Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus

© INSTITUTPASTEUR/ELSEVIER Paris 1991

Res. ViroL 1991, 142, 25-31

Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus M. da Gloria da Costa Carvalho (*) and M. Vasconcellos Fournier

Lab. do Controle da Express~o G~nica, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941, Rio de Janeiro, R.J. (Brazil)

SUMMARY

Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 rain (heat shock conditions), tho synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2} the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells.

Key-words: Aedes albopictus, RNA, Mayaro virus, Heat shock; Cell culture, Protein s,'nthesis, Gene expression.

INTRODUCTION

The response of cells to high temperature is characterized by specific alterations in gene expression. The synthesis of a group of highly evolutionary conserved proteins, referred to as heat shock proteins (HSP), is induced by the treatment (Schlesinger et al., 1982; Lindquist, !986; Craig, 1985). Recent studies have shown that the infection of cells by several viruses also induces the synthesis of cellular proteins related to HSP (Nevins, 1982; Notarianni et al., 1982; Garry et al., 1983 ; Collins and Hightow-

er, 1982; Khandjian and Turler, 1983). Viral replication itself represents a stress for the host cells. Both conditions of stress (heat s h o o ' o r viral infection) involve the reprogramming of gene expression in host cells. Recently, we have investigated the effect of high temperature on the replicat, on of Mayaro virus. Approximately, a 1OO-fold reduction of virus titre was observed in Aedes albopictus cells maintained at 37°C, as compared to those kept at 28°C (Carvalho et al., 1987). Mayaro virus (alphavirus genus, family Toga¢iridae) was first

Submitted September 9, 1989, accepted November 19, 1990. (*) Corresponding author.

26

M. DA GLORIA DA COSTA C A R V A L H O A N D M. VASCONCELLOS FOURNIER

isolated in Trinidad in 1954 from the b l o o d o f f e b ~ e patients (Anderson et al., 1957). In Brazil, it was isolated during an outbreak in a rural community (120 miles east o f the city o f Belem) (Causey and Maroja, 1957). Togaviruses have an icosahedral capsid envelope containing a positive single-stranded R N A ( K a ~ a i n e n and Soderlund, 1978). In this communication, we extended our studies of the high temperature effect in cells infected with Mayaro virus. We show that the transcription of the heat shock genes for 82- and 70-kDa H S P , and translation o f the respective m R N A , are not modified in A. aibopictus cells previously programmed for translation o f Mayaro viral messengers.

MATERIALS AND METHODS

Infection of cells Confluent monolayers of 2 × 106 A. albopictus cells growing in scintillation vials or 1.5 x 107 cells growing in bottles were infected with Mayaro virus at a multiplicity of 10 PFU/ceI1. After the adsortion period, the inoculum was aspirated and growth medium was added to each culture dish. Cells were incubated at 28 or 37°C, respectively. Labelling of cultures with 3sS-methionine A. albopictus cells growing in scintillation vials were preincubated for 30 rain at 28 or 37°C, in Eagles' minimal essential medium in the absence of serum and methionine. After this period, the medium was supplemented with 35S-methionine and the incubation continued. One hour later, the medium was removed and the monolayers resuspended directly in 80 ~1 of electrophoresis loading buffer (62.5 mM Tris-HCl pH 6.8, 2 % sodium dodecyl sulphate, 10 070glycerol, 5 % 2 - ~ r e a p t o e t h ~ o l aad 0.001% bromophenol blue).

Cell cultures A. albopictus cells, clone C6/36 (Igarashi, 1978), a gift from Dr. R.E. Shope, Arbovirus Research Unit, Yale University (USA) were used. Cells were grown in 60-cm 2 glass bottles at 28°C. The growth medium consisted of Dulbecco's modified Eagle's medium supplemented with 0.2 mM non-essential amino acids, 2.25 070 NaHCO3, 2 % foetal calf serum, penicillin (500 U/nil), streptomycin (100 gg/ml) and amphotericin B (fungizone, 2.5 gg/ml). For subcultivations, confluent monolayers containing 1.5 x 107 cells/bottle were washed gently with Dulbecco's phosphate-buffered saline (PBS), and after a brief trypsinization, cells were suspended in the culture medium. Monolayers, grown in scintillation vials, were seeded with I x 105 cells and the culture incubated at 28°C in an atmosphere of 5 070 CO 2. Virus

Analysis of 3sS-methionine-labeiled proteins by polyacrylamide gel electrophoresis Protein samples labelled with 35S-methionine (20 ~tCi/ml) were heated for 5 min at 95°C and subjected to electrophoresis on 1-dimensional 12.5 % polyacrylaraide gels (Laemmli, 1970). The dried gels were exposed to "Kodak X-Omat" (YAR-S) film. The molecular weight of the proteins were determined by coelectrophoresis of standard proteins (Pharmacia): phosphorylase b, 94,000; bovine serum albumin, 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; soybean trypsin inhibitor, 20,000; and et-lactalbumin, 14,000. An eqaal amount of protein was applied to each gel lane. RNA extraction and hybridization

Mayaro virus was obtained from American Type Culture Collection, Rockville, MD (USA). The virus stock was prepared from BHK-21 cells and stored at - 60°C.

Total RNA was extracted according to Holmes and Booner (1973). It was denatured by the formamide-formaldehyde method, size fractionated on 1.2 070agarose formaldehyde gel (Lehrach et ai., 1977) and transferred to nitrocellulose filters as

HSP = heat shock protein. LMW = low molecular weight. PFU -- plaque-formingunit.

SDS-PAGE= sodium dodecyl sulfate/polyacrylamidegel electrophoresis.

M A Y A R O I, IRUS, H E A T S H O C K A N D G E N E E X P R E S S I O N I N AEDES described by Thomas (1980). The nucleic acid bound to nitrocellulose filters was baked for 2 h at 80°C in a vacuum oven and prehybridized in a medium consisting o f 50 % formamide, 100 i~g/ml o f denatured salmon sperm DNA, 5 x SSC, 5 x Denhardt's solution and 50 m M Na2HPO 3 buffer pH 6.8 (Rondinelli et al., 1986). Blots were hybridized with nick-translated plasmids containing genes coding for Drosophila H S P 82 or 70 (Hackett and Lis, 1983; Livack et al., 1978). Hybridizations were performed at 37°C for 48 h. Filters were washed 3 times in 0 . 1 x S S C , 0.1 °70 SDS at room temperature for 15 min and autoradiographed with " K o d a k " X-ray film using an intensifying screen (Lightning Plus, Du Pont) at - 70°C.

Quantification of macromolecular synthesis For quantification of macromolecular synthesis, densitometric tracings of the autoradiograms were made in a " L K B 2202 Ultroscan Laser" densitome-

34

27

ter. The percentage of synthesis of macromolecules (RNA or protein) was determined by calculating the areas of the defined peaks.

RESULTS

Quantitative analysis of heat shock proteins synthesized in A. albopictus cells infected with Mayaro In p r e v i o u s w o r k ( C a r v a l h o et al., 1987), we s h o w e d t h a t when A . albopictus cells infected with M a y a r o virus g r o w i n g at 2 8 ° C were heated t o 3 7 ° C f o r 90 min, t h e synthesis o f t w o m a j o r H S P (82 a n d 70 k D a ) was induced. C o n c o m i t a n t l y , strong i n h i b i t i o n o f n o r m a l a n d viral p r o t e i n synthesis was observed. In o r d e r t o

50 62

i

B

I 2

//:: -

Fig. 1. Densitometer tracings (A) and autoradiogram (B) of proteins synthesized in cultured ofA. albopictus cells infected with Mayaro virus at an input of I0 PFU/celI. Cells were labelled with 35S-methionine and protein synthesis analysed by SDS-PAGE and autoradiography as described in "Materials and Methods". 1) (A and B), represents extracts from mock-infected cells incubated at 28°C. 2) (A and B), represents extracts from cells 48 h postinfection with Mayaro virus, incubated at 28°C. Equal amounts of labelled proteins were applied to each gel lane. The positions of Mayaro virus proteins of 62, 50 and 34 kDa are indicated.

28

M. DA GLORIA DA COSTA C A R V A L H O A N D M. VASCONCELLOS FOURNIER

investigate whether the control of gene expression o f HSP was modified by infection o f the cells with Mayaro virus, the amount o f HSP RNA in mock or infected cells and the rate o f synthesis o f HSP were studied in the following experiments. A. albopictus cells growing in scintillation vials were infected with Mayaro virus at 10 PFU/cell and incubated at 28°C for 48 h. Cells were then labelled with 35S-methionine and analysed in SDS-PAGE. The results o f figure 1B show that 3 major Mayaro virus proteins of 62, 50 and 34 kDa were detected after 48 h post-infection. Autoradiographs were

scanned (fig. 1A) to quantitate the virus proteins synthesized. Viral proteins o f 62, 50 and 34 kDa constituted 5.5, 7 and 10 % of the total protein, respectively. When the infected cells were shifted to 37°C for 90 min, the synthesis o f 82- and 70-kDa HSP was induced (fig. 2B). The densitometric quantitation (fig. 2A) o f the proteins showed that during heat shock, a decrease o f 50 °70 in the synthesis o f the 34-kDa viral protein was observed. Virus proteins o f 50 and 62 k D a were not detected. The distribution of the radioactivity showed that 11 and 10 % resides in the high-molecular weight 82- and 70-kDa H S P , respectively. Six

70 82

l! B I 2 LMW

34

10

MW

Fig. 2. Effect of heat shock on virus protein synthesisin A. albopictuscellsinfectedwith Mayaro virus. A. aibopictuscellswereinfected with Mayaro virus at a multiplicityof 10 PFU/cell, and incubated at 28°C for 48 h. Then, cells were shifted to 37°C, labelled with 35S-methionineand analysed in SDS-PAGE and autoradiographed as described in "Materials and Methods". 1) Densitometerictracing (A) and autoradiogram (]3)of extracts from heat-shockedmock-infected cells. 2) Densitometer tracing (A) and autoradiogram (B) of extracts from heat-shocked infected cells. HSP of high molecular weight (82 and 70), LMW and also the 34-kDa viral protein are indicated.

MA YARO VIRUS, H E A T SHOCK AND GENE EXPRESSION IN AEDES

percent of the labelling was present in another group o f HSP o f low molecular weight (LMW). Quantitation o f the H S P synthesized in mockinfected cells and submitted to heat shock showed a distribution o f 11 and 10 % for HSP o f 70 and 82 kDa, and 7.5 % for the group o f L M W . These results showed that infected cells synthesized a similar quantity o f HSP as mockinfected cells.

A1

Kb

2.8

2

3

•

B

1

4

5

29

Quantification of RNA for HSP 70 and 82 in mock and infected cells at 28°C under heat shock Although infected cells presented the same a m o u n t o f H S P synthesized as mock infected cells, we must consider that the amount o f hsp RNA could be different and present different efficiencies o f translation. To investigate these possibilities, total cell RNA was isolated from mock and infected cells maintained at 28°C or submitted to heat shock. R N A was size fractionated by gel electrophoresis, blotted onto nitrocellulose filters and hybridized to nick-translated genomic probes for HSP 82 or 70 from Drosophila, as described in "Materials and Methods". As can be seen in figure 3A, the probes for HSP 70 hybridized to an R N A species of 2.5 kb. These hybridizations occurred only with RNA from m o c k or infected cells when submitted to heat shock. The weight corresponding to densitometric tracing areas o f the autoradiograms from heat-shocked mock or infected cells were: 25 and 30 lng, respectively (fig. 3B, lines 2 and 4). When the probe for HSP 82 was used (fig. 4A), an RNA species o f 2.6 kb was detected upon heat shock, from mock or infected cells (fig. 4A, lines 3 and 5). The weight of the corresponding areas o f the densitometric tracing of the autoradiograms were found to be 30 and 25 mg respectively (rig. 4B, lines 1 and 2). Since these values were very close, these results show that the presence o f Mayaro viruses in the cells does not inhibit the transcription o f these genes.

•

2

Fig. 3. Northern blot analysis of HSP 70 mRNA. Total RNA (10 Ixg)were applied to each lane and fractionated by gel electrophoresis. RNA from mock-infected cells incubated at 28°C (1A), and sabmitted to heat shock (2A). RNA from cells infected with Mayaro virus at 10 PFU/cell and incubated at 28°C for 48 h (3A), and submitted to heat shock (4A). After autoradiography, the region of the blot containing the labelledbands was scanned in a laser densitometer as described in "Materials and Methods". BI) Densitometertracing of the autoradiogram 2A. B2) Densitometer tracing of the autoradiogram 4A.

DISCUSSION In the present study, we observed that A. albopictus cells previously programmed for translation o f Mayaro virus messengers (fig. 1), when submitted to a heat shock, synthesized the same amounts o f HSP as mock-infected cells. This results is very intriguing since Mayaro virus modifies the translational machinery of the host cell so that it translates principally its mRNA. It is important to note that these modifications do not interfere in the translation of heat-shock messengers. It is not known what are the modifi-

30

M. DA GLORIA DA COSTA C A R VALHO A N D M. VASCONCELLOS FOURNIER

cations induced by Mayaro virus in the translational machinery of the host cells. The switchover from host to virus protein synthesis is generally caused by the ability o f viral RNA to outcompete cellular m R N A for a component necessary for initiation (Lodish and Porter, 1980). Recently, we have shown that the translation o f Mayaro virus messengers could be modulated by temperature (Carvalho et al., 1989). The results presented in figure 3 show that the m o u n t of HSP 70 RNA in heat-shocked infected cells is approximately 1.2-fold greater than that observed in mock-infected cells. However, the same amount o f HSP 70 protein was synthesized in both cells (mock or infected heatshocked cells). These results suggest that in mock infected cells, the efficiency of translation o f HSP 70 messengers is higher than ip infected cells. Apparently the presence of the virus can interfere in the expression o f the HSP 82 gene, since a decrease o f 1.2-fold in the HSP transcript was observed in heat-shocked infected cells when compared with mock-infected cells. Although the amount of synthesis of HSP 82 was equal in mock and infected cells, these results suggest that the efficiency o f translation of 82 RNA was higher in infected cells. Quantitative diffordanr'oc ; n thJa exrnth,ac;~ o f LMW HSP was ,~h_ served in mock and infected cells (fig. 2). As can be seen, a decrease o f 1.25-fold in the synthesis o f this HSP group was observed in infected cells. Data from the literature (Schlesinger et al., 1982) have shown that transcription o f the HSP is coordinated. As no inhibition o f the translation of high molecular weight HSP was observed in heat-shocked infected cells, the results in relation to the quantitative differences in the synthesis of the L M W HSP between mock and infected cells (fig. 2), may be the result o f either a higher degradation rate of these R N A species in infected cells or inhibition o f translation, resulting from modifications in the translational machinary by virus infection. These studies are under investigation in our laboratory. Taken together, these results show that M a y a r o virus does not interfere in the reprogrammation of gene expression induced by heat. However, different kinds of control oper-

A

1

2

3

4

Qq)

Kb 2.5

13

1

2

Fig. 4. Northen blot analysis of HSP 82 mRNA. Total RNA (10 ~g) were applied to each lane and fractionated by gel electroph6resis. RNA from cells infected with Mayaro virus for 24 h (IA), and 48 h at 28°C (2A). RNA from cells infected with Mayaro virus 48 h and submitted to heat shock (3A). RNA from mock-infected cells incubated at 28°C (4A). RNA from mock-infected cells submitted to heat shock (SA). B1) Densitometer tracing of the autoradiogram 3A. B2) Densitometer tracing (2B) of the autoradiogram 5A.

ate in high and LMW H S P in Mayaro-virusinfected cells.

Acknowledgements We thank P.S. Lopes for excellenttechnical assistance and Dr. E. Rondinellifor criticalreading of the manuscript. The research was supported by Conselho Nacional de DesenvolvimentoCientifico e Tecnologico(CNPq), Financiadora de Estudos e Projetos (FINEP) and Funda~;aode Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

AIA Y A R O VIRUS, H E A T S H O C K A N D G E N E E X P R E S S I O N I N AEDES

Effet d ' u n choc thermique sur l'expression des g~nes des cellules de A e d e s albopictus infect6es par le virus Mayaro Trois prot6ines majeures de 62, 50 et 30 k D a du virus M a y a r o ont 6t~ mises en 6vidence dans les cellules de A e d e s albopictus expos6es/t 28°C pendant 48 h apr~s l'infe~ion. Quae.d les cellules infect6es ont 6t~ expos6es/t une temp6rature variant de 28 h 37°C pendant 90 min (conditions du choc thermique) la synth~se de 2 H P S majeures (heat shock protein) de 82 et 70 kDa a 6t~ induite parall~lement h une inhibition virale 6levee et une synth~se normale de prot6ines. L ' A R N cellulaire total a ~t6 isol~ de cellules ~tt6moins >>et de cellules infect~es incub6es h 28°C ou soumises au choc thermique. L'analyse ~tNorthern blot >>/l l'aide de sondes g6nomiques pour des H S P de l'esp~ce Drosophila montrent (1) que la sonde H S P 82 s'hybride avec un A R N de 2,6 kb pr6sent seulement dans les cellules ayant re~u un choc thermique, et (2) la sonde H P S 70 s'hybride avec des types d ' A R N de 2,5 kb, pr6sents seulement sur les A R N des cellules ayant re~u un choc thermique. Ces r6sultats montrent que le virus Mayaro n'est pas capable d'alt~rer la reprogrammation de l'expression des g~nes induite par le choc thermique, dans les cellules de A . albopictus. Mots-clds: A e d e s albopictus, A R N , Virus M a y a r o , Choc t h e r m i q u e ; Culture cellulaire, Synth~se de prot~ines, Expression g~nique.

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