Table 1. Sugar uptake in human embryo fibroblasts infected with human cytomegalovirus .... As shown in Table 3, both Vero cells and HEF treated with Ara-C.
J. gen. Virol. (1984), 65, 1229 1232. Printed in Great Britain
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Key words: HCMV/glucose uptake~transcription
Early Enhanced Glucose Uptake in Human Cytomegalovirus-infected Cells By M. P. L A N D I N I Institute of Microbiology, Medical School, University of Bologna, St Orsola General Hospital, Via MassarentL 9, 1-40138 Bologna, Italy
(Accepted 2 April 1984)
SUMMARY
The uptake rate of some sugars was demonstrated in human embryo fibroblasts following infection by human cytomegalovirus (HCMV) and a significant increase in glucose uptake was demonstrated. Quantitative and sequential analysis of glucose uptake during the HCMV replication cycle showed that the enhanced uptake began during the first 20 h after infection and occurred even when a high glucose level was cbnstantly present in the medium. The increase in sugar uptake requires an active viral gen0me, de novo protein synthesis and seems to be dependent only on the early transcription of the viral genome, as it did occur when replication of the viral genome was limited (non-permissive cells or cells infected in the presence of D N A synthesis inhibitors). The increase in glucose uptake on malignant transformation, in contrast to its decrease in slowly growing cells, is a well-known phenomenon (Hatanaka, 1974; Bose & Zlotnick, 1973). Alterations in the characteristics of sugar transport have been shown in cells transformed by avian sarcoma viruses, where a significant increase in glucose and mannose uptake was evident at the same time as changes in cell morphology (Hatanaka & Hanafusa, 1970). The increase in glucose uptake in Rous sarcoma virus (RSV)-transformed cells has been correlated with both an increase in the number of glucose receptors on the plasma membrane and with their greater affinity for the sugar (Lee & Lipmann, 1977). Identical modifications in membrane polypeptides have been described in RSV-transformed cells and in cells deprived of glucose (Shiu et al., 1977). Maintenance of a high glucose level in the medium decreases the synthesis of these polypeptides both in glucose-starved and in RSV-transformed cells. Thus, their enhancement in the latter appears to be secondary to the increased transport of glucose (due to their higher metabolism) rather than being a transformation-specific event (Shiu et al., 1977). Since human cytomegalovirus (HCMV) stimulates cellular macromolecular syntheses (St. Jeor et al., 1974; Tanaka et al., 1975; Estes & Huang, 1977; Furukawa et al., 1976) and is suspected to be potentially oncogenic, it was of interest to determine the effect of H C M V infection on sugar uptake. Human embryo fibroblasts (HEF) (obtained from Istituto Zooprofilattico, Brescia, Italy) were grown as described previously (Landini et al., 1979). BALB/3T3 cells (ATCC) were grown in Dulbecco's MEM supplemented with 5% (v/v) foetal calf serum (FCS). African green monkey kidney cells (Vero) were grown in RPMI 1640 with 5 % (v/v) FCS. The Towne strain of HCMV (kindly made available by M. Stinski, University of Iowa, Iowa City, Iowa, U.S.A.) was used in all the experiments. The virus was propagated as described by Stinski (1978) and the virus titre was determined as previously described (Landini & Baldassarri, 1982). In all the experiments confluent cells were infected at a m.o.i, of 1 to 3 p.f.u./cell. After 1 h adsorption at 37 °C in a humidified CO2 incubator, the inoculum was removed and replaced with fresh growth medium. In some experiments 25 mM-glucose (final concentration) was added to normal medium every 12 h to maintain a high glucose concentration (Shiu et al., 1977). 0022-1317/84/0000-6065 $02.00© 1984 SGM
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Table 1. Sugar uptake in human embryo fibroblasts infected with human cytomegalovirus Substrate D-Glucose 2-Deoxy-D-glucose D-Mannose D-Fructose Sucrose D-Galactose
Sugar uptake (ratio to control) at 50 h 2.62 2"24 0-69 0.90 0.98 0-86
Table 2. Glucose uptake in HEF cell cultures infected in the presence of cycloheximide or with
u.v.-inactivated virus Cells infected with -* HCMV U.v.-treated HCMVt HCMV with cycloheximide:~
Glucose uptake (ratio to mock-infected cells) at 50 h 1.0
2.62 1.1 0.6
* Mock-infected cells. t The virus stock was spread on a Petri dish and left 15 min at 8 cm under a germicidal lamp. :~The cells were kept in the presence of 50 ~tg/ml cycloheximide from I h before infection until 50 h after. A t each experimental point sugar uptake was determined following the procedure described by Lee & L i p m a n n (1977). Briefly, after the culture medium had been drained off, the culture dishes were washed with sugar-free medium and were then incubated for 15 rain at 37 °C in a humidified CO2 incubator with 2 g c i / m l radioactive sugar dissolved in sugar-free medium. At the end of the incubation period, the radioactive solution was drained and the dishes were washed once with medium containing 1 ~ (w/v) of the sugar being tested and twice with sugarfree medium. The cells were then counted, dissolved in 0.1 M-NaOH and the radioactivity was determined. As shown in Table l, 50 h after infection D-glucose and 2-deoxy-D-glucose uptake in infected cells was found to be more than twice that in uninfected cells. In other experiments in which very young fibroblasts (4 to 7 passages) were infected, the increase in glucose uptake was 4.9 to 5.3 times greater (data not shown). The data presented in Table 1 are the m e a n values obtained in three different experiments performed using H E F from the 15th to 20th passage. Mannose, fructose, sucrose and galactose uptakes were essentially unchanged after infection. Glucose uptake was then followed at various times of the viral replication cycle, maintaining the cells in normal growth medium or in the presence of high glucose concentration. As shown in Fig. I, a marked increase (over twofold) in the uptake was evident in infected cells at 20 h after infection. This was even more marked later in the viral replication cycle and it seems to be dependent on de novo protein synthesis (Table 2), being absent in infected cells maintained in the presence of protein synthesis inhibitors such as cycloheximide. Moreover, an active viral genome is required, because the phenomenon was not detectable in cells infected with u.v.inactivated H C M V (Table 2). Similarly, a virus stock neutralized by a pool of human sera having a high titre against H C M V was unable to induce such a stimulation (data not shown). These results suggest that there is a selective increase in glucose uptake in HCMV-infected cells, probably due to virus-induced stimulation in cellular macromolecular syntheses and to the subsequent increase in the need for energy-yielding reactions. This could lead to a rapid depletion of glucose from the medium and to an increased number of glucose receptors on the plasma membrane, as has been described for RSV-transformed cells (Lee & Lipmann, 1977). However, at least part of the H C M V - i n d u c e d increase in glucose uptake seems to be independent of the sugar level in the medium. There m a y therefore be a specific virus-induced modification in the plasma membrane, independent of glucose starvation, as described for paramyxovirus infection (Peluso et al., 1978).
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Fig. 1. Rate of D-glucose uptake in HEF infected with HCMV or mock-infected. 0 , ©, HCMVinfected cells; II, f-q, mock-infected cells; 0 , II, maintained in normal growth medium; O, [-1, maintained in growth medium containing a high concentration of glucose. Table 3. Glucose uptake in permissive and non-permissive systems for human
cytomegalovirus replication Glucose uptake (ratio to control)
Percent of infected cells showing HCMV-specific antigens*
,k
Infected cells HEF HEF + Ara-C BALB/3T3 Vero
20 h 2.06 NDt 1.01 1.79
~k
50 h 2.62 i '92 0.80 2.06
IEA 100 100 2-5 20-30
LA 50 0 0 0
* Immediate-earlyantigens (IEA) were detected by indirect immunofluoresence (IIF) 12 h after infection, using monoclonal antibodies (E3) to the 72K immediate-early protein (kindly provided by Genetic Systems, Seattle, Wash., U.S.A.). Late antigens (LA) were detected by IIF 48 to 72 h post-infection, using selected human sera. t ND, Not determined.
Other experiments were carried out to investigate whether the increase in glucose uptake was dependent on early transcription of the viral genome, since the stimulation occurred early in the viral replication cycle. Two different non-permissive cell lines were infected together with permissive H E F maintained in the' presence of 1-/3-D-arabinofuranosylcytosine (Ara-C), an inhibitor of D N A replication. As shown in Table 3, both Vero cells and H E F treated with A r a - C showed the increase in glucose uptake 50 h after infection. The presence of HCMV-specific immediate-early antigens (IEA) was detected in 100 ~o of H E F infected in the presence of A r a - C and in at least 2 0 ~ of infected Vero cells. In BALB/3T3 cells, the presence of HCMV-specific I E A was detected only occasionally and in no more than 2 to 5 ~o of the infected cells, suggesting a very low efficiency o f viral infection and/or viral genome expression in this type o f cell. N o increase in glucose uptake was detected in BALB/3T3 cells. Although the latter finding needs further investigation, these results suggest that the stimulation in glucose uptake is only dependent upon early transcription of the H C M V genome. W o r k aimed at identifying the membrane receptors involved in this event and their molecular characterization is in progress.
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This work was partially supported by C. N. R. Progetto Finalizzato 'Controllo delle Malattie da infezione' grant no. 83.00662.52 and from the Italian Ministry of Education (40~). REFERENCES BOSE, B. K. & ZLOTNICK, B. J. (1973). Growth and density dependent inhibition of deoxy-glucose transport in Balb 3T3 cells and its absence in cells transformed by murine sarcoma virus. Proceedingsof the National Academy of Sciences, U.S.A. 70, 2374-2378. ESTES, J. E. & HUANG, E. S. (1977). Stimulation of cellular thymidine kinase by human cytomegalovirus. Journal of Virology 24, 13-21. FURUKAWA, T., SAKUMA, S. & PLOTKIN, S. A. (1976). Human cytomegalovirus infection of WI 38 stimulates mitochondrial D N A synthesis. Nature, London 262, 414-416. I-~TANAKA, M. (1974). Transport of sugars in tumor cell membranes. Biochimica et biophysica acta 355, 77-104. r~TANAKA, M. & HhNAFUSA, H. (1970). Analysis of a functional change on membrane in the process of cell transformation by Rous sarcoma virus: alteration in the characteristics of sugar transport. Virology41, 647652. LANDINI, M. P. & BALDASSARRI,B. (1982). Early inhibition of human cytomegalovirus by novobiocin. Journal of Antimicrobial Chemotherapy 10, 533-537. LANDINI, M. P., MUSIANI, M., ZERBINI, M., FALCIERI, E. & LA PLACA, M. (1979). Inhibition of a complete replication cycle of human cytomegalovirus in actinomycin pre-treated cells. Journal of General Virology 42, 423-428. LEE, S. G. & LIPMANN,F. (1977). Isolation from normal and Rous sarcoma virus transformed chicken fibroblasts of a factor that binds glucose and stimulates its transport. Proceedingsof the National Academy of Sciences, U.S.A. 74, 163-167. PELUSO, R. W., LAMB, R. A. & CHOPPIN, P. W. (1978). Infection with paramyxoviruses stimulates synthesis of cellular polypeptides that are also stimulated in cells transformed by Rous sarcoma virus or deprived of glucose. Proceedings of the National Academy of Sciences, U.S.A. 75, 6120-6124 ST. JEOR, S. C., ALBRECHT, T., FUNK, F. D. & RAPP. F. (1974). Stimulation of cellular D N A synthesis by human cytomegalovirus. Journal of Virology 13, 353-362. SHIU, R. P. C., POUYSSEGUR, J. & PASTAN, I. (1977). Glucose depletion accounts for the induction of two transformation-sensitive membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts. Proceedings of the National Academy of Sciences, U.S.A. 74, 3840-3844. STINSKI, M. F. (1978). Sequence of protein synthesis in cells infected with human cytomegalovirus: early and late virus-induced polypeptides. Journal of Virology 26, 686-701. TANAKA, S., FURUKAWA, T. & PLOTKIN, S. A. (1975). Human cytomegalovirus stimulates host cell R N A synthesis. Journal of Virology 15, 297-304.
(Received 19 January 1984)
