-permissive and permissive cells between Ad 40 and Ad 5dl 312 or dl 1520, mutants deleted in E 1 A and E 1 B regions, respectively, to test the ability of Ad 40 to complement these mutants and vice versa. Ad 40 could enhance Ad 5dl 312 DNA synthesis in HRT 18 and HeLa cells, although its own DNA disappeared in the presence of this mutant in HRT 18 cells. In coinfection with dl 1520, Ad 40 DNA synthesis was inhibitied by dl 1520 in HRT 18 cells and dl 1520 DNA synthesis was inhibited by Ad 40 in 293 cells. This might reflect the presence of unusual products encoded by Ad 40 E 1 B region.

Adenoviruses are important agents of infantile viral gastroenteritis and serotypes 40 and 41 (Ad40 and Ad41) are responsible for a large majority of cases [4] .

In spite of this pathogenicity, Ad 40 and Ad 4t have been discovered later than most of the serotypes due to their difficulty to be propagated in vitro. Contrary to conventional human adenoviruses, the so-called fastidious adenoviruses could not be serially passaged in primary cells such as human embryonic kidney cells (HEK) or human diploid fibroblasts [1] . However, successful growth of Ad 41 in primary cells in defined conditions has been recently reported [24] . Ad 40 and Ad 41 can replicate with variable success in human cell lines such as HeLa, Hep-2, KB and A 534 cells [7, 23, 25, 37] Replication of fastidious adenoviruses in human cell lines is thought to be grown to some extent in the Ad 5-transformed human embryonic kidney cell line 293 [16, 28] , which contains and expresses the Ad 5 early region E 1 [12] . This has led to the hypothesis that these viruses were defective in early regions E 1 A and E 1 B or in one of them and could be complemented by early gene functions of Ad 5 integrated in the genome of these cells [28, 19] .

The Ad2 E 1 A region gives rise to two major mRNAs (12S and 13 S). The polypeptides encoded by these mRNAs are identical except for an internal domain of 46 aminoacids within the largest product due to differential splicing of RNAs. Adenovirus mutants in E 1 A region that express only the 13 S gene product, replicate normally, are capable of trans-activating viral gene transcription and can immortalize primary rodent cells. Adenovirus mutants expressing only the 12 S gene product do not replicate in HeLa cells at tow multiplicity of infection (for review see [3, 11] ).

Ad 2 E 1 B region governs the synthesis at early times of two major polypeptides. A 22 S mRNA encodes a 19 k and a 55 k polypeptides. A 13 S mRNA encodes only the 19 k polypeptide. The 55 k polypeptide is essential for a complete lytic cycle in HeLa cells and is involved in accumulation of viral mRNA during productive infection. E 1 B mutants in 55 k polypeptide are altered in expression of early mRNAs and in DNA synthesis. Mutants in 19k cause a rapid cytopathic effect and induce degradation of host and viral DNAs [9, 27, 36] .

The nucleotide sequence of early regions E 1 A and E 1 B of fastidious adenoviruses has been determined [14, 34] . There is no major difference in the structure of E 1 A and E 1 B regions of nonfastidious and fastidious adenovir u s e s .

An increase of the replication of fastidious adenoviruses when cultivated in the presence of nonfastidious adenoviruses has been observed. 31] determined that Ad 2 could complement the growth of Ad41 in Chang conjunctival and HEF cells by measuring the rate of late antigens with monoclonal antibodies specific to Ad41.

For Ad 40, Mautner et al. [19] found that Ad 40 could be complemented in HeLa cells by E 1 B 55 K protein of nonfastidious adenoviruses.

In this study, we investigated the possible helper function of early regions E 1 A or E 1 B of nonfastidious adenoviruses in complementing DNA replication of Ad 40. We show that Ad 40 E 1 A region is functional. Coinfection assays of Ad40 with a nonfastidious adenovirus deleted in E 1 B region (d11520) led to an inhibition of the synthesis of both DNAs.

Human cell lines used were 293, HRT 18, an intestinal cell line [17] and HeLa. Monolayer cultures from these cell lines were grown in Dulbecco's medium containing 10% foetal bovine serum and divided twice a week at an appropriate subculture ratio.

Ad5d13t2 is a deletion mutant lacking E 1 A activity [15] . d11520 is a hybrid Ad 2/Ad 5 E 1 B mutant which does not synthesize E 1 B 55 K protein and shows a reduced rate of E 1 B 19K protein synthesis [2] .

Stocks ofdl 1520 and Ad 5 dl 312 were prepared in 293 cells, stock ofAd 2 WT (prototype strain provided by Dr. J. F. Williams, Pittsburg, Pa. ) was made in HeLa cells. These viruses were purified as previously described [10] . Stock of Ad40 (prototype strain Dugan) was obtained in HRT 18 cells after 10 passages of the virus in this cell line. Stock of Ad 40 was maintained as a crude extract after freezing and thawing of cell cultures three times.

Titers of Ad 40, d11520 and Ad 5 d1312 stocks on 293, HRT 18 and HeLa cells, were determined at 48 h post-infection, by a fluorescent focus assay as previously described [10] , using as first antibody a rabbit polyclonal anti-Ad 2 raised against purified virions and as a second antibody, fluorescein conjugated sheep anti-rabbit immunoglobulin. Titers were expressed as fluorescent focus units per ml (FFU/ml).

Ad 40 infected cultures were freezed and thawed three times then serially diluted and tested for adenovirus antigens by ELISA as previously described [22] .

Cell monolayers of 293, HRT 18 or HeLa grown in 25cm 2 flasks were single or double infected with virus stocks. The input virus concentration, expressed in FFU/cell, was variable. Virus inocula were adsorbed for 2 h at 37 °C. After this time, medium containing 2% foetal bovine serum was added. Each infected culture was incubated at 37 °C until total cytopathic effect (CPE). In cases of absence of CPE, infected cells were recovered after 8 days.

Viral DNAs were extracted from 5 x 106 infected cells using the Hirt [13] procedure modified by Wadelt and de Jong [35] . Half of the extracted DNA was digested by Hind III restriction endonuclease and electrophoresed in a 0.8% agarose horizontal slab gel in Tris-Borate buffer at 50 V. Gels were stained in ethidium bromide and photographed under short UV light.

Total RNA was prepared from mock-infected and from Ad 40 infected HRT 18 cells at 24 h post-infection by the acid guanidinum thiocyanate extraction method [6] .

cDNA synthesis and PCR amplification cDNA synthesis was performed using random primers (Gene Amp RNA PCR kit, Perkin Elmer Cetus). PCR amplification was carried out with oligodeoxynucleotides representing nucleotides 2485 to 2506 (5' end) and the complementary sequence from nucleotides 3018 to 3039 (3' end) of the Ad 40 sequence [34] . These primers were purchased from Eurogentec S. A. and purified by HPLC chromatography. The amplification reaction was performed with Taq DNA polymerase (Promega) in 40 cycles of DNA denaturation (94 °C, 1 min), annealing (55 °C, t rain) and elongation (72 °C, 3 rain). Amplified DNAs were electrophoresed in a 2% agarose gel.

Human cell lines 293, HRT 18, and HeLa were tested for their capability to support growth of Ad40. HRT 18 is a cell line derived from human rectal adenocarcinoma [321 and was chosen because it is permissive to bovine enteric fastidious coronavirus [17] . To estimate virus growth in each cell line, the rate of Ad antigens in cultures was measured after three serial passages by ELISA. In 293 and HeLa cells, antigen levels were very low and the virus was not readily propagated. HRT 18 cell line gave a higher level of Ad40 antigens, although Ad 40 grew more slowly than nonfastidious adenoviruses. Total cytopathic effect was observed after 6 -7 days post-infection. A reference stock of the virus was then prepared in this cell line after several additional passages.

Reference stocks of mutants Ad 5 dl 312 [15] and dl 1520 [21 showing large deletions in transcription units E 1 A and E 1 B, respectively, were prepared in 293 cells.

Each reference stock was titrated on 293, HRT 18, and HeLa cell lines. Results are shown in Table 1 . The titer of Ad 5 dl 312 mutant of HeLa cells was about 105-fold lower than on 293 cells. This was in agreement with the results published by Jones and Shenk [15] . The titers of Ad 5 dl 312 mutant in HRT 18 and HeLa cells were of the same order of magnitude. The titer of E 1 B mutant dl 1520 was 6-fold and 100 fold lower on HRT 18 and HeLa cells, respectively, than on 293 cells. For Ad 40, the difference between the titers obtained in the three cell lines during the first passage was less significant. Ad 40 behaved neither as an adenovirus mutated in E 1 A 13 S mRNA nor as an E 1 B mutant lacking 55 k protein.

To determine if a complementation could occur between Ad 40 and Ad 5 dl 312 or d11520, coinfection assays with Ad40 and deletion mutants were carried out. The synthesis of viral DNAs in infection and coinfection assays was vis- Viruses were titrated at 48 h post-infection by fluorescent focus assay [10] . Titers are expressed in focus forming units per ml (FFU/ml) ualized after electrophoresis on agarose gel of Hirt extracted DNAs digested by Hind III restriction enzyme.

The cells were first single infected at a multiplicity of infection (m.o.i.) of 1 FFU/cetl. This m.o.i, was calculated from reference stocks titrated in the most permissive cells, i.e., HRT 18 for Ad40, 293 for the mutants. At this m.o.i., a clear restriction pattern of Hirt extracted DNAs was seen only with the most permissive cell line (Fig. 1) .

Coinfection assays of Ad 40 and deletion mutants were performed in HRT 18, 293 and HeLa cells. from 1 FFU/cell (Fig. 2 A) to 10,000 FFU/cell (Fig. 2 B and C) . After infection of 1 FFU/cell of Ad 5 dl 312 in HRT 18 cells, the restriction pattern of this virus was not visible (Fig. 2 A, lane 1) . Restriction patterns obtained in Ad 40 single infection (Fig. 2 A, lane 3) and in coinfection Ad 40 + Ad 5 dl 312 (Fig. 2 A, lane  2) were identical. At low m.o.i., no effect of one virus on DNA viral synthesis of the other virus was therefore observed. With 10,000 FFU/cell of Ad 5 d1312 in HRT 18, the restriction pattern of Ad 5 d1312 was visible in single infection (Fig. 2 B, lane 1) . Although the cells were washed before the viral DNA extraction, it could not be ruled out that the inoculum could be responsible for visible restriction pattern. As a control, a similar experiment was performed in which viral DNA extraction was made 1 h post infection. In this case, no visible band was seen after digestion with restriction enzyme. In coinfection assays, the restriction pattern of Ad 5 dl 312 was enhanced but Ad 40 DNA was absent (Fig. 2 B, lane 2) . With 10,000 FFU/ cell of Ad5d1312 in HeLa cells, the same phenomenon of enhancement of Ad 5 dl 312 DNA was observed by coinfection assays (Fig. 2 C, lane 2) although the restriction pattern of Ad 40 was not visible in single infection (Fig. 2C, lane 3).

These experiments indicated that Ad 40 could complement E 1 A functions from deletion mutant Ad5d1312 in HRT 18 and HeLa cells, and that this complementation was dependent on the input concentration of Ad 5dl 312. At high m.o.i, ofAd 5 dl 312, Ad 40 was able to stimulate the synthesis ofAd 5 dt 312 DNA although its own DNA disappeared in coinfection assays. In HeLa cells the restriction pattern of Ad 40 could not be visualized, but a similar enhancement of the synthesis of Ad 5 dl 312 DNA was observed when the two viruses were present.

To determine if Ad 40 could also stimulate the growth of a mutant deleted in the E 1 B region, coinfection assays of Ad 40 with dl t 520 were performed. The m.o.i, of Ad 40 and d11520 was 1 FFU/cell. A clear restriction pattern ofd11520 was visible when the virus was infected in 293 cells (Fig. 3 A, lane 1) . The bands visible on Fig. 3 B, lane 1 (dl 1520 grown in HRT 18 cells) result from leakage of this mutant (see Table 1 ). For Ad 40, a clear restriction pattern was observed in HRT 18 (Fig. 3 B, lane 3) . In coinfection assays with dl 1520 and Ad 40 both DNAs disappeared in 293 and HRT 18 cells (Fig. 3 A and B, lanes 2) .

In a similar manner, coinfection assays with Ad 2 and dl 1520 were performed in HRT 18 cells to determine if d11520 was able to inhibit DNA synthesis of nonfastidious adenovirus or if this inhibition was specific of fastidious Ad 40. Results of this assay are shown in Fig. 4 , lane 2. No inhibition of Ad 2 DNA by d11520 was observed. A faint band of DNA visible under Hind III-B band of Ad 2 revealed the presence of d11520 DNA. As expected, this virus was therefore complemented by Ad 2. 

In the present study, we were able to cultivate Ad 40 in a human intestinal cell line, H R T 18, which has not been transformed in vitro by adenovirus genes.

From an Ad40 stock obtained in H R T 18 cells, there was a modest difference for antigen production during the first passage between H R T 18, HeLa and 293 cell lines (Table 1) Mautner et al. [19] have detected by slot blot hybridization a complementation of Ad40 growth by Ad5d1312 and vice versa in HeLa cells. Due to relatively low yields of Ad40, these authors were not able to obtain visible restriction patterns of Hirt extracted DNA. We also used slot blot hybridization (results not shown) and found an amount of Ad 40 DNA lower in cells coinfected by Ad 5 d1312 and Ad 40 than in cells infected with Ad40 only. A possible explanation for this discrepancy is that the Ad40 reference stocks have not been obtained in the same cell line.

To know if Ad 40 could complement a nonfastidious adenovirus mutated in E 1 B transcription unit, coinfection of Ad 40 and d11520 was performed. Surprisingly, this coinfection led to an inhibition of DNA synthesis of both viruses. As a control, cells were coinfected with dl 1520 mutant and Ad 2 wild type. No inhibition of Ad 2 DNA synthesis was observed. Furthermore, dl 1520 was complemented by Ad 2 (Fig. 4, lane 2) . We do not presently know the reasons of the mutual inhibition between Ad 40 and dl 1520. Interactions between two adenoviruses lacking E 1 B products should not lead to an inhibition of the DNA synthesis of both viruses. Several findings have suggested that Ad40 E 1 B region is transcribed in an unusual manner [18, 26] although it is structurally similar to E 1 B region of nonfastidious adenoviruses [341. The presence of unusual E 1 B proteins of Ad 40 associated with viral or cellular factors might explain the inhibition of d11520 DNA synthesis in coinfection experiments. Our PCR results show that at least one mRNA is synthesized in a region corresponding to the 55 k messenger of nonfastidious adenoviruses. To know why dl 1520 DNA synthesis is inhibited by Ad 40, further experiments are necessary to detect and characterize the Ad 40 E 1 B mRNAs and proteins.

Enteric adenoviruses. Brief review

Adenovirus proteins from both E 1 B reading frames are required for transformation of rodent cells by viral infection and DNA transfection

Adenovirus promoters and E 1 A transactivation

Adenoviruses and pediatric gastroenteritis

Comparative epidemiology of two rotavirus serotypes and other viral agents associated with pediatric gastroenteritis

Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction

Canditate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool

Genetic expression of human adenoviruses in simian cells. Evidence for interserotypic inhibition of viral DNA synthesis

Characterization of an early temperature-sensitive and cytocidal double mutant of adenovirus type 2

Boulanger P (t978) Adenovirus type 2 assembly analyzed by reversible cross-linking of labile intermediates

Adenovirus E 1 A protein paradigm viral transactivator

Characteristics of a human cell line transformed by DNA from human adenovirus type 5

Selective extraction of polyoma DNA from infected mouse cell cultures

Characterization of adenovirus type 40 E 1 region

I979) Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells

In vitro growth of some fastidious adenovirus from stool specimens

Une lign6e cellulaire particuli6rement sensible /t la r6plication du coronavirus ent~ritique bovin

Enteric adenovirus type40: expression of E 1 B mRNA and proteins in permissive and nonpermissive cells

Complementation of enteric adenovirus type 40 for lyric growth in tissue culture by E 1 B 55 K function of adenovirus types 5 and 12

Mecanism of activation of early viral transcription by the adenovirus E 1 A gene product

Direct detection and differentiation of fastidious and nonfastidious adenoviruses in stools by using a specific nonradioactive probe

A combined enzyme immunoassay for rotavirus and adenovirus (EIARA)

Isolation and propagation of enteric adenoviruses in HEp-2 cells

Enteric adenovirus 41 requires low serum for growth in primary cells

Differential growth of human enteric adenovirus 41 (TAK) in continuous cell lines

Enteric adenovirus type 40: E 1 B transcription map and identification of novel E 1 A-E 1 B cotranscripts in lyrically infected cells

Functions of adenovirus E 1 B tumour antigens

Propagation and in vitro studies of previously non-cultivable enteral adenoviruses in 293 cells

Early replicative block prevents the efficient growth of fastidious diarrhea-associated adenoviruses in cell culture

Helper function of adenovirus 2 for adenovirus 41 antigen synthesis in semi-permissive and non-permissive cells

Adenovirus growth in semi-permissive cells shows multiple-hit kinetics

Culture and antigenic properties of newly established cell strains derived from adenocarcinomas of human colon and rectum

Ad40 growth in HRT 18 cells

Importance of enteric adenovirus 40 and 41 in acute gastroenteritis in infants and young children

Structure and organization of the left-terminal DNA regions of fastidious adenovirus types 40 and 41

Restriction endonucleases in identification of a genome type adenovirus 19 associated with keratoconjunctivitis

Mutation in the gene encoding the adenovirus E 1 B 19 K tumour antigen causes the degradation of chromosomal DNA

Comparison of enteric adenovirus infection in various human celt lines

We are grateful to Dr. H. G. Pereira and to C. Cousin for critical reading of the manuscript, to Drs. T. Shenk and A. Berk for providing us Ad 5 d1312 and d11520, respectively, to Dr. T. H. Flewett for the gift of Ad 40, to N. Helbecque for oligonucleotide purification, and to D, Petite for cell culture assistance. S. A. Gomes is a recipient of a fellowship from CNPq (Brazil).

Received April 30, 1991