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Journal of Virology, March 2008, p. 2575-2579, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.00962-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Heat Shock Perturbs TRIM5 Restriction of Human Immunodeficiency Virus Type 1

Jenny L. Anderson, Edward M. Campbell, Anna Figueiredo, and Thomas J. Hope^*

Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Received 3 May 2007/ Accepted 28 November 2007

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TRIM5 restriction factors protect target cells from retroviruses by blocking infection prior to the accumulation of viral reverse transcription (RT) products. Here, we demonstrate that heat shock perturbed owl monkey TRIMCyp and rhesus TRIM5-mediated restriction of human immunodeficiency virus type 1 (HIV-1) late RT products and 2-long terminal repeat circles. Heat shock partially rescued HIV-1 infection from TRIMCyp restriction, and this rescue became more profound when combined with the presence of the proteasome inhibitor MG132. This indicates that viral RT products rescued from restriction by either heat shock treatment or the presence of MG132 are on a productive pathway, supporting a model in which TRIM5 proteins restrict retroviruses in multiple phases that are differentially sensitive to heat shock and proteasome inhibitors.

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Members of the TRIM5 family of restriction factors protect target cells from retroviruses originating from other host species (4, 6, 18). For example, rhesus monkey TRIM5 (rhTRIM5) and owl monkey TRIMCyp proteins protect target cells from human immunodeficiency virus type 1 (HIV-1) infection (16, 18). In most instances, TRIM5 proteins impair retroviral infection early after entry into target cells, blocking viral reverse transcription (RT) products from accumulating (10, 14, 18). However, the precise mechanism by which TRIM5 proteins prevent infection remains poorly understood.

Previously, we reported that proteasome inhibitors perturb TRIM5 proteins, preventing them from restricting their target retroviruses (1, 21). These inhibitors abrogate restriction of viral RT products by TRIM5 proteins, allowing integration-competent viral cDNA to accumulate, although TRIM5 restriction of viral infection persists. This indicates that TRIM5 proteins restrict retroviruses in multiple steps. The first step involves recognition of the viral core by TRIM5 and is sufficient to prevent the nuclear translocation of the viral cDNA. The second step prevents the accumulation of viral RT products and is sensitive to the presence of proteasome inhibitors (1, 21).

To extend these findings, we decided to examine the impact of an early block in the ubiquitin-proteasome pathway on TRIM5 restriction. For this purpose, we initially used E36 (E36TC) and ts20 (ts20TC) hamster cell lines obtained from Paul Bieniasz (Aaron Diamond AIDS Research Center, New York, NY) that stably expressed owl monkey TRIMCyp (14). Ts20 cells derive from E36 cells (7) but contain temperature-sensitive alleles of the E1 ubiquitin-activating enzyme (12), which is critical for initiating the ubiquitin-proteasome cascade. Elevating the temperature of ts20 cells from 30°C to 42°C for 5 h eliminates E1 protein expression and impairs the ubiquitin-proteasome pathway whereas E1 levels in the E36 cells remain unaffected (14). However, when we used these cells to examine the role of E1 inactivation in TRIMCyp restriction, we observed that incubation at 42°C affected restriction in both ts20TC cells and E36TC control cells, which do not contain a temperature-sensitive allele of E1 (data not shown). While these data complicated our analysis of the effect of E1 depletion and an early block in the ubiquitin-proteasome pathway on TRIMCyp restriction of HIV-1, they inadvertently revealed a novel 42°C temperature treatment that could perturb TRIMCyp restriction.

To assess whether the change in the TRIMCyp restriction of HIV-1 caused by 42°C treatment was an anomaly of the hamster cells or also pertained to TRIMCyp in its native cellular context, we analyzed the impact of the 42°C treatment on HIV-1 restriction in owl monkey kidney (OMK) cells endogenously expressing TRIMCyp. At 37°C, HIV-1 RT product and infection levels were restricted in the OMK cells compared to the results seen with the cyclosporine (CsA) control which blocks restriction (19). Also, the proteasome inhibitor MG132 efficiently rescued the HIV-1 late RT products but not 2-long terminal repeat (2-LTR) circles or infection (Fig. 1A to C) as expected (1). Note that 2-LTR circles form when cellular nonhomologous end-joining proteins ligate the LTR ends of the viral cDNA together, and they can mark nuclear entry of the viral cDNA. However, treating the OMK cells at 42°C perturbed the restriction of HIV-1, partially rescuing HIV-1 late RT products and 2-LTR circles at levels approximately 15- and 10-fold below those seen with the unrestricted CsA counterpart at 42°C (Fig. 1A and B). Moreover, combining MG132 with the 42°C treatment substantially rescued HIV-1 late RT products and 2-LTR circles, resulting in levels 1.6-fold and 4.7-fold below the levels seen with the unrestricted CsA counterpart (Fig. 1A and B). Surprisingly, when infection was analyzed, the 42°C treatment also rescued a modest level of infection that increased substantially when combined with the presence of MG132 (Fig. 1C).

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FIG. 1. Heat shock partially rescues HIV-1 cDNA products and infection from endogenous owl monkey TRIMCyp restriction. OMK cells were incubated 16 h at 37°C (white bars) or 42°C (gray bars) before infection with vesicular stomatitis virus G protein-pseudotyped R7envGFPHIV-1 at the same temperature. Comparative samples lacking virus (mock) or containing virus and MG132 (HIV.MG), CsA (HIV.CsA), or nevirapine (HIV.Nev) were included. (A and B) At 14 h into infection, either cellular DNA was collected and analyzed for levels of late RT products (A) or 2-LTR circles (B) per cell (β-actin) via real-time PCR, or the virus was removed from duplicate samples and all cells were returned to 37°C and analyzed for infection 48 h later using flow cytometry to detect the green fluorescent protein (GFP) reporter. (C) The percentages of GFP-positive cells are shown. Error bars indicate standard deviations of triplicate values. (D) Western analysis of OMK and HeLa cells either before (0 h) or after incubation at 37°C or 42°C for 16 h. Samples with equivalent cell numbers were loaded onto gels, and blots were probed with anti-CypA antibody (upper panel) to detect TRIMCyp and then stripped and reprobed with anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) antibody (lower panel) as a loading control. Twofold dilutions of the OMK sample at 0 h were included for comparison of protein levels.

Incubating cells at elevated temperatures, such as 42°C for cells normally cultured at 30°C or 37°C, triggers a heat shock response to help cells recover from damage induced by the temperature stress (8, 13, 15). This heat shock response has important consequences with respect to protein folding, aggregation, and turnover by cellular proteasomes (2, 3, 8, 9, 20), potentially including TRIMCyp. However, the ability of the 42°C "heat shock" treatment to affect TRIMCyp restriction of HIV-1 (Fig. 1A to C) was not due to a heat shock-induced loss of TRIMCyp steady-state levels, as observed from the Western blot results seen with cell lysates collected 16 h after incubation at 37°C or 42°C and probed using an anti-CypA antibody (Fig. 1D).

We also examined the ability of 42°C treatment to relieve restriction mediated by rhTRIM5. RhHA HeLa cells stably expressing hemagglutinin (HA)-tagged rhTRIM5 protein (rhTRIM5.HA; 18) and control HeLa cells were infected with HIV-1 at 37°C or 42°C. At 37°C, HIV-1 was restricted in the RhHA cells relative to the unrestricted HeLa control results (Fig. 2A to C). Moreover, MG132 treatment rescued HIV-1 late RT products and, to a lesser extent, 2-LTR circles from rhTRIM5.HA restriction, while HIV-1 infection remained impaired (Fig. 2A to C). Treatment at 42°C also perturbed the rhTRIM5.HA restriction of HIV-1 in the RhHA cells, partially rescuing HIV-1 late RT products and 2-LTR circles (Fig. 2A and B). While treatment at 42°C alone did not relieve the restriction of HIV-1 infection (Fig. 2C), combining the 42°C treatment with MG132 treatment allowed nearly complete rescue of the HIV-1 late RT products and 2-LTR circles from rhTRIM5.HA restriction (Fig. 2A and B). Unfortunately, effects of the combination of 42°C treatment and MG132 treatment on rhTRIM5.HA restriction of HIV-1 infection could not be measured due to the long-term toxicity of this combination upon both RhHA and HeLa cells. Again, we examined the effect of the 42°C "heat shock" treatment on rhTRIM5.HA protein expression by use of an anti-HA antibody (Fig. 2D). No marked loss in steady-state rhTRIM5.HA expression levels was observed after 16 h of 42°C treatment compared to the results seen with cells treated at 37°C. Additionally, we determined the impact of 42°C on the rhTRIM5.HA turnover rate by treating cells with cycloheximide and assaying protein expression at several time points as previously described (5). The approximate half-life of rhTRIM5.HA in cells incubated at 37°C was 85 min (Fig. 2E), which is consistent with our previous data (21). A similar degradation rate of 71 min was observed at 42°C, indicating that the 42°C treatment did not significantly alter the turnover rate of rhTRIM5.HA (Fig. 2E). Thus, the ability of heat shock to perturb rhTRIM5.HA restriction does not appear to be a consequence of aberrant expression or turnover of rhTRIM5.

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FIG. 2. Heat shock elevates HIV-1 cDNA levels but not infection during rhesus TRIM5 restriction. HeLa (white bars) and rhTRIM5-stable HeLa cells (black bars) were incubated 16 h at 37°C or 42°C and then infected with vesicular stomatitis virus G protein-pseudotyped R7envGFPHIV-1 for 14 h at the same temperature. Samples lacking virus (mock) or containing virus with MG132 (HIV MG) or nevirapine (HIV Nev) were included for comparison. Cellular DNA was extracted and analyzed for levels of late RT products (A) or 2-LTR circles (B) per cell (β-actin) by real-time PCR, or the virus was removed from duplicate samples and all cells were returned to 37°C and analyzed 48 h later for infection by use of flow cytometry for determination of green fluorescent protein (GFP) levels (in panel C, the percentage of GFP-positive cells is graphed). A representative experiment is shown, with the error bars reflecting standard deviations of triplicate values. (D) Western analysis showing steady-state levels of rhTRIM5 in the same cell types either before (0 h) or after incubation at 37°C or 42°C for 6 h or 16 h. Samples with equivalent cell numbers were loaded onto gels, and blots were probed with anti-HA antibody (upper panel) to detect the HA-tagged rhTRIM5 and then stripped and reprobed with anti-GAPDH antibody (lower panel) as a loading control. Twofold dilutions of the rhTRIM5-stable HeLa cells at 0 h were included for a comparison of protein levels. (E) rhTRIM5-stable HeLa cells were treated with 100 µg/ml cycloheximide after 16 h of incubation at 37°C or 42°C. Cells were lysed at the indicated times after the initiation of cycloheximide treatment. Cell lysates were analyzed by Western blotting with anti-HA antibody (right panels). The density of each band detected by the anti-HA antibody, normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; detected using anti-GAPDH antibody), was quantified using a ChemiImager 4400 low-light imaging system (Alpha Innotech Corporation) and AlphaEase software (version 5.0). The logarithms of the percentages of the initial amounts of protein versus time are plotted (left panel). These data represent the average results of three individual experiments. Data from 37°C treatment are shown using black squares; data from 42°C treatment are shown using gray triangles.

To further investigate the impact of heat shock on rhTRIM5.HA protein behavior, we examined protein localization by use of immunofluorescence (Fig. 3). RhHA cells cultured for 16 h at either 37°C or 42°C were fixed and stained for HA, DNA, and filamentous actin. At 37°C, rhTRIM5.HA was diffusely distributed throughout the cytoplasm and also localized into punctate accumulations or cytoplasmic bodies (Fig. 3A) as previously reported (18, 21). In contrast, the 42°C heat shock treatment altered the appearance of rhTRIM5.HA bodies in RhHA cells. We observed an increase in the number of cells either without obvious bodies or with one or two large accumulations of rhTRIM5.HA protein that differed in size and appearance from the more typical cytoplasmic bodies seen at 37°C (Fig. 3A). To quantify these changes, cells were imaged in panels and manually counted, and the percentages of cells without distinct bodies, with regular-sized bodies, or with one or two large bodies were calculated. The average percentage of each cell type, as derived from four separate experiments in which a total of 3,026 cells were counted, is shown (Fig. 3B). The 42°C heat shock reproducibly increased the number of cells without distinct bodies or with one or two large bodies compared to cells treated at 37°C (Fig. 3B).

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FIG. 3. Heat shock alters rhesus TRIM5 bodies in HeLa cells. (A) Immunofluorescence analysis of HeLa cells stably expressing HA-tagged rhTRIM5 either maintained at 37°C (left panels) or shifted to 42°C for 16 h (right panels). Fixed samples were stained for HA-tagged rhTRIM5 (red), DNA (blue), and filamentous actin (F-actin [green]) by use of anti-HA antibody plus Cy3-conjugated anti-mouse antibody, Hoechst staining, and Oregon Green-conjugated phalloidin, respectively. Representative, deconvolved images taken with a 100x objective lens are shown. Thin and thick white arrows indicate regular-sized and large bodies, respectively. (B) The percentages of cells containing no rhTRIM5 bodies, regular-sized bodies, or one or two large bodies were quantified for cells cultured at 37°C (gray bars) or 42°C (black bars) for 16 h. A total of 3,026 cells from four independent experiments was imaged in panels and manually counted. The percentages of cells in each category were averaged for the four independent experiments and graphed, with error bars indicating the standard errors of the means. Heat shock increased the percentages of cells with large bodies or without bodies. (C) HeLa cells stably expressing rhTRIM5.HA were incubated 16 h overnight at 37°C (top panel), at 42°C (middle panel), or at 37°C (bottom panel) in the presence of the proteasome inhibitor MG132. Cells were then fixed and stained with anti-HA antibody (green) and an antibody for human vimentin (red).

Cytoplasmic bodies are reported to possess features of aggresomes (5). We therefore used immunofluorescence to examine whether the large rhTRIM5.HA cytoplasmic accumulations observed after 42°C heat shock exhibited vimentin caging, a characteristic feature of aggresomes (11). No evidence of rhTRIM5.HA cytoplasmic bodies surrounded by vimentin cages was observed in cells cultured overnight at 37°C or 42°C (Fig. 3C). In contrast, overnight treatment with MG132 induced large perinuclear cytoplasmic bodies that exhibited obvious vimentin caging consistent with the presence of aggresomes (Fig. 3C). Thus, while the cytoplasmic localization of rhTRIM5.HA altered following 42°C heat shock treatment, these accumulations of protein do not represent classic aggresomes as defined for vimentin caging. Therefore, in summary, the 42°C heat shock treatment perturbed rhTRIM5.HA cytoplasmic body formation whereas levels of rhTRIM5.HA expression remained largely unaltered.

Conclusions. While we originally sought to examine the role of the ubiquitin-proteasome pathway in HIV-1 restriction mediated by TRIM5 proteins, our investigation revealed an unappreciated ability of 42°C heat shock to perturb the retroviral restriction mediated by TRIM5 proteins. Moreover, the heat shock was sufficient to rescue viral cDNA in the cytoplasm and nuclei of restricted cells to various extents, as measured by examination of late RT products and 2-LTR circles, respectively (Fig. 1A and B and Fig. 2A and B). While the precise mechanism by which heat shock perturbs TRIM5 restriction remains unclear, this is the second time this pathway has been observed to affect the cellular biology of TRIM5. Song et al. previously reported that treating cells with the heat shock protein inhibitor geldanamycin induces a loss of rhTRIM5 cytoplasmic bodies that does not relieve rhTRIM5-mediated restriction of HIV-1 infection (17). Here we report that heat shock can reduce the degree to which rhTRIM5 forms cytoplasmic bodies (Fig. 3B). Moreover, this state correlates with a loss in the extent of restriction of HIV-1 2-LTR circles (Fig. 1B and 2B) and infection in the case of endogenous TRIMCyp restriction (Fig. 1C). Thus, the heat shock pathway can clearly impact the biology of TRIM5 proteins. While we have previously reported that proteasome inhibitors do not relieve TRIM5-mediated restriction with respect to 2-LTR circles and infection (1, 21), proteasome inhibition in combination with heat shock relieved TRIMCyp (Fig. 1B) and rhTRIM5 (Fig. 2B) restriction of 2-LTR circles at various magnitudes. Moreover, while heat shock partially rescued HIV-1 infection from TRIMCyp restriction, this rescue became more profound when combined with MG132 treatment (Fig. 1C). This indicates that the viral RT products rescued from restriction by either heat shock or MG132 treatment are on a productive pathway, thus supporting a model in which TRIM5 proteins restrict retroviruses in multiple phases differentially sensitive to heat shock and proteasome inhibitors. Therefore, this work reinforces the idea of a role of the ubiquitin-proteasome pathway in TRIM5 restriction and also demonstrates a previously unappreciated ability of heat shock to relieve TRIM5-mediated restriction of HIV-1. Thus, heat shock may serve as a useful tool in future studies delineating the role of the ubiquitin pathway in TRIM5 restriction.

 
We thank Paul Bieniasz (Aaron Diamond AIDS Research Center, New York, NY) and Joseph Sodroski (Harvard Medical School) for generously providing cell lines.

This work was supported by National Institutes of Health grant R01 AI47770 to T.J.H., who is also an Elizabeth Glaser Scientist.

 
* Corresponding author. Mailing address: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Ward 8-140, 303 East Chicago Avenue, Chicago, IL 60611. Phone: (312) 503-1360. Fax: (312) 503-7912. E-mail: thope{at}northwestern.edu

Published ahead of print on 12 December 2007.

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Journal of Virology, March 2008, p. 2575-2579, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.00962-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Anderson, J. L. Articles by Hope, T. J. Search for Related Content PubMed PubMed Citation Articles by Anderson, J. L. Articles by Hope, T. J.