HSC70 interactions with SV40 viral proteins differ between permissive ...

Cell Stress & Chaperones (2000) 5 (2), 132–138 Q Cell Stress Society International 2000 Article no. csac. 2000.188

HSC70 interactions with SV40 viral proteins differ between permissive and nonpermissive mammalian cells Ioannis Sainis, Charalambos Angelidis, Gerasimos N. Pagoulatos, and Ioannis Lazaridis Laboratory of General Biology, Medical School, University of Ioannina, GR 45110 Ioannina, Greece

Abstract SV40 belongs to a group of DNA tumor viruses which induce the expression of the 70 Kd heat shock proteins, but the meaning of this induction remains unclear. Investigating the role of hsc70 in the SV40 life cycle, we found that the protein translocates to the nucleus late in infection of permissive CV1 cells, in contrast to infected nonpermissive BALB/3T3 and NIH/3T3 cells in which hsc70 remains cytoplasmic. Moreover, the pattern of hsc70 nuclear staining was diffused and clearly distinguishable from that observed after heat shock. In addition hsc70 late in infection coimmunoprecipitated with the viral capsid protein VP1, suggesting a role in the process of viral packaging. Interactions of hsc70 with the early viral oncoprotein T antigen were observed only in nonpermissive cells, indicating that the binding of the above proteins is specific to cells that do not support viral propagation. Finally, treatment of permissive CV1 cells with interferon g, a known antiviral cytokine, resulted in hsc70 binding to T antigen. Our results suggest that the role of hsc70 in the process of SV40 infection is directly related to the ability of the host cells to support viral propagation and is clearly different between permissive and nonpermissive cell lines.

INTRODUCTION

The 70 Kd heat shock proteins (hsp70s) are cellular stress response proteins that are induced by a variety of physical or chemical stress stimuli (Lindquist and Craig 1988). They were shown to function as molecular chaperones facilitating the proper folding of nascent, denatured, or misfolded proteins (Hendrick and Hartl 1993; Becker and Craig 1994), and were implicated in a number of cellular processes such as protein translocation (Chirico et al 1988; Shi and Thomas 1992), acquisition of thermotolerance (Angelidis et al 1991; Li et al 1991), prevention of protein aggregation (Pelham 1986; Skowyra et al 1990), and degradation of short-lived proteins (Terlecky et al 1992). Induction of hsp70s was observed after infection of cells with a variety of DNA viruses including adenovirus (Nevins 1982), vaccinia virus (Sedger and Ruby 1994), cytomegalovirus (Santomenna and Colberg-Poley 1990), simian virus 40 (SV40) and polyomavirus (Khandjian and Turler 1983; Sainis et al 1994). The above induction was found to be mediated by the early viral gene products at Received 29 June 1999; Revised 18 November 1999; Accepted 18 November 1999. Correspondence to: I. Lazaridis, Tel: 130 651 97568; Fax: 130 651 97863; E-mail: [email protected].

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the level of transcription (Wu et al 1986; Taylor et al 1989) but its biological significance remains unclear. Physical association between the T antigen of SV40 and the cellular hsc70 was detected in mouse transformed cell lines (Sawai and Butel 1989), and the region of the oncoprotein responsible for the above interaction was recently identified as the HPDK/R motif (Kelley and Georgopoulos, 1997; Campbell et al 1997) which is present in all polyomavirus tumor antigens (residues 42–45 in SV40 T antigen). The same sequence is invariant in the J-domains of DnaJ proteins which act as co-chaperones stimulating the ATPase activity of specific hsp70 members (Kelley and Landry 1994; Tsai and Douglas 1996). These findings combined with the observation that the amino terminal domain of T antigen can functionally substitute the J domain of the DnaJ, led to the suggestion that some aspects of the viral life cycle are the results of the T antigen/hsc70 interactions (Kelley and Georgopoulos 1997). More specifically, these interactions were proposed to play a role in the processes of eukaryotic DNA replication (Campbell et al 1997), the rearrangement of multiprotein complexes (Srinivasan et al 1997), and the inactivation of Rb family proteins (Zalvide et al 1998). Complexes between hsc70 and capsid proteins have

Hsc70 interactions with SV40 viral proteins

been detected during cell infection with a number of viruses including adenovirus type 5 (Macejak and Luftig 1991), vaccinia virus (Jindal and Young 1992), poliovirus (Macejak and Sarnow 1992), and polyomavirus (Cripe et al 1995). The significance of the interactions was attributed to the active participation of hsc70 late in viral infection and it was proposed that hsc70 acting as a cellular chaperone facilitates virion assembly (Jindal and Young 1992; Cripe et al 1995). The evidence indicating a role for hsc70 in the viral life cycle is strengthened by the fact that T antigen and E1A expressing transformed cells produce elevated levels of hsp70s (Sainis et al 1994; Phillips et al 1991). Given that the oncoproteins induce cell entry into the S phase of the cell cycle, which coincides with the maximum accumulation of the cell cycle regulated expression of hsp70s (Sainis et al 1994; Milarski and Morimoto 1986), it is reasonable to speculate (Cripe et al 1995) that overexpression of hsp70s represents a strategy to induce chaperone proteins for use at later times in the virus life cycle. In this report we show that hsc70 binds to T antigen only in nonpermissive cells, which are unable to support productive infection. In addition we show that in permissive cells, hsc70 acts late in infection by forming complexes with the viral capsid protein VP1. Our results indicate that the role of hsc70 in the viral life cycle depends on the permissiveness of the infected cell.

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for 10 minutes. The cells were then permeabilized by incubation with 0.4% Triton X-100 in PBS for 5 minutes at room temperature and subsequently incubated with blocking buffer (20 mM Hepes pH 7.9, 0.25 M KCL, 1% BSA, 0.1% gelatin, 0.02% NaN3 and 0.1% Triton X-100) for 20 minutes at room temperature. Primary antibodies directed against hsc70 (SPA 815, StressGen, British Columbia, Canada) or T antigen (Pab 419, Oncogene Science, Uniondale, NY, USA) were diluted 1:25 in blocking buffer, added to the cells and the monolayers were incubated for 60 minutes. The cells were then washed 3 times (5 minutes each time) with blocking buffer, and incubated for 60 minutes with the appropriate fluorescein isothiocyanate-conjugated secondary antibody (Sigma, St Louis, MO, USA) diluted 1:50 in blocking buffer. Cells were then washed 3 times as described above and the cellular immunostaining was examined and photographed on Fujicolor 400 film with a Nikon Microphot-FXA immunofluorescence microscope. For double immunofluorescence the cells were incubated with a mixture of the primary antibodies and consequently exposed to a mixture of secondary antibodies (anti-rat-FITC and anti-mouse-CYE; Jackson ImmunoResearch, West Grove, PA, USA) under the experimental conditions described. Cellular immunostaining was examined on a Zeiss immunofluorescence microscope. Immunoprecipitation analysis

MATERIALS AND METHODS Cells and viruses

Monkey kidney CV1 cells and COS cells (CV1 cells stably transformed with SV40 and expressing T antigen) and mouse fibroblast BALB/3T3 and NIH/3T3 cells were grown as monolayers in DMEM medium supplemented with 10% fetal calf serum at 378C. Infections were carried out using wild type SV40 virus at a multiplicity of 50 pfu/cell in DMEM without serum. After 90 minutes of virus absorption, the cells were washed, supplemented with complete medium and allowed to grow. Interferon treatment of CV1 cells was achieved by the addition of 500 units/mL interferon g to the culture medium of infected cells immediately after virus absorption. The culture media supplemented with interferon g were changed after 24 hours of incubation. Thermal treatment was performed by placing the cells in a water bath set at 438C for 90 minutes. Immunofluorescence

Cell monolayers growing on glass coverslips were washed twice with phosphate buffer saline (PBS) and subsequently fixed in ethanol/acetone (1:1, v/v) at 2208C

Confluent cell monolayers were washed twice with PBS and lysed immediately by incubation on ice for 20 minutes in lysis buffer containing 20 mM Tris-HCL pH 8.0, 5 mM EDTA, 150 mM NaCL, 1% NP40, 10% glycerol (Campbell et al 1997) supplemented with 10 mg/mL leupeptin, 10 mg/mL pepstatin, 0.1 mM PMSF and 10 mg/ mL apyrase. The cell lysates were then cleared by centrifugation at 13 000 g for 5 minutes at 48C and incubated with 30 mL nonimmune serum for 2 hours with continuous rocking at 48C. Following incubation with 30 mL protein G Plus-agarose for 60 minutes the beads were collected by brief centrifugation and examined by Western blotting for nonspecific binding of Tag, hsc70 and VP1. Under the conditions described, we did not observe any such nonspecific binding. The supernatants (precleared lysates) were then incubated with the relevant antibodies as follows: In the case of T antigen 50 mL tissue culture supernatant containing the monoclonal Pab 419 anti-T antigen antibody were used, in the case of hsc70 5 mL of anti-hsc70 specific monoclonal antibody (SPA 815, StressGen) were used and in the case of VP1 10 mL of anti-VP1 rabbit polyclonal antibody were used. Incubation was carried out for at least 2 hours with continuous rocking at 48C, and then 30 mL of protein G Plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Cell Stress & Chaperones (2000) 5 (2), 132–138

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were added to the samples. Incubation was continued for 60 minutes and the beads were collected by brief centrifugation. The immune complexes were washed 4 times with lysis buffer and then released from the beads by boiling in 30 mL Laemmli sample buffer. Analysis of the immune complexes was performed by SDS-PAGE in 10% polyacrylamide gels. The resolved proteins were transferred to nylon membranes (Hybond-C, Amersham, Buckinghamshire, UK) which were then blocked for 2 hours with 5% nonfat dry milk in PBS. The immunoblots were incubated with either 1:2500 dilution of anti-hsc70 antibody, or 1:50 dilution of anti-T antigen antibody, or 1:100 dilution of anti-VP1 antibody for 2 hours at room temperature and the appropriate horseradish peroxidaseconjugated secondary antibody was subsequently added for an additional 60 minutes incubation. Finally the immunoblots were washed with TBS-T (Amersham) and developed using the enhanced chemiluminescence method (ECL, Amersham) according to the manufacturers. It should be noted that the amount of lysates used for all immunoprecipitations derived from 2.5 3 106 cells, compared to 104 cells used to prepare lysates for direct detection of Tag, hsc70, and VP1. RESULTS Differential localization of hsc70 in SV40 infected permissive and nonpermissive cells.

To investigate the intracellular localization of hsc70 in SV40-infected cells we decided to utilize cell systems that are both permissive and nonpermissive for viral propagation. When the CV1 cell system that is permissive for viral propagation was used, a dramatic nuclear translocation of hsc70 was observed late in infection (Fig 1B). The intracellular redistribution of hsc70 was found to be dependent on the progression of viral infection reaching its maximal nuclear accumulation 48 hours after virus absorption (data not shown). Moreover, the nuclear staining pattern of hsc70 late in infection of permissive cells was predominantly diffused or punctate in contrast to the nucleolar staining observed in the same cells after heat shock treatment (data not shown). An analogous translocation was detected in COS cells which although permissive to the wild type SV40 virus they constitutively express T antigen. In these cells hsc70 is predominantly cytoplasmic but moves to the nucleus after infection (data not shown). Based on the above findings, we concluded that hsc70 translocates to the nucleus of SV40 infected permissive cells as a consequence of cell infection and not as a result of the viral oncoprotein expression. However, when cell systems nonpermissive for viral propagation, such as BALB/3T3 and NIH/3T3, were used, hsc70 was found to remain cytoplasmic even after Cell Stress & Chaperones (2000) 5 (2), 132–138

48 hours from virus absorption (Fig 1E). Again T antigen expression did not affect the intracellular distribution of hsc70 which was determined solely by the cell permissiveness to viral propagation. Coimmunoprecipitation of hsc70 with the capsid protein VP1 during SV40 infection of permissive CV1 cells

Having determined that hsc70 translocates to the nucleus of infected permissive cells, we decided to investigate the possibility of hsc70 participation in the process of viral maturation, since it is known that SV40 assembles in the nuclei of infected cells. Cellular extracts of SV40 infected CV1 cells were prepared at 0 and 48 hours post-infection and subsequently immunoprecipitated with an anti-hsc70 specific antibody. The late viral capsid protein VP1 was found to be readily detectable in the immunoprecipitates, indicating its association with hsc70 (Fig 2A). Verification of the physical association between hsc70 and VP1 was achieved by immunoprecipitating the same extracts with an anti-VP1 antibody. Again hsc70 and VP1 were found to be present in the immunoprecipitate (Fig 2B), indicating that the 2 proteins, in addition to their intracellular colocalization, bind to each other late in infection of permissive CV1 cells. Association of hsc70 with T antigen in infected nonpermissive cells and interferon g treated permissive CV1 cells

Having shown that hsc70 remains cytoplasmic in SV40 infected nonpermissive cells (ie, BALB/3T3 and NIH/ 3T3 cells) and in stably transformed with T antigen CV1 cells (ie, COS cells) in contrast to infected permissive cells (ie, CV1 cells) in which hsc70 translocates to the nucleus, we decided to investigate the possible interactions between T antigen and hsc70. We prepared cellular extracts from SV40 infected CV1 and BALB/3T3 cells at 0 and 48 hours post-infection and subjected them to immunoprecipitation with an anti-T antigen specific antibody. The presence of hsc70 in the immunoprecipitates was subsequently determined by immunoblotting using an antihsc70 specific antibody. As shown in Figure 3A, hsc70 was found in association with T antigen only in the SV40infected nonpermissive BALB/3T3 cells. In contrast, no association between T antigen and hsc70 was detected in SV40-infected permissive CV1 cells (Fig 3A). In order to verify our findings we proceeded in immunoprecipitating the same extracts with an anti-hsc70-specific monoclonal antibody. Again the physical association of hsc70 with T antigen was readily detectable in infected nonpermissive BALB/3T3 cells but not in infected permissive CV1 cells (Fig 3B). The possibility that hsc70 binds to T antigen


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Fig. 1. Intracellular localization of hsc70 in SV40 infected permissive and nonpermissive cells. The intracellular localization of hsc70 was monitored in SV40 permissive CV1 cells (A) and nonpermissive BALB/3T3 cells (D). The cell lines were infected with SV40 and 48 hours post-infection were examined by double immunofluorescence for the presence of hsc70 and SV40 T antigen. The intracellular localization of hsc70 and T antigen was monitored in CV1 cells (B and C, respectively) and BALB/3T3 cells (E and F, respectively). The size bar corresponds to 20 mm.

only transiently in infected CV1 cells was also examined by immunoprecipitating under the same conditions cellular extracts prepared at various times ranging from 0 to 50 hours post-infection. In all cases examined we were unable to detect even traces of hsc70 in the anti-T antigen antibody immunoprecipitates (data not shown). Since our results indicated that the hsc70 binding to T antigen is dependent on the cell permissiveness to virus infection, we examined these associations in transformed and stably expressing T antigen permissive cells (COS cells). As shown in Figure 4, hsc70 was not found in the anti-T antigen antibody immunoprecipitates of COS cell extracts, and when the same extracts were immunoprecipitated with an anti-hsc70 specific antibody, no detectable amounts of T antigen were observed in the precipitate. Based on these findings and the findings presented in Figure 3, we concluded that hsc70 binds to T antigen

only in a cellular environment which does not support viral propagation. Inhibition, at least partial, of viral propagation is known to be achieved by the well-characterized cytokine interferon g (Pestka et al 1987), which was also found to stimulate the expression of hsp70s (Pine et al 1988). We therefore decided to investigate the effects of interferon g on the binding status of hsc70 with T antigen. Interferon g treated and mock treated CV1 cells were infected with SV40 virus and cellular extracts were prepared at 0 and 48 hours post-infection. The extracts were then subjected to immunoprecipitation with an anti-hsc70 specific antibody and the presence of T antigen in the precipitates was examined by immunoblotting. Although, as expected, no detectable amounts of hsc70 were found in the nontreated extracts of infected CV1 cells, T antigen was readily detectable in the anti-hsc70 immunoprecipitates Cell Stress & Chaperones (2000) 5 (2), 132–138

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Fig. 2. Association of hsc70 with SV40 capsid protein VP1 during virus infection. (A) Cellular extracts of SV40 infected CV1 cells were prepared and immunoprecipitated with an anti-hsc70 specific antibody. The presence of hsc70 and VP1 was monitored by immunoblotting total lysates at 0 hours post-infection (lane 1), total lysates at 48 hours post-infection (lane 2), immunoprecipitates of the lysates at 0 hours post-infection (lane 3) and immunoprecipitates of the lysates at 48 hours post-infection (lane 4). (B) The same cellular extracts as in (A) were immunoprecipitated with an anti-VP1 specific antibody and the presence of hsc70 and VP1 was monitored by immunoblotting total lysates at 0 hours post-infection (lane 1), total lysates at 48 hours post-infection (lane 2), immunoprecipitates of the lysates at 0 hours post-infection (lane 3) and immunoprecipitates of the lysates at 48 hours post-infection (lane 4). The prominent bands on lanes 3 and 4 focusing above VP1 correspond to the IgG heavy chain.

of the interferon g treated and infected CV1 cells (Fig 5). Therefore we concluded that interferon g induces the T antigen/hsc70 binding in treated CV1 cells, which otherwise are unable to support association of these proteins. DISCUSSION

The role of hsc70 in the viral life cycle remains controversial. Association of hsc70 with early viral oncoproteins has been reported on numerous occasions (Sawai and Butel 1989; Campbell et al 1997; White et al 1988; Pallas et al 1989; Sheng et al 1997) but the significance of these interactions is still obscure. In the case of SV40 T antigen, binding to cellular hsc70 was reported in transformed cells which do not support viral propagation (Sawai and Butel 1989; Campbell et al 1997). In this study we demonstrate that hsc70 indeed forms a complex with T antigen but only in infected nonpermissive cells and not in infected permissive cells. Our results clearly show that the interaction of hsc70 with T antigen is specific to transformed cells that do not permit SV40 viral maturation. Therefore, our findings indicate that the binding of T anCell Stress & Chaperones (2000) 5 (2), 132–138

Fig. 3. Association of hsc70 with T antigen in permissive and nonpermissive for viral propagation cells. (A) Cellular extracts of SV40 infected nonpermissive BALB/3T3 cells and permissive CV1 cells were prepared and immunoprecipitated with an anti-T antigen specific antibody. The presence of hsc70 and T antigen was monitored by immunoblotting total lysates at 0 hours post-infection (lane 1), total lysates at 48 hours post-infection (lane 2), immunoprecipitates of the lysates at 0 hours post-infection (lane 3) and immunoprecipitates of the lysates at 48 hours post-infection (lane 4). (B) The same cellular extracts as in (A) were immunoprecipitated with an antihsc70 specific antibody and the presence of hsc70 and T antigen was detected by immunoblotting total lysates at 0 hours post-infection (lane 1), total lysates at 48 hours post-infection (lane 2), immunoprecipitates of the lysates at 0 hours post-infection (lane 3) and immunoprecipitates of the lysates at 48 hours post-infection (lane 4).

tigen to hsc70 is somehow related to the transforming ability of the oncoprotein, since it is not observed in infected permissive cells destined to undertake the pathway of cell lysis. It was recently shown that the presence of the T antigen hsc70 binding site (HPDK/R tetrapeptide) is necessary in order for the oncoprotein to downregulate the tumor suppressor activities of the Rb family members (Zalvide et al 1998; Sheng et al 1997). It is therefore possible that the hsc70 binding to T antigen in nonpermissive cells mediates the oncoprotein function leading to cellular transformation. On the other hand, the inability of permissive cells to support formation of a complex between T antigen and hsc70 probably allows the Rb family proteins to exert their antitumor activities. According to our findings, the viral propagation in permissive cells seems to be mediated by the prevention of hsc70 binding to T antigen. Recent findings indicate that the existence of an intact hsc70-binding site on T antigen is necessary for efficient viral DNA replication (Campbell et al 1997). Our results


Fig. 4. Hsc70 does not bind to T antigen in T antigen expressing permissive COS cells. (A) Cellular extracts of SV40 infected COS cells were prepared and immunoprecipitated with an anti-T antigen specific antibody. The presence of hsc70 and T antigen was monitored by immunoblotting lysates at 0 hours post-infection (lane 1), lysates at 48 hours post-infection (lane 2), immunoprecipitates of the lysates at 0 hours post-infection (lane 3) and immunoprecipitates of the lysates at 48 hours post-infection (lane 4). (B) The same extracts as in (A) were immunoprecipitated with an anti-hsc70 specific antibody and the presence of hsc70 and T antigen was monitored by immunoblotting as described in (A).

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T antigen and hsc70 may play a critical role in inhibiting viral propagation. The observed interferon g stimulation of complex formation between hsc70 and T antigen in permissive cells strengthens our hypothesis that this binding is related to an inhibitory pathway for viral maturation. It is possible that the observed induction of T antigen binding to hsc70 in interferon g treated infected permissive cells represents a step in the viral strategy to overcome the cellular antiviral defense mechanism. Nuclear translocation of hsc70 late in infection of permissive cells has been already reported for adenovirus and polyomavirus (White et al 1988; Cripe et al 1995). In this study we show that SV40 infection of permissive cells also induces nuclear translocation of hsc70 in a manner almost identical to polyomavirus. Our findings that hsc70 translocates to the nucleus as a result of virus infection coupled with the nonnucleolar distribution of the protein indicate that the function of hsc70 in infected permissive cells is clearly different from the protective function exerted by the protein when the cells are submitted to heat treatment. Indeed the observed binding of hsc70 to the late viral capsid protein VP1 suggests that hsc70 contributes actively in the SV40 viral maturation. Therefore we suggest that, in the case of permissive cells, hsc70 is recruited late in infection and by translocating to the nucleus it participates in the virus assembly. In conclusion, our results indicate that the permissiveness of the cellular environment to SV40 propagation determines the interactions of hsc70 with the viral proteins. ACKNOWLEDGMENTS

Fig. 5. Interferon g induces complex formation between T antigen and hsc70 in SV40 permissive cells. Cellular extracts of SV40 infected CV1 cells treated (1 IFN) or nontreated (2 IFN) with interferon g were prepared and subjected to immunoprecipitation with an anti-hsc70 specific antibody as described in Materials and Methods. The presence of T antigen and hsc70 was monitored by immunoblotting lysates at 0 hours post-infection (lane 1), 48 hours postinfection (lane 2), immunoprecipitates of the 0 hours post-infection lysates (lane 3) and immunoprecipitates of the 48 hours post-infection lysates (lane 4).

suggest that although the intact T antigen hsc70 binding site may be necessary for viral DNA replication, the actual binding of the 2 molecules is promoted only in cells unable to support viral propagation. Therefore, we believe that the permissiveness of the cellular environment is the determining factor in regulating whether or not hsc70 will bind to T antigen. The presence of an intact T antigen is obviously necessary for viral replication (Campbell et al 1997), but our results indicate that in nonpermissive cells the observed complex formation between

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