JOURNAL OF VIROLOGY, May 1990, p. 2033-2040 0022-538X/90/052033-08$02.00/0 Copyright ) 1990, American Society for Microbiology
Vol. 64, No. 5
Induction of Cellular hsp7O Expression by Human Cytomegalovirus LINDA D. SANTOMENNA AND ANAMARIS M. COLBERG-POLEY* Central Research & Development, E. I. du Pont de Nemours & Co., Inc., Experimental Station, P.O. Box 80328, Wilmington, Delaware 19880-0328 Received 24 October 1989/Accepted 8 January 1990
Expression of the celular heat shock protein 70 gene (hsp70) is transiently induced by human cytomegalovirus (HCMV) infection of permissive human diploid fibroblasts. Induction of the cellular heat shock response during critical times of infection had previously been reported to alter the growth of HCMV in vitro. Thus, a potential interaction between heat shock proteins and HCMV expression was indicated. HCMV dramatically increased expression of hsp70 RNA within 8 h of infection. hsp70 RNA remained elevated at 24 and 48 h postinfection and decreased to low levels of 72 h postinfection. Induction of HSP70 protein occurred more slowly; inducible HSP70 protein encoded by this RNA increased within 16 h postinfection and continued to increase throughout infection until 72 h postinfection, when the highest abundance of inducible HSP70 protein was observed. Cells that received both heat (43°C for 70 min) treatment and HCMV infection expressed hsp70 RNA to levels above the sum of levels present in cells given either treatment alone. Furthermore, hsp70 RNA induction occurred earlier and remained elevated longer than in cells infected with HCMV alone or in cells treated with heat alone, respectively. Nevertheless, the pattern of HCMV immediate-early transcript expression at 2, 4, and 6 h postinfection appeared to be unchanged by this prior heat treatment. Our results suggest that heat shock treatment and HCMV infection can act additively in stimulating hsp70 RNA expression. The previously reported stimulation of HCMV growth in vitro following the heat shock response apparently does not result from alterations in the steady-state expression of HCMV immediate-early transcripts. members of the hsp70 family, including promotion of protein folding (35), acquisition of thermotolerance (31), participation in the recovery of nucleoli after heat stress (35), initiation of bacteriophage lambda DNA replication (28), degradation of abnormal proteins in E. coli (35, 42), and facilitation of transport of proteins across membranes (4, 10). Induction of the cellular heat shock response during critical times of infection appears to alter the HCMV growth cycle, which is protracted in vitro. HCMV has a narrow host range; it grows slowly but well only in human diploid fibroblasts. Mild heat shock treatment (10 min, 44°C) of permissive human embryo fibroblasts immediately before or 24 h after HCMV infection shortened the viral eclipse period, enhanced viral replication, and increased the number of cells that expressed HCMV early and late antigens (53). These effects were not seen, however, when HCMV-infected cells were heat treated at later times of infection. A potential target for regulation of HCMV expression during the heat shock response was proposed by Geelen and co-workers (13), who found that a transcriptionally silent HCMV major immediate-early (IE) locus which was integrated into nonpermissive Rat-9G cells was activated following heat shock treatment. Homology between the consensus sequence of the heat shock response element and the HCMV major IE enhancer was also noted. These studies suggested a potential interaction between heat shock proteins and HCMV expression and led us to examine in more detail the induction of cellular hsp70 by HCMV during permissive infection. Previously, we had observed that HCMV increases the expression of several growth-regulated genes during permissive infection of human diploid fibroblasts (6). In particular, we had noted increased expression of hsp70, ornithine decarboxylase gene (odc), thymidine kinase gene, and brain creatine kinase gene transcripts but not of 1-actin or metallothionein IIA gene transcripts. After HCMV infection,
Expression of the heat shock protein 70 gene (hsp7O), the major heat shock gene, is under complex regulation at both the transcriptional and translational levels (reviewed in references 2, 7, and 29). During the heat shock response, expression of hsp70 and other heat shock genes is selectively enhanced while that of most cellular genes is inhibited. The transcription rate of hsp70 is rapidly and transiently increased at elevated temperatures, and the resulting transcripts show increased stability. Translation of hsp70 RNA proceeds with equal efficiency under normal and heat shock conditions, whereas translation of other cellular RNAs is repressed (44). A wide variety of stimuli, including heat shock (2, 7, 29), heavy metals (50), growth factors (51), and infection with some viruses, such as adenovirus (21, 33), simian virus 40 (23), polyomavirus (23), herpes simplex virus type 2 (HSV-2; 26), and human cytomegalovirus (HCMV; 6), results in increased hsp70 expression. Expression of the human hsp70 gene is also tightly regulated during the cell cycle, increasing in expression upon entry of cells into the S phase (30). hsp70 is a highly conserved gene; human HSP70 is 73% homologous to Drosophila HSP70 and 47% homologous to Escherichia coli DnaK (18). HSP70 proteins are essential for viability of the yeast Saccharomyces cerevisiae, even at normal temperatures (29, 40), and there is mounting evidence that heat shock proteins play key roles in the viability of mammalian cells following heat treatment. Microinjection of rat embryo fibroblasts with antibodies to HSP70 demonstrated that functional HSP70 is required for survival of these cells during and after thermal treatment (36). Amplification of integrated copies of the hsp70 control region fused to the gene for dihydrofolate reductase resulted in decreased expression of HSP70 and increased thermosensitivity of the modified cells (19). Several roles have been proposed for *
Corresponding author. 2033
2034
J. VIROL.
SANTOMENNA AND COLBERG-POLEY L
u.s
f I 0.1
0
0.2
0.3
O.S
0.4
0
0
0
0.6
0.7
0.8
0.9
1.0
mu
pp65, 71 probe
0.24 mu
IE1,2
0.87 mu
IE2a
IE2b
IEI
k
__ _
3.4
0
0.89
23
-
0.74
1.7
-
0.74
-
0.58
-
0.44
1.9
-40..
.-_ _
-_
IA
1.7
1.7 2.3
-.
1.65
Vol. 64, No. 5
Induction of Cellular hsp7O Expression by Human Cytomegalovirus LINDA D. SANTOMENNA AND ANAMARIS M. COLBERG-POLEY* Central Research & Development, E. I. du Pont de Nemours & Co., Inc., Experimental Station, P.O. Box 80328, Wilmington, Delaware 19880-0328 Received 24 October 1989/Accepted 8 January 1990
Expression of the celular heat shock protein 70 gene (hsp70) is transiently induced by human cytomegalovirus (HCMV) infection of permissive human diploid fibroblasts. Induction of the cellular heat shock response during critical times of infection had previously been reported to alter the growth of HCMV in vitro. Thus, a potential interaction between heat shock proteins and HCMV expression was indicated. HCMV dramatically increased expression of hsp70 RNA within 8 h of infection. hsp70 RNA remained elevated at 24 and 48 h postinfection and decreased to low levels of 72 h postinfection. Induction of HSP70 protein occurred more slowly; inducible HSP70 protein encoded by this RNA increased within 16 h postinfection and continued to increase throughout infection until 72 h postinfection, when the highest abundance of inducible HSP70 protein was observed. Cells that received both heat (43°C for 70 min) treatment and HCMV infection expressed hsp70 RNA to levels above the sum of levels present in cells given either treatment alone. Furthermore, hsp70 RNA induction occurred earlier and remained elevated longer than in cells infected with HCMV alone or in cells treated with heat alone, respectively. Nevertheless, the pattern of HCMV immediate-early transcript expression at 2, 4, and 6 h postinfection appeared to be unchanged by this prior heat treatment. Our results suggest that heat shock treatment and HCMV infection can act additively in stimulating hsp70 RNA expression. The previously reported stimulation of HCMV growth in vitro following the heat shock response apparently does not result from alterations in the steady-state expression of HCMV immediate-early transcripts. members of the hsp70 family, including promotion of protein folding (35), acquisition of thermotolerance (31), participation in the recovery of nucleoli after heat stress (35), initiation of bacteriophage lambda DNA replication (28), degradation of abnormal proteins in E. coli (35, 42), and facilitation of transport of proteins across membranes (4, 10). Induction of the cellular heat shock response during critical times of infection appears to alter the HCMV growth cycle, which is protracted in vitro. HCMV has a narrow host range; it grows slowly but well only in human diploid fibroblasts. Mild heat shock treatment (10 min, 44°C) of permissive human embryo fibroblasts immediately before or 24 h after HCMV infection shortened the viral eclipse period, enhanced viral replication, and increased the number of cells that expressed HCMV early and late antigens (53). These effects were not seen, however, when HCMV-infected cells were heat treated at later times of infection. A potential target for regulation of HCMV expression during the heat shock response was proposed by Geelen and co-workers (13), who found that a transcriptionally silent HCMV major immediate-early (IE) locus which was integrated into nonpermissive Rat-9G cells was activated following heat shock treatment. Homology between the consensus sequence of the heat shock response element and the HCMV major IE enhancer was also noted. These studies suggested a potential interaction between heat shock proteins and HCMV expression and led us to examine in more detail the induction of cellular hsp70 by HCMV during permissive infection. Previously, we had observed that HCMV increases the expression of several growth-regulated genes during permissive infection of human diploid fibroblasts (6). In particular, we had noted increased expression of hsp70, ornithine decarboxylase gene (odc), thymidine kinase gene, and brain creatine kinase gene transcripts but not of 1-actin or metallothionein IIA gene transcripts. After HCMV infection,
Expression of the heat shock protein 70 gene (hsp7O), the major heat shock gene, is under complex regulation at both the transcriptional and translational levels (reviewed in references 2, 7, and 29). During the heat shock response, expression of hsp70 and other heat shock genes is selectively enhanced while that of most cellular genes is inhibited. The transcription rate of hsp70 is rapidly and transiently increased at elevated temperatures, and the resulting transcripts show increased stability. Translation of hsp70 RNA proceeds with equal efficiency under normal and heat shock conditions, whereas translation of other cellular RNAs is repressed (44). A wide variety of stimuli, including heat shock (2, 7, 29), heavy metals (50), growth factors (51), and infection with some viruses, such as adenovirus (21, 33), simian virus 40 (23), polyomavirus (23), herpes simplex virus type 2 (HSV-2; 26), and human cytomegalovirus (HCMV; 6), results in increased hsp70 expression. Expression of the human hsp70 gene is also tightly regulated during the cell cycle, increasing in expression upon entry of cells into the S phase (30). hsp70 is a highly conserved gene; human HSP70 is 73% homologous to Drosophila HSP70 and 47% homologous to Escherichia coli DnaK (18). HSP70 proteins are essential for viability of the yeast Saccharomyces cerevisiae, even at normal temperatures (29, 40), and there is mounting evidence that heat shock proteins play key roles in the viability of mammalian cells following heat treatment. Microinjection of rat embryo fibroblasts with antibodies to HSP70 demonstrated that functional HSP70 is required for survival of these cells during and after thermal treatment (36). Amplification of integrated copies of the hsp70 control region fused to the gene for dihydrofolate reductase resulted in decreased expression of HSP70 and increased thermosensitivity of the modified cells (19). Several roles have been proposed for *
Corresponding author. 2033
2034
J. VIROL.
SANTOMENNA AND COLBERG-POLEY L
u.s
f I 0.1
0
0.2
0.3
O.S
0.4
0
0
0
0.6
0.7
0.8
0.9
1.0
mu
pp65, 71 probe
0.24 mu
IE1,2
0.87 mu
IE2a
IE2b
IEI
k
__ _
3.4
0
0.89
23
-
0.74
1.7
-
0.74
-
0.58
-
0.44
1.9
-40..
.-_ _
-_
IA
1.7
1.7 2.3
-.
1.65
