Hsp27 and Hsp70) Expression in CD4/ Lymphocytic Cells

VIROLOGY 233, 364–373 (1997) ARTICLE NO. VY978618

Modulation of Stress Protein (hsp27 and hsp70) Expression in CD4/ Lymphocytic Cells

View metadata, citationFollowing and similar papers Acute at core.ac.uk Infection with Human Immunodeficiency Virus Type-1 brought to you by CORE provided by Elsevier - Publisher Connector Zev Wainberg, Maureen Oliveira, Sharon Lerner, Yuzhen Tao, and Bluma G. Brenner*,†,1

*McGill AIDS Centre, Lady Davis Institute, Jewish General Hospital, and †Department of Experimental Surgery, McGill University, Montreal, Quebec, Canada Received December 5, 1996; returned to author for revision January 13, 1997; accepted May 5, 1997

This study was designed to assess the impact of acute human immunodeficiency virus (HIV-1) infection on host intracellular expression of the heat shock family of stress proteins (hsps). Experimental conditions were established wherein CD4/ lymphocytic cell lines undergo a synchronous HIV-1 infection cycle. During the early phase of infection, HIV-1 mRNA expression was restricted to singly and multiply spliced subspecies, with no genomic viral RNA present until 30 hr following infection. In contrast, hsp27 and hsp70 mRNA transcription appeared as early as 3–8 hr following viral infection. No corresponding induction was observed in mock-infected cells. Notably, hsp27 and hsp70 mRNA transcripts were down-regulated by 24 hr, concomitant to the first appearance of full-length genomic HIV-1 mRNA. Hsp27 and hsp70 mRNA transcripts reemerged at end stages of the viral replicative cycle, coincident to virion release and CD4 cell death. Similarly, a transient induction of de novo hsp27 protein expression occurred between 12 and 24 hr. The generated hsp27 stress response was viral dose-related, suppressed by heat-inactivation of virus, and abrogated by neutralizing antibodies to HIV-1. Acute infection did not alter levels of hsp60, hsp70, and hsp90 protein synthesis. However, two-dimensional Western blot analysis did show the appearance of novel hsp70 homologues between 6 and 24 hr following infection. CEM.NKR, Jurkat, H9, and MT-2 cells showed similar patterns of viral-associated modulation of host hsp27 and hsp70 protein and RNA expression. Thus, host hsp27 and hsp70 stress pathways are selectively implicated in the HIV- 1 viral life cycle. ᭧ 1997 Academic Press

INTRODUCTION inducible hsp subspecies (up to 100 subspecies per cell) play diverse roles in cellular function. Under physiologi- Infection with human immunodeficiency virus type 1 cal conditions, constitutively synthesized hsp cognates (HIV-1), the etiologic agent of acquired immunodeficiency act as molecular chaperones modulating protein–protein syndrome (AIDS), results in protracted latency periods interactions. Hsps regulate the folding, assembly, traf- between viral exposure and disease manifestation. Viral ficking, function, and degradation of oligomeric protein persistence clearly plays a major role in progressive complexes (Craig et al., 1994; Gething and Sambrook, depletion of CD4-expressing cells. Nevertheless, host 1992). Cellular stress increases expression of inducible CD4 cells show dynamic capacities to contain, block, or hsp subspecies by complex regulatory controls at tran- fuel HIV-1 viral replication depending on viral phenotype, scriptional and translational levels (Arrigo and Landry, cell lineage, and/or biological situation (Butera et al., 1994; Belka et al., 1994; Morimoto, 1993). 1994; Ho et al., 1995; McCune, 1995; Wei et al., 1995). Cumulative findings suggest an intermediary role of Investigating the interactions of the virus and the host stress proteins in viral replication. Acute infection of cells cell machinery is important to establish how cellular pro- with a variety of RNA and DNA viruses result in induction teins regulate the viral replicative cycle. Host cell factors in stress protein expression (Andrews et al., 1995; Jindal may elicit defensive responses that disrupt HIV-1 viral and Malkovsky, 1994; Santoro, 1994). The transient asso- replication; conversely, HIV-1 may utilize cellular path- ciation of hsps with viral proteins and the presence of ways to maximize its own innate survival and infectivity. hsps in virions have been described (Hu and Seeger, Host intracellular responses to metabolic or environ- 1996; Nagata et al., 1992; Santoro, 1994). In this regard, mental stresses are mediated by the heat shock family the chaperonin, cyclophilin has been shown to be incor- of proteins (hsp27, hsp60, hsp70, hsp90) (DeNagel and porated into HIV-1 virions and is required for HIV-1 viru- Pierce, 1993; Morimoto, 1991). Constitutive and stress- lence (Bratten et al., 1996; Franke et al., 1994 and 1995; Thali, et al., 1994). Our previous studies demonstrated differential 2- to 1 To whom correspondence and reprint requests should be ad- dressed. Lady Davis Institute, Jewish General Hospital, 3755 Cote Ste. 15-fold increases in heat-inducible hsp27 production in Catherine Road, Montreal, Quebec, Canada. Fax: (514) 340-7537. mdbl chronically HIV-infected monocytic and lymphocytic CD4 @musica.mcgill.ca. cell lines as compared to their uninfected counterparts

0042-6822/97 $25.00 364 Copyright ᭧ 1997 by Academic Press All rights of reproduction in any form reserved.

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(Brenner et al., 1995). The goal of the present study was sis as previously described (Gryllis et al., 1992). Intracel- to investigate variations in patterns of stress protein ex- lular HIV-1 p24 was quantified by flow cytometric analysis pression during acute HIV-1 infection using Northern and using modifications to described methods (Walker et al., Western analysis. We were able to synchronously infect 1985). Briefly, cells (2 1 106) were incubated for 5 min lymphocytic cell lines with HIV-1 and show that hsp27 in ice-cold 50% ethanol in RPMI-1640. The cells were and hsp70 mRNA and protein subspecies are selectively washed three times in RPMI and exposed for 30 min to modulated at early and end stages of the viral replicative 1:20 anti-viral p24 monoclonal antibodies (clone 39/5.4A, cycle. Cellular Products Inc., Buffalo, NY). The cells were subse- quently washed three times and incubated for 30 min MATERIALS AND METHODS with a 1:20 dilution of FITC-labeled goat anti-mouse Ig (Cedarlane Labs. Ltd., Hornby, Ontario, Canada). Follow- Cells and viruses ing three washes, cells were subjected to cytofluorome- CEM.NKR, H9, Jurkat, and MT2 cells were obtained tric analysis using an EPICs analyzer (Coulter Electron- from the American Type Culture Collection (Rockville, ics, Burlington, Ontario). MD) and were passaged twice weekly in complete RPMI- 1640 medium containing 10% FCS, 2 mM glutamine, 100 Western blot analysis of stress and viral protein U/ml penicillin, 100 mg/ml streptomycin, and 10 mM expression HEPES. The IIIB isolate of HIV-1 was kindly supplied by Dr. R. C. Gallo (National Institute of Health, Bethesda, Five-milliliter aliquots of cell cultures collected at des- MD) and used to chronically infect H9 cells (Brenner et ignated time intervals were harvested and counted. Cell al., 1995). viabilities were confirmed by trypan blue staining. Cyto- plasmic cell extracts were prepared by lysing 5 1 106 Viral time course cultures cells in 100 ml lysis buffer containing 20 mM Tris-buffered saline (TBS, pH 8.0), 2% sodium dodecyl sulfate (SDS), 2 The 50% tissue culture infective dose (TCID ) of IIIb 50 mM ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, viral stocks on MT2 cells were adjusted to 1–2 1 106 100 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml aproti- TCID /ml. Aliquots of cell cultures (5 1 106 cells/ml in 50 nin, and 1 mg/ml leucopeptin. Protein concentrations complete growth medium) were centrifuged and cell pel- were determined using the Bio-Rad D protein assay kit lets were separately treated, as indicated: (i) resus- c (Hercules, CA). Cell extracts were analyzed for cyto- pended in 1 ml infectious virus stock at a multiplicity of plasmic stress and HIV-1 viral protein expression by infection (m.o.i.) of 0.2 (i.e., 106 TCID /5 106 cells); (ii) 50 1 Western blot analysis. resuspended in the same amount of virus stock inacti- Briefly, polypeptides present in extract samples (25 vated for 1 hr at 56 ; (iii) mock-infected by resuspending Њ mg) of various cytoplasmic lysates were separated by in 1 ml of conditioned clarified medium from uninfected SDS–polyacrylamide gel electrophoresis in 12% acryl- H9 cells or ultracentrifuged (35,000 rpm for 1 hr in a amide gels as previously described (Brenner et al., 1995). Beckman Ti45 rotor) medium from infected H9 cells; (iv) Following electrophoresis, gels were transferred to nitro- resuspended in serial dilutions of virus stock (m.o.i. Å cellulose membranes, blocked with 5% milk in TBS, con- 0.2, 0.067, and 0.02); (v) resuspended in viral stocks that taining 0.05% Tween 20, and probed for various hsps had been preincubated with high titer HIV-1 neutralizing using hsp27, hsp60, hsp70, and hsp90 class-specific antibodies for 1 hr at 4 ; (vi) incubated with 20 mg of anti- Њ monoclonal antibodies (SPA-800, SPA-806, SPA-820, and human CD4 (Cedarlane Labs., Hornby, Ontario, Canada). SPA-830, StressGen Biotechnologies Corp., Victoria, Cells were incubated for 3 or 5 hr at 37 and then washed Њ B.C., Canada) and HIV-1 using anti-viral p24 monoclonal twice by low speed centrifugation, resuspended to the antibodies (clone 39/5.4A, Cellular Products Inc, Buffalo, original 5 105 cells/ml aliquots in fresh growth medium, 1 NY). The enhanced chemiluminescent (ECL) detection and incubated at 37 for specified time intervals (3 hr, 5 Њ procedure was used to detect stress and viral protein hr, 8 hr, 12 hr, 24 hr, 48 hr, 5, 6, 7, 9, 10, 12, or 14 days expression on radiographic film (Amersham Canada Ltd., after infection). Cells were routinely split weekly on Days Oakville, Ontario, Canada). Levels of enhanced chemilu- 4 and 7. Samples of culture harvested at designated minescence were quantified using the Bio-Rad GS-363 intervals were assayed for the release of extracellular molecular PhosphorImager (Bio-Rad Inc., Mississauga, viral particles by reverse transcriptase and p24 viral anti- Ontario, Canada) using a GS-250 chemoluminescent gen capture assays (Boulerice et al., 1990). screen. In two-dimensional Western blot analysis, nondena- Flow cytometric analysis for cell surface CD4 and tured protein extracts were separated according to pI intracellular virus following infection (pH 3–10) and then according to molecular weight using Levels of cell surface CD4 in acutely and chronically precast gel kits and manufacturer’s protocols (Novex infected cells were determined by cytofluorometric analy- Technology, San Diego, CA).

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FIG. 1. Flow cytometric analysis of intracellular HIV-1 viral p24 expression. (A) cells were mock-infected with spent media of uninfected H9 cells. (B) Cells were isolated 48 hr following acute exposure to HIV-1. (C) Cells isolated from chronically infected H9 cells.

Northern blot analysis of stress and viral protein posed to viral concentrates for 3 or 5 hr after which expression unbound virus was washed away. Flow cytometric analysis of intracellular viral p24-associated fluorescence was For Northern blot analysis, total RNA was isolated from 6 used to determine the proportion of cells that were in- 5to10110 cells using TRIzol total RNA isolation kits fected by 48 hr following viral exposure. Mock-infected (Life Technologies, Burlington, Ontario, Canada). The cells showed less than 5% background fluorescence (Fig. quality and intactness of RNA were verified by ethidium 1A). In contrast, significant viral p24 immunofluorescence bromide staining. Samples of 10 g total RNA were elec- m was present in ú95% of acutely infected cells by 48 hr trophoresed on 1.2% agarose-formaldehyde gels and (Fig. 1B). In addition, ú50% of these cells showed down- then transferred onto nitrocellulose membranes regulated cell surface CD4 while õ10% expressed cell (Schleicher & Schuell, Keene, NH). RNA blots were pre- surface virus (unpublished results). hybridized in 50% formamide, 51 SSPE (11 SSPE Å 180 Northern blot analysis was utilized to determine levels mM NaCl, 20 mM sodium phosphate, 1 mM EDTA, pH of HIV-1 viral mRNA transcription at different times fol- 8.0), 51 Denhardt’s solution, 0.2% SDS, and 100 mg/ml lowing acute infection of H9 cells. As early as 3 hr follow- salmon sperm DNA at 48Њ for 2 hr. Hybridizations were ing infection, singly spliced 2-kb and multiply spliced 4.3- performed using hsp27-, hsp60-, hsp70-, hsp89-specific kb viral mRNA transcripts were detected intracellularly cDNA probes (Stress Gen Biotechnologies Corp., Victo- (Fig. 2). However, whole 9.2-kb genomic virus transcripts ria, B.C., Canada), HIV-1- and actin-specific probes were not apparent until 24 to 30 hr following infection (kindly provided by Mark Wainberg at our Institute), which 32 (Fig. 2). It has been reported that a temporal appearance were [ P]-labeled using nick translation labeling kits of HIV mRNA subspecies suggests synchronous, single (Boehringer Mannheim, GmbH, Germany). At the end of cycle, or nonproductive viral infection (Bartz et al., 1996; the hybridization periods, the blots were washed up to Kim et al., 1989; Pomerantz et al., 1990). Viral transcription a stringency of 0.11 SSPE, 0.1% SDS at 48Њ. The blots reached approximate levels of expression observed in were autoradiogaphed at 070Њ using Fuji scientific im- chronically infected cell lines by 5 to 7 days (Fig. 2B). aging film and levels of RNA expression were quantified 32 Thus, flow cytometric analysis and Northern blot analy- in a phosphorimager using a Bio-Rad GS-250 P-detec- sis show that we have developed an effective system for tion screen. monitoring stress protein expression during a relatively synchronous infection of all T cells. RESULTS Acute infection of CD4 lymphocytic cell lines Northern blot analysis of stress protein mRNA expression in lymphocytic cells following HIV-1 This study was designed to monitor quantitative and infection qualitative changes in stress protein expression in CD4/ lymphocytic cells following acute exposure to HIV-1. HIV- Northern blot analysis was used to identify variations IIIb was isolated from chronically infected H9 cells of in levels of stress protein mRNA expression during acute which ú95% were infected and õ5% expressed surface infection. Total RNA was isolated from virally infected CD4 (Fig. 1C, unpublished results). Viral stocks were nor- and mock-infected H9 cells. Steady state levels of hsp27 6 6 malized so as to infect cells at 10 TCID50/5 1 10 cells and hsp70 mRNA transcripts were barely detectable in (m.o.i. Å 0.2), unless otherwise indicated. Cells were ex- uninfected H9 cells (Fig. 3, CON lanes). These findings

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FIG. 2. (A) Northern blot analysis of total RNA extracted from H9 cells at various times following acute infection (m.o.i. Å 0.34) and probed with 32P-labeled HIV-1 cDNA. (B) Phosphorimager analysis of various HIV mRNA subspecies. Values are expressed as a percentage of the HIV 9.2-kb intensity (100%) observed for chronically infected H9 cells. are consistent with other published reports (Hickey et coincident to maximal levels of HIV-1 RNA expression, al., 1986; Jindal and Young, 1992; Theodorakis and Mori- and the onset of massive CD4 cytolysis (Fig. 3B). moto, 1987). Increases in hsp27 mRNA transcripts were In contrast, there was abundant constitutive expres- observed 3 hr following infection, attaining peak levels sion of hsp60, hsp90, and actin mRNA transcripts in H9 by 8 hr, and rapidly declining to steady state levels by cells. Levels of transcription of these latter host proteins 24 hr (Figs. 3A and 3B). Maximal levels of hsp27 mRNA remain unchanged during infection (Fig. 3A, unpublished were 6.4-fold higher than basal levels. There was a lesser results). These findings indicate that acute HIV-1 infec- induction of Hsp70 mRNA that peaked at 5 hr and disap- tion leads to the selective modulation of hsp27 and hsp70 peared by 8 hr following infection with a weak secondary mRNA transcription. peak between 24 and 48 hr. (Figs. 3A and 3B). Transcrip- To ensure that the induction of stress protein mRNA tional modulation of hsp27 and hsp70 occurred prior to was viral-associated and not due to cellular stress, a the first appearance of genomic HIV-1 viral transcripts mock infection was carried out in parallel using the same that appeared 30 hr following infection (Fig. 3B). virus stock depleted of virus by ultracentrifugation. Substantial reemergence of hsp27 and hsp70 mRNA Whereas the original virus stock (m.o.i. Å 0.34) induced transcription occurred 5 and 7 days following infection, hsp27 and hsp70 mRNA transcription, the parallel mock

FIG. 3. (A) Northern blot analysis of total RNA extracted from H9 cells at various times following acute infection and probed with 32P-labeled hsp27, hsp70, and hsp89b cDNA. The negative (CON) and positive (HS) control represent RNA extracts of uninfected H9 cells and heat shocked chronically infected H9 cells. (B) Relative levels of hsp27 and hsp70 mRNA transcripts in H9 cells as determined by phosphorimager analysis of signal intensities. The open arrow shows the time point of first detection of the 9.2-kb viral mRNA subspecies. The closed arrow shows the time point of a 50% drop in H9 survival relative to mock-infected cells.

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FIG. 4. Northern blot analysis of total RNA extracted from mock- infected (m.o.i. Å 0.005) and virally-infected (m.o.i. Å 0.34) H9 cells at various times following infection probed with 32P-labeled hsp27 and hsp70 cDNA. In this experiment, mock infection was performed with the viral stock that was ultracentrifuged to deplete HIV-1. infection with ultracentrifuged stock (m.o.i. õ 0.005) failed to yield hsp27 and hsp70 mRNA induction (Fig. 4). These findings indicate that HIV-1 infection leads to the selective modulation of hsp27 and hsp70 transcription. Levels of mRNA transcripts were high at early latent stages, declined during productive stages, and increased at lytic end stages of the viral replicative cycle. FIG. 5. Western blot chemoluminescent detection of hsp27 and hsp70 expression. Purified hsp27 and hsp70 protein standards were Stress protein expression in lymphocytic cells probed with hsp-specific antibodies and detection was performed us- following HIV-1 infection ing the ECL chemoluminescent kit. (A) Immunoblots exposed to radiographic film for 1 min. (B) Phosphorimager quantification of chemolum- Intracellular stress protein (hsp27, hsp60, hsp70, and inescent intensity of generated hsp signals. Lines of best fit were com- hsp90) and intracellular HIV-1 viral (p24- and p55-gag puter-generated (r Å 0.996 for both lines). precursor) protein expression were monitored by West- ern blot analysis using chemoluminescent detection pro- early during primary HIV infection. There were 6.4 { 1.7 cedures. To ensure that chemoluminescence would pro- (mean { SEM)-fold increases in hsp27 peak signals in vide accurate quantitative assessment of intracellular infected as compared to mock-infected cells. In all exper- stress protein levels, serial dilutions of hsp27 and hsp70 iments, peak hsp27 inductions were rapidly down-regu- standards were subjected to Western blot analysis. Lin- ear chemoluminescent signals were generated between 5 and 100 ng of hsp per well (Fig. 5). Viral-associated changes in stress protein expression were initially monitored in CEM.NKR cells since this cell line lacks constitutive hsp27 cognates. As depicted in Fig. 6, de novo hsp27 protein synthesis occurred following HIV-1 infection. No such response was observed in mock-infected cells. In contrast, levels of hsp60, hsp70, and hsp90, remained constant throughout infection (Fig. 6). Maximal induction of hsp27 protein synthesis occurred 24 hr following infection prior to viral synthesis, as indicated by the absence of viral p55-gag precursor protein (Fig. 7). The presence of viral p24-env without p55-gag between 5 and 24 hr represents intracellular virion trapping (Fig. 7). There was a down-regulation of hsp27 at 48 hr, associated with initiation of viral synthesis (Fig. 7). FIG. 6. Western blot analysis of hsp27, hsp60, hsp 70, and hsp90 Summary data of seven independent experiments protein expression during the course of lytic HIV infection. CEM cells were mock-infected (no infection) or acutely infected with HIVIIIb (m.o.i. show variations in CD4 cell survival, intracellular hsp27, Å 0.2). Cellular extracts were isolated at designated time intervals and viral p55-gag protein at various times following initial and probed with relevant hsp subspecies. Chronically infected CEM HIV-1 exposure (Fig. 8). Hsp27 levels were modulated extracts are presented for comparative purposes.

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FIG. 7. Western blot analysis showing relative levels of hsp27 and viral expression during the course of infection with virus (m.o.i. Å 0.2). Cellular extracts were isolated at designated time intervals following infection and probed for hsp27 or HIV-1 p24 and p55 antigenic determinants. FIG. 9. Levels of intracellular hsp27 following (i) mock infection; (ii) mock infection with anti-CD4 antibodies; (iii–v) infection with serial dilutions of virus stock (m.o.i. Å 0.2); (vi) infection with heat-inactivated lated within 2 to 3 days of infection, accompanied by viral stock (56Њ for 1 hr); and (vii and viii) infection with virus stock pretreated with neutralizing antibody and soluble CD4 prior to infection. concomitant initiation of de novo viral protein synthesis. Generated chemoluminescent phosphoromager intensities of hsp27 There were no significant differences in cell proliferation signals at 24 hr were expressed as a percentage of the observed or cell viabilities between infected and mock-infected signals of the viral stock at 24 hr. The data represent averages of 2 to cells until 6 days of infection. The culmination of viral 5 experiments. infection was associated with substantial increases in viral protein and viral RNA synthesis, a dramatic decline sponses were significantly reduced when viral inocula in CD4 cell survival, and a corresponding reappearance (m.o.i. 0.2) were heat-treated (56 for 1 hr) prior to of a hsp27 stress response (Fig. 8). Å Њ infection. Furthermore, serial dilution of viral inocula re- Inducible hsp27 protein synthesis was viral dose- duced and attenuated hsp27 expression, accompanied dependent and abrogated by neutralizing antibodies by corresponding lags in intracellular viral protein syn- and heat inactivation of virus thesis (Fig. 9). In addition, hsp27 induction was abrogated when viral isolates were pretreated with neutraliz- Several experiments, summarized in Fig. 9, show that ing antibodies prior to infection. As expected, HIV-1 neu- early hsp27 responses were directly attributable to HIV- tralizing antibody blocked viral infection (Fig. 9). 1. Viral infections led to 6-fold increases in levels of intra- Cross-linking of cell surface CD4 moieties with soluble cellular hsp27 as compared to minor responses in mock- CD4 antibodies was insufficient to trigger hsp27 stress infected controls. These viral-associated hsp27 re- responses in uninfected cells (Fig.9). Pretreatment of cells with CD4 antibodies before infection suppressed hsp27 induction and viral infection. Thus, hsp27 responses cannot be simply ascribed to cell surface pertur- bations. Similar induction of hsp27 subspecies was observed in Jurkat, H9, and MT2 lymphocytic cell lines (data not shown). These cumulative findings indicate that hsp27 induction is associated with viral infection, occurring subsequent to HIV-1 binding to CD4, but before the initiation of viral mRNA and protein synthesis.

Determination of hsp isoforms induced following HIV- FIG. 8. Average data based on seven experiments of cell survival, intracellular levels of hsp27, and viral p24. Cells were mock-infected 1 infection or infected with HIV-1 (m.o.i. Å 0.2). Infected cell numbers at each time point are expressed as a percentage of corresponding mock-infected The abundant constitutive expression of hsp60, hsp70, cell numbers. Individual phosphorimager hsp27 and viral p55 intensi- and hsp90 may have masked detection of their modula- ties are expressed as percentages of respective peak intensities. tion during acute HIV-1 infection. Two-dimensional West-

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maximal viral production and CD4 cytolysis. To our knowledge, this is the first study to identify selective changes in hsp27 subspecies during an acute viral replicative cycle. Evidence presented, herein, indicates that the increase in hsp27 expression did not reflect a generalized stress response but was directly associated with HIV-1 viral infection. Inducible hsp27 protein synthesis was viral dose-dependent, inhibited by heat inactivation of virus, and blocked by neutralizing antibodies to HIV-1. In- fections with viral stocks depleted of HIV-1 by ultracentrifugation also failed to induce Hsp27. Hsp27 induction was observed in all tested lymphocytic cell lines. It is difficult to attribute a precise role of hsp27 in HIV- 1 viral infection since relatively little is known about the functional and molecular basis of hsp27 action in com- parison to that of other hsp subspecies (Arrigo and Landry, 1994; Ciocca et al., 1993). Hsp27 is a member of a family of small heat shock proteins related to a- crystallins. While high levels of hsp27 expression have FIG. 10. CEM cell extracts were isolated at various times following been found in epithelial and steroid-responsive tissues, mock-infection or HIV-1 infection (m.o.i. Å 0.2). Extracts were subjected this protein is generally expressed at low levels in eu- to isoelectric focusing (left to right, pI Å 10.0 to 3.0) in the first dimension and SDS–PAGE in the second dimension. (A) Mock-infected extracts karyotic cells. Following heat shock, steroid hormone isolated 24 hr following infection and probed for hsp27 and hsp70. treatment, or chemical stress, hsp27 levels can increase Similar patterns were observed at 48 hr and 6 days (data not shown). and accumulate, correlating with cellular differentiation, (B and C) HIV-1-infected extracts were probed for hsp27 and hsp70 oncogenic status, and acquired thermotolerance (Arrigo expression at 24 and 48 hr postinfection. At 6 days, the pattern of and Landry, 1994; Ciocca et al., 1993). hsp27 and hsp70 in infected cells resembled that observed with mock infection (data not shown). Transcriptional activation of hsp27 is thought to be the major regulatory mechanism wherein hsp27 accumulation confers thermoresistance (Arrigo and Landry, 1994; ern blot analysis was performed to determine if isoforms Knauf et al., 1994). In acquired thermotolerance, hsp27 of these latter hsps changed during acute infection. Cel- has been postulated to prevent protein aggregation and lular extracts from mock- and virally infected cells were actin polyermization (Arrigo and Landry, 1994). subjected to isoelectric-focusing (pI ranging from 3.0 to In contrast, a variety of T cell modulators, including 10) followed by SDS-electrophoresis. As shown in Figs. interleukins (IL-1 and IL-2), tumor necrosis factor a 10A and 10B, the transient emergence of novel hsp27 (TNFa), and interferon are potent stress protein agonists and hsp70 subspecies was observed 24 hr following HIV- that induce posttranslational phosphorylation of hsp27 1 exposure, absent in mock-infected cells at the same species (Knauf et al., 1994, Landry et al., 1992). Native time point. The novel hsp27 and hsp70 isoforms gener- hsp27 is found as 500,000- to 800,000-kDa macromolecu- ated by viral infection disappeared by 48 hr (Fig. 10C). lar cytosolic complexes. Upon stress exposure, hsp27 Other experiments indicated that novel hsp70 homo- polypeptides are phosphorylated and posttranslationally logues were generated as early as 6 and 12 hr postinfec- modified to yield multiple hsp27 homologues. Hsp27 ki- tion (data not shown) but disappeared by 48 hr. There nase pathways appear to be distinct from mitogen-acti- were no changes in hsp60 or hsp90 homologues vated kinase (MAPK) cascades (Belka et al., 1995; throughout infection (data not shown). Freshney et al., 1994; Rouse et al., 1994; Saklatvala et al., 1991). Hsp27 species represent components of signal DISCUSSION transduction pathways between mitogens and cytoskele- tal networks, similar to GTP-binding proteins (Arrigo and Acute HIV-1 infection causes a unique hsp27 stress Landry, 1994; Ciocca et al., 1993). response in CD4-expressing lymphocytic cells. This re- There is less of a documented role of hsp27 in viral sponse is characterized by early six-fold increases in infection. An inverse relationship has been found be- hsp27 mRNA and protein levels. These initial stress re- tween hsp27 expression and the oncogenic potential of sponses are short-lived and decline coincident to the different adenovirus serotypes (Zantema et al., 1989). Ex- initiation of de novo viral synthesis. Hsp27 mRNA and pression of hsp27 was not found to be related to estrogen protein synthesis remain down-regulated until late and progesterone status in human papilloma viruses and stages of infection where elevations in hsp27 parallel hepatitis virus infections (Ciocca et al., 1993).

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Therefore, our findings of transcriptional and transla- blot analysis reveals that the isoforms of hsp70 homo- tional changes in hsp27 expression at discrete stages logues induced at end lytic phases of infection resem- in the HIV-1 replicative cycle are note-worthy. Our results bled constitutive hsp70 species rather than the novel demonstrate that the kinetics of elevations in hsp27 and patterns of hsp70 subspecies observed 24 hr following hsp70 mRNA expression precede their enhanced protein infection. This suggests that stress proteins are involved expression, suggesting that hsp-induced synthesis is in different steps in the viral replicative cycle. due primarily to transcriptional regulation. The dynamics of stress protein expression may reflect Our studies show, however, that hyperthermia and vi- host–virus interactions. The fact that hsp27 and hsp70 ral infection show distinct impacts on stress protein in- homologues are generated early in infection suggests duction, cell proliferation, and HIV-1 viral expression that hsps may be involved in early events of the replica- (Brenner et al., 1995). Short febrile stimuli (45Њ for 15 min tive cycle prior to reverse transcription as has been de- or 42Њ for 2 hr) of chronically infected lymphocytic and scribed for cyclophilins (Braaten et al., 1996; Thali et al., monocytic cell lines induced hsp27 responses of similar 1994). It is also interesting to note that heat stress has magnitude to that observed in the present study. The been shown to transcriptionally activate not only the production of hsp27 protein in chronically infected CEM hsp70 promotor but also the HIV-1 long terminal repeat cells, however, was sustained for 2 to 4 days, and was (Geelen et al., 1988; Kretz-Remy et al., 1994). The decline accompanied by abrogated cell proliferation and a sus- of stress responses presents the possibility that viral tained block in intracellular viral synthesis (Brenner et gene products may be involved in the down-regulation of al., 1995). Moreover, the isoforms of hsp70 subspecies the early host stress responses. The induction of stress induced with acute infection are quite distinct from those protein expression at late stages of infection may indi- hsp70 homologues that result from heat shock or TNFa cate that these proteins serve a role in viral protein or exposure (unpublished results). virion assembly. Our preliminary findings indeed suggest The transiency of stress protein expression and its that both hsp27 and hsp70 are associated with virion rapid down-regulation concomitant to cellular viral syn- particles. This may directly or indirectly contribute to the thesis has been described in a number of other viral viruses’ ability to produce virions in a manner consistent systems. Infections with adenovirus, Epstein–Barr virus, with infectiousness. and herpes simplex virus, show transient induction of Cellular expression of hsp27 and hsp70 during infec- hsp70 or hsp90 as one of the earliest changes in gene tion is consistent with other studies showing an in- expression induced by viruses in newly infected host creased cell surface localization of hsp27 and hsp70 in cells (Oroskar and Read, 1989; Phillips et al., 1991; Strom HIV-infected cells (DiCesare et al., 1992; Poccia et al., and Frenkel, 1987). Induction of hsp70 in one study was 1991). These results have led to a postulated role of limited to those DNA viruses that encode immediate early stress proteins in the clearance of HIV-infected CD4 cells proteins with transactivating capabilities (Phillips et al., by the immune system (DiCesare et al., 1992; Gryllis et 1991). The rapid down-regulation of hsp70 responses al., 1992; Poccia et al., 1991). In this regard, hsps are has been linked to viral-induced shutoff of host gene highly antigenic determinants that evoke B and T host expression that enables viral genes to be selectively tran- immune responses to a variety of parasitic infections scribed as infection progresses (Oroskar and Read, 1989; (Mahvi et al., 1993; O’Brien et al., 1991). Conversely, in- Strom and Frenkel, 1987). This shutoff has also been duction of stress protective responses at end stages of attributed to virion structural proteins causing disaggre- infection may render cells less susceptible to viral, immu- gation of cellular polyribosomes and degradation of host nologic, and apoptotic cytolytic events (Mehlen et al., RNAs (Strom and Frenkel, 1987). 1995; Mosser and Martin, 1992; Samali and Cotter, 1996). Our findings with hsp70 are consistent with other re- To summarize, we show that hsp27 and hsp70 path- ports that show nuclear hsp70 translocation at early ways are selectively affected during lytic HIV-1 infection. times following either HIV-1 infection or gp120 binding The interplay of virus and cellular stress machinery may to the CD4 receptor (Furlini et al., 1994). In our study, be implicated in the life–death balance of HIV-1 in lym- cross-linking of CD4 with antibodies did not trigger hsp27 phocytes. On the one hand, host stress responses may or hsp70 induction. Thus, hsp27 and hsp70 subspecies allow cells to cope with infection and on the other hand, arose subsequent to viral binding events but prior to de they may play a critical role in the viral replicative pro- novo viral synthesis. During productive stages of viral cess. A further understanding of viral-induced intracellu- infection, hsp27 and hsp70 mRNA and protein synthesis lar stress responses may lead to novel strategies for declined. At late stages of infection, there were second- control of HIV-1 viral infection. ary inductions of hsp27 and hsp70 mRNA levels that were likely associated with necrotic and/or apoptotic lytic ACKNOWLEDGMENTS events rather than viral-induced effects (Li et al., 1996; Mosser and Martin, 1992; Samali and Cotter, 1996; Suto This study was made possible by grants from Health and Welfare and Srivastava, 1995). Indeed, two-dimensional Western Canada and CANFAR (Canadian Foundation for AIDS Research).

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