Virus shedding by SV40-transformed human cells.

Intervirology 9: 39-47 (1978)

Virus Shedding by SV40-Transformed Human Cells C hristopher A. L omax1, J ean-P aul T hirion and D anielle BourgauxR amoisy Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Qué.

Key Words. Transformation of human cells • Papovavirus SV40 • Shedding of virus by transformed cells • SV40-transformed human cell cultures • Temperature-sensitive mutant of SV40, cell transformation • tsA30, SV40 ts mutant Summary. Cultures of human cells transformed by SV40 were found to release infectious virus, even after several passages in vitro. Virus shedding by these cultures did not depend on propagation of virus from cell to cell, as it was not affected by anti-SV40 antiserum that could effectively block virus propagation in acutely infected cells. Cells transformed by the ‘early’ temperature-sensitive mutant tsA30, and maintained at the restrictive temperature of 39°, shed virus in reduced amount. Finally, xeroderma pigmentosum cells transformed by SV40 were also found to release virus, indicating that the enzymes of excision and repair of UV-induced damage to DNA probably were not involved in the molecular mechanism underlying virus shedding.

Human cells are considered semi-permissive for SV40 replication, as they yield smaller amounts of this virus than the fully permissive simian cells, after acute infection. Human cells can also be transformed by SV40 at a relatively high frequency [1]. The SV40/human cell system is therefore ideal to study the factors influencing the balance between viral replication and transformation. The transformation event believed to result from the integration of the SV40 genome into cellular DNA [2] is accompanied by the expression of the

Received: October 4,1976; revised: March 1, 1977.

Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/24/2019 1:57:16 PM

1 Present address: 3 Bailey Fold, Westhaughton Bolton BL5 3H4 (England). Address inquiries to: Dr. D anielle Bourgaux-R amoisy, Département de Microbio logie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4 (Canada)

40

L om ax/T hirion /B ourg aux-R amoisy

'early’ but not the ‘late’ functions [3], Thus, most cell lines transformed by SV40 fail to release infectious virus [4], although the latter can sometimes be rescued by fusion with permissive simian cells [5,6], Some transformed human and rabbit cells, however, yield virus spontaneously [7-9]. In this paper, we describe further characteristics of virus shedding by SV40-transformed human cultures. Our data suggest that this phenomenon, rather than being one mani festation of a carrier culture state, results from the activation of latent viral genomes.

Origin o f cells and culture conditions. The untransformed human cell cultures M l, M2 and M5 were derived from primary cultures of skin fragments from three male donors [10], The cell strain GM30 from a patient with xeroderma pigmentosum was obtained from the Human Genetic Mutant Cell Repository, Institute for Medical Research, Camden, N.J. These cell strains were grown at 37“ in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% fetal bovine serum (Flow Labs., Inc., Rockville, Md.) in a humidi fied atmosphere of 95% air and 5% COe. Transformed cells were cultured in DMEM con taining 10% fetal bovine serum. Transformation o f human cells. Confluent monolayers of human cells were infected with SV40 at a multiplicity of about 20 PFU/cell, and immediately sub-cultured in DMEM containing 10% fetal bovine serum and 0.25% anti-SV40 antiserum (Flow Labs.; titer 640 vs. 100 TCD 50). An incubation temperature of 37° was then maintained for the cells infected with wild type SV40, or 39° for those infected with tsA30 [10]. Transformants able to form colonies over the monolayer of normal cells were thus selected. Individual foci were picked and sub-cultured. This was performed in medium containing anti-SV40 anti serum, at 37“ for wild type transformants, and 39° for tsA30 transformants. Viruses. Wild type and tsA30 mutant of SV40, kindly provided by P. T egtmeyer, were grown in CV-1 cells from an MOI of 0.01 PFU/cell, at 37° for the wild type and 33“ for the mutant, as described elsewhere [10]. Titration o f virus stocks. Virus stocks were titrated by plating at high dilution on con fluent monolayers of CV-1 cells that were then incubated under Eagle’s medium plus 10% calf serum, solidified with 0.9% agar. Fresh medium was added at 5-day intervals. After 14 days of incubation at 37“, or 21 days at 33“, the cells were stained by adding medium con taining 0.01 % neutral red. Plaques were counted after a further 24 h incubation. Detection o f virus shedding. About half a million of the cells to be tested, together with the culture medium, were disrupted by three freeze-thaw cycles. After clarification at 3,000 rpm for 10 min, this extract was plated directly onto CV-1 cells. The development of a cytopathic effect within 14 days was interpreted as an indication of the presence of infectious particles in the extract. Virogenic cultures could often be detected by infecting CV-1 cells with the medium in which the culture had been growing. Quantitation o f infectious virus in a culture. The culture medium was removed and the monolayers were washed twice with phosphate-buffered saline (PBS). The cells were then detached with trypsin-versene solution, centrifuged and resuspended in 1 ml of Tris-


Materials and Methods

Virus Shedding by Transformed Cells

41

Table I. Frequency of occurrence of virus shedding1 Cell type

Transforming virus

Number of transformed colonies

Number of virogenic colonies

M5 M2 Ml GM30

tsA30 tsA30 WT WT

14 4 3 5

12 2 2 3

1 Transformed cells growing in DMEM + 10% fetal bovine serum, at 37° for wild type (WT) transformants, or 33° for tsA30 transformants, were lysed in their culture medium by three freeze-thaw cycles. After centrifugation the lysate was used to infect confluent CV-1 cells. The development of a cytopathic effect was taken to indicate that the extract came from a virogenic colony.

buffered saline. After counting, the cells were sonicated for 10 sec. Virus titer was then determined by plaque titration on CV-1 cells [10]. Quantitation o f virogenic celts in a culture. Various numbers of cells (up to 106) to be tested were added to a suspension of 10° CV-1 cells. This mixture was then plated in one 6-cm dish in 5 ml DMEM, containing 10% fetal bovine serum and 0.25% anti-SV40 anti serum. The next day, this medium was removed. The cells were washed with PBS and then covered with 5 ml of DMEM supplemented with 10% fetal calf serum and 0.9% agar. The cells were stained 14 or 21 days later by adding medium containing 0.01% neutral red.

For several human cell lines transformed by SV40, we observed that a cell-free extract, prepared as described in ‘Materials and Methods’, produced a cytopathic effect in CV-1 cells. The agent responsible for this effect was completely neutralized by anti-SV40 antiserum. Also, plaque formation by cell-free extracts of tsA30-transformed cells was as temperature-sensitive as by the mutant virus itself. It could thus be concluded that these human lines were releasing the agent used to transform them, even though they had been selected in a medium containing antiserum. Frequency o f occurrence o f virus shedding. In order to estimate the frequency of occurrence of the phenomenon, transformed colonies were isolated and tested for virus shedding (table I). It can be seen that virus shedding occurred in almost 75% of the cultures examined. Shedding was not cell- or virus-


Results

Lomax/T hirion/B ourgaux-R amoisy

specific, since it was observed in cells from four different sources, and occurred with both wild type and mutant virus. Time-course o f virus shedding. Infectious virus was measured after each weekly passage in one transformed cell line, lineT227, by extracting a known number of washed cells and assaying the extract on CV-1 cells (table II). At early passages, the cells contained large quantities of virus. The yield of virus, however, decreased at each passage, until no virus could be detected in the culture. A cell line derived from another transformed colony, line T233, behaved differently. It was found to contain undiminished amounts of virus for at least eight passages (table III). Effect o f anti-SV40 antiserum. One of the simplest explanations for virus shedding is that of the carrier state, in which a small number of cells harbor the replicating virus and subsequently reinfect other cells. Since reinfection is essential for the maintenance of a carrier state, incubation in anti-SV40 antiserum might neutralize it. This hypothesis was tested by incubating SV40transformed human cells in medium containing anti-SV40 antiserum (table III). The effect of antiserum on the virus content of the cells was minimal, though a small and consistent reduction was seen at every passage level. In contrast, anti-SV40 antiserum acted promptly on control human cells infected with wild type virus. All detectable virus was removed after four cell passages. Thus, the persistence of virus shedding by a culture does not require virus spreading through the medium. The small reduction in virus yield by antiSV40 antiserum may result from the neutralization of some cell-associated particles, either before or after virus harvest, or alternatively indicate that some reinfection does indeed occur which can be blocked by antiserum. Quantitation o f virogenic cells in a culture. The limited amount of infectious material obtained from any transformed line suggested that the majority of the cells in every culture were non-shedders. In order to estimate the propor tion of cells synthesizing virus, whole transformed cells were tested for their ability to induce the formation of virus plaques on lawns of CV-1 cells. Such experiments indicated that for a specific culture, at any particular passage level, less than 1% of the cells were actually shedding virus. Effect o f temperature on virus shedding. The mutant tsA30 was shown to be temperature-sensitive for viral DNA replication [11]. Virus shedding in cells transformed by wild type and tsA30 viruses was therefore studied as a function of temperature (table IV). At early passages of the wild type transformant T227, there was a 15- to 20-fold greater yield of virus at 39° compared to 33°. These cells could not be studied beyond passage 6, as they ceased shedding virus. In contrast, cells transformed by tsA30 yielded an approximately 30-fold


42


43

Table II. Infectious virus released by a virogenic culture1 Passage No.

PFU/108 cells

3 4 5 6 7 8

6.8 x 10‘> 2.0 xlO4 4.0 x 102 0 0 0

1 T227 cells, transformed by wild type virus, were passaged from inocula of around 2 x 105 cells per 60-mm dish, and allowed to grow to confluence. The cells were washed, detached, counted and lysed in 1 ml Tris-saline. The cell-free extract was assayed for virus titer on CV-1 cells.

Table III. Effect of anti-SV40 antiserum on virus shedding8 Cells

T233

Ml infected with WT SV40

Passage No.

PFU/106 cells + ASV

—ASV

3 4 5 6 7 8 9

3.0X106 4.3 x 102 7.5 xlO 2 3.5x103 3.0x103 1.0 x 102 15

6.5 x 10» 3.0 x 103 1.9x103 4.8 x 10» 2.2 x 104 n.d.b n.d.

8 9 10 11

47 7 8 0

1.4 x 104 9.1 x 103 3.5 x 104 2.5 x 104

Ratio - ASV/+ ASV

2.2 7.0 2.5 1.4 7.3 -

3.0 x 102 1.3X103 4.4 x 103 _


a T233 cells, transformed by wild type (WT) virus, were passaged in DMEM with 10% fetal bovine serum in the presence ( + ASV) or absence (-A S V ) of anti-SV40 antiserum. Cell extracts were made as described in the footnote of table II and assayed on CV-1 cells. b n.d. = Not determined.

44

Lomax/T hirion/B ourg aux-R amoisy

Table IV. Effect of incubation temperature on virus shedding by cells transformed by wild type (WT) and tsA30 viruses1 Cells

T227

T307

Transforming Passage virus No.

PFU/IO0 cells 33°

39°

WT

4 5 6

1.81 x 102 0.72 x 102 1.30 xlO2

3.3 x 102 1.1 xlO 3 1.3 x 102

3 4 5

5.3 xJO“ 4.5 xlO* 1.3 x 105

1.6 xlO 3 1 .3 x l0 3 4.5 x 104

tsA30

Ratio 33°/39°

5.5 x 10-2 6.5 x 10-'2

1.0 33 35 29

greater amount of virus at 33° than at 39°. Taking into account the 20-fold increase from 33 to 39° for wild-type transformants, the reduction in yield is about 2.5 logs at the restrictive temperature for tsA30 transformants. This difference is consistent with a 2-log difference in yield following infection of normal human cells at 33 and 39° [10]. Virus shedding by SV40-transformed xeroderma pigmentosum cells. We speculated that the excision of the integrated viral genome could possibly represent the primary molecular mechanism of virus shedding. Cells from xeroderma pigmentosum could perhaps provide us with a suitable model to test this hypothesis, as these cells are known to lack the enzymes required for the excision and repair of DNA after ultraviolet light-induced damage [12]. Colonies of SV40-transformed xeroderma pigmentosum cells (GM30) were thus produced. Five of them were picked, sub-cultured and examined for their capacity to release infectious virus (table I, bottom line). The cell lines derived from three of these colonies were found to shed virus. Therefore, we concluded that the enzymes of excision-repair of UV-induced damage proba bly are not involved in virus shedding. If involved at all in virus shedding, the excision of the integrated viral DNA would thus be performed by distinct enzymes.


1 Transformed cells were plated at 2 x 105 cells per 60-mm dish and incubated at 33 or 39° for 7 days. Cell extracts were made as described in the footnote to table II. The extracts were titrated on CV-1 cells at 33° for tsA30 or 37° for wild type virus.


45

This paper further documents the shedding of infectious SV40 by SV40transformed human cells [7]. The use we made of a virus carrying a tempera ture-sensitive mutation enabled us to demonstrate unambiguously that the SV40 released by the cells was the same as that originally used to effect trans formation. Nearly 75% of all cell lines tested were capable of releasing virus at some stage in the life of the cultures. The mechanism by which such transformed cells continually shed virus is unknown, but some possibilities can be excluded. The lack of a strong effect of anti-SV40 antiserum on virus shedding eliminates one possible mechanism, reinfection by virus through the medium. It is still conceivable, however, that virus could pass directly from one cell to another, if these two are in intimate contact. Since the untransformed cell cultures were never cloned, they may be heterogeneous and contain a population of cells, permissive for SV40 replication, distinct from the much larger population of non-permissive cells. However, the transformed cells were isolated as individual colonies which had overgrown a confluent monolayer of non-dividing cells. They were thus likely to derive from single cells. Furthermore, various transformed lines derived independently from one single colony were all found to be virogenic [Lomax, unpublished results]. Excision of the transforming genome, followed by its replication, of course provides the most attractive explanation for virus shedding. The results we registered with xeroderma pigmentosum cells, however, suggest that the enzymes which would perform such an excision most probably are not those involved in the excision and repair of UV-induced damage to DNA. Although the majority of transformed lines examined were virogenic, 25% of them did not release detectable virus at any stage in cultivation. It is possible that the transforming genome present in these cells, being defective, could not replicate autonomously. In keeping with this idea, we should mention repeated failures to induce virus replication from non-shedding cells by Sendai-induced fusion to CV-1 cells [Lomax, unpublished results]. Alternatively, the non-shedding cultures could contain non-defective viral genomes integrated in some unique way, which would render excision impossible. Indeed, W atkins [9] has sug gested that transformed cells may exist in three interchangeable states, one of which is virogenic. Our results suggest that virus shedding may occur at an early stage with respect to the transformation event, and that the transition to a non-virogenic state is irreversible. In this respect, it is interesting that, by fusion to simian cells, virus may still be rescued from cells which have


Discussion

46

L omax/T hirion /B ourgaux-R amoisy

just passed through the shedding stage to become non-virogenic [L om ax , unpublished results]. An understanding of the mechanism of SV40 shedding is of great importance, as this phenomenon is reminiscent of the release of Epstein-Barr virus by transformed peripheral lymphocytes [13]. Finally, we should stress the observation that cells transformed by the tsA30 mutant, while remaining virus shedders, produce reduced amounts of infectious virus at the temperature selected as restrictive. Such an inhibition of virus replication, similar to that observed in acutely infected permissive cells, demonstrates that the temperature-sensitive mutation is indeed expressed in the transformed human cells. That these cells would still behave as trans formed cells at the same temperature may indicate that the tsA30 mutation affects the gene A function(s) to a lesser extent in the maintenance of trans formation than in the initiation of viral DNA replication. This aspect of our results is discussed more extensively elsewhere [10].

A cknowledgments This work was supported by the National Cancer Institute of Canada. J.-P.T. is a Scholar of the Medical Research Council of Canada. We thank P eter T egtmeyer for the generous gift of wild type SV40 and mutant tsA30, Pierre Bourgaux and J oseph W eber for stimulating discussion and critical review of the manuscript.

1 A aronson, S.A. and T odaro, G .J.: SV40 T-antigen induction and transformation in human fibroblast cell strains. Virology 36: 254-261 (1968). 2 Sambrook, J.; W estphal, H.; S rinivasan, P.R., and D ulbecco, R.: The integrated state of DNA in SV40-transformed cells. Proc. natn. Acad. Sci. USA 60: 1288-1295 (1968). 3 K houry, G.; M artin , M. A.; L ee, T .N .H ., and N athans, D.: A transcriptional map of the SV40 genome in transformed cell lines. Virology 63: 263-272 (1975). 4 G erber, P. and K irschstein, R. L. : SV40-induced ependymomas in newborn hamsters. I. Virus-tumor relationships. Virology 18: 582-588 (1962). 5 K oprowski, H.; J ensen, F.C., and Steplewski, Z.S.: Activation of production of infectious tumor virus SV40 in heterokaryon cultures. Proc. natn. Acad. Sci. USA 58: 127-133 (1967). 6 W atkins, J. F. and D ulbecco, R. : Production of SV40 virus in heterokaryons of trans formed and susceptible cells. Proc. natn. Acad. Sci. USA 58: 1396-1403 (1967). 7 G irardi, A .J.; J ensen, F.C., and K oprowski, H.: SV40-induced transformation of human diploid cells: crisis and recovery. J. cell. comp. Physiol. 65: 69-84 (1965).


References

Virus Shedding by Transformed Ceils

9 10 11 12 13

N oordaa, J. and P auw, W .: The production of infectious virus by SV40transformed human renal cells. J. gen. Virol. 18: 201-202 (1973). W atkins, J. F .: Studies on a virogenic clone of SV40-transformed rabbit cells using cell fusion and in situ hybridization. J. gen. Virol. 21: 69-81 (1973). Lomax, C.A.; B radley, E.; W eber, J., and Bourgaux, P.: Transformation of human cells by temperature-sensitive mutants of simian virus 40. Intervirology 9: 28-38 (1978). T egtmeyer, P.: Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J. Virol. 10: 591-598 (1972). C leaver, J.E .: Defective repair replication of DNA in xeroderma pigmentosum. Nature, Lond. 218: 652-656 (1968). E pstein, M. A. and A chong , B .G .: The EB virus. A. Rev. Microbiol. 27:413-437(1973).

van der


8

47

Virus precursor shedding from mammary tumour cells.

Ganglioside GD3 shedding by human gliomas.

Shedding of the glycoprotein from vesicular stomatitis virus-infected cells.

Transformation of human cells by SV40 virus.

Shedding of Hepatitis C Virus in Semen of Human Immunodeficiency Virus-Infected Men.

Spontaneous shedding of plasma membrane fragments by human cells in vivo and in vitro.

Shedding of tumor necrosis factor receptors by activated human neutrophils.

Ocular shedding of herpes simplex virus.

Shedding of human neuroblastoma gangliosides.

Enhanced shedding of cytomegalovirus in semen of human immunodeficiency virus-seropositive homosexual men.

Non-producer human cells induced by murine sarcoma virus.

Antigen shedding and metastasis of tumour cells.

Shedding light on biology of bacterial cells.

Shedding of infectious virus and virus antigen during acute infection with respiratory syncytial virus.

CD32 molecules by human Langerhans cells: a subtle balance between shedding and secretion?

Extended viral shedding of a low pathogenic avian influenza virus by striped skunks (Mephitis mephitis).

Shedding of Ebola Virus in an Asymptomatic Pregnant Woman.

Influenza A Virus Shedding and Infectivity in Households.

Membrane association and shedding of the GPI-anchored Ca-MOv18 antigen in human ovary carcinoma cells.

Inhibition of human immunodeficiency virus infection in human colon epithelial cells by recombinant interferon-gamma.

Human cytomegalovirus inhibits human immunodeficiency virus replication in cells productively infected by both viruses.

In vitro suppression of normal human bone marrow progenitor cells by human immunodeficiency virus.

Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.

Differential mucin expression by respiratory syncytial virus and human metapneumovirus infection in human epithelial cells.