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The Journal of Immunology, 2007, 179, 7262 -7269
Copyright © 2007 by The American Association of Immunologists, Inc.
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CUGBP1 Is Required for IFNβ-Mediated Induction of Dominant-Negative CEBPβ and Suppression of SIV Replication in Macrophages1

Justyna M. Dudaronek*, Sheila A. Barber{dagger} and Janice E. Clements2,*

* Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and {dagger} Booz Allen Hamilton, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Productive HIV replication in the CNS occurs very early after infection, yet HIV-associated cognitive disorders do not typically manifest until the development of AIDS, suggesting that mechanisms exist in the CNS to control HIV replication and associated virus-induced pathological changes during the acute and asymptomatic stages of disease. Using an established SIV/macaque model of HIV dementia, we recently demonstrated that the mechanisms regulating virus replication in the brain at these stages involve the production of IFNβ, which induces the truncated, dominant-negative isoform of C/EBPβ, also referred to as LIP (liver-enriched transcriptional inhibitory protein). Alternative translation of C/EBPβ mRNA and increased production of LIP can be mediated by CUGBP1 (CUG-repeat RNA-binding protein 1). Because IFNβ induces the inhibitory C/EBPβ in macrophages, we considered the possibility that IFNβ signaling regulates the activity of CUGBP1, resulting in increased expression of LIP and suppression of SIV replication. In this study, we report that IFNβ induces LIP and suppresses active SIV replication in primary macrophages from rhesus macaques. Further, we demonstrate that IFNβ induces the phosphorylation of CUGBP1 and the formation of CUGBP1-C/EBPβ mRNA complexes in the human monocytic U937 cell line. Finally, we demonstrate that CUGBP1 is not only required for IFNβ-mediated induction of LIP but also for IFNβ-mediated suppression of SIV replication. These results suggest that CUGBP1 is a previously unrecognized downstream effector of IFNβ signaling in primary macrophages that likely plays a pivotal role in innate immune responses that control acute HIV/SIV replication in the brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type I IFNs are among the first effector molecules induced during innate immune responses to viral pathogens. IFNβ, an integral mediator of innate immunity and the predominant type I IFN that is expressed in the CNS in response to many viral infections (1), inhibits active replication of HIV as well as SIV in macrophages, which are the major sources of productive HIV/SIV replication in the brain (2, 3, 4, 5). Although HIV-associated cognitive disorders do not typically manifest until the development of AIDS, existing data have documented that HIV can invade the CNS early during infection (6, 7). Similarly, we have demonstrated by using our rapid and consistent SIV/macaque model of HIV CNS disease (8, 9, 10) that, although neurocognitive impairment typically occurs between 56 and 84 days when all infected macaques exhibit AIDS (9), viral RNA can be detected in the brain as early as 7 days postinoculation (11, 12). Acute SIV replication in the brain is suppressed between 14 and 21 days postinoculation in this model as evidenced by undetectable levels of SIV RNA (although SIV DNA levels remain unchanged; Ref. 13). That the expression of IFNβ is also induced in the brain as early as 7 days postinoculation (12) suggests that innate immune responses involving IFNβ contribute significantly to mechanisms that suppress acute SIV replication in the CNS.

Production of IFNβ is an early and effective antiviral response of macrophages to HIV infection (14, 15, 16). The mechanism by which IFNβ inhibits active HIV replication in primary macrophages involves increased expression of the truncated, dominant-negative form of the transcription factor C/EBPβ (2, 12, 17). This truncated isoform is translated from the third in-frame AUG in C/EBPβ mRNA such that it maintains the DNA binding domain but lacks most of the transactivation domain, thereby antagonizing C/EBPβ-mediated transcription via dominant-negative heterodimeric complexes with full-length C/EBPβ or by competing for DNA binding sites as a homodimeric inhibitory complex (18). The elegance of this inhibitory mechanism can be appreciated through studies demonstrating that C/EBPβ is a crucial activator of the HIV-1 long terminal repeat (LTR)3 and that the active replication of HIV in primary macrophages and differentiated promonocytic cell lines in vitro (but not in lymphocytes/lymphocytic cells lines) requires at least one C/EBP binding site in the LTR (19, 20, 21). Studies from our laboratory demonstrated further that the truncated dominant-negative C/EBPβ, which is also referred to as LIP (liver-enriched transcriptional inhibitory protein) (22), suppresses C/EBPβ-mediated SIV LTR activity (12) and that the increased expression of LIP in the brain parallels the increase in IFNβ during the suppression of acute SIV replication in the CNS (4).

Our studies on the regulation of the SIV LTR in the brain of infected macaques recently led to the following molecular model to describe the control of acute SIV/HIV replication in the CNS: SIV/HIV invade the CNS during acute infection when full-length C/EBPβ predominates at the LTR and, together with chromatin remodeling events, mediates LTR-dependent transcription resulting in the production of full-length viral RNA. Concomitantly, acute virus infection triggers the production of IFNβ in the brain that induces the alternative translation of C/EBPβ mRNA, resulting in increased expression of LIP between 7 and 21 days postinoculation. At this time LIP predominates at the LTR, chromatin-remodeling events subside, and the production of full-length SIV RNA becomes undetectable (12, 13).

What remains to be determined is the mechanism by which IFNβ induces the alternative translation of C/EBPβ mRNA in macrophages favoring the expression of LIP. To date a single RNA binding protein, CUGBP1 (CUG-repeat RNA-binding protein 1), has been linked to LIP expression (23, 24, 25). CUGBP1 binds to upstream AUG regions in the 5' untranslated region of the C/EBPβ mRNA and regulates a leaky ribosomal scanning mechanism favoring the translation of LIP (23, 24, 25, 26, 27). In the present study, we demonstrate that IFNβ induces the activation of CUGBP1 and the formation of CUGBP1-C/EBPβ mRNA complexes in macrophages. Furthermore, we demonstrate that the depletion of CUGBP1 by using small interfering RNA (siRNA) inhibits IFNβ-mediated induction of LIP and the suppression of SIV replication. Thus, we conclude that CUGBP1 is a previously unrecognized downstream target of IFNβ signaling and that CUGBP1 and undoubtedly LIP are essential for IFNβ-mediated suppression of SIV and HIV replication in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

U937 cells were maintained in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS, 10 mM HEPES (Invitrogen Life Technologies), 2 mM L-glutamine (Invitrogen Life Technologies), and 0.5 mg/ml gentamicin (Invitrogen Life Technologies). Cells were differentiated overnight with PMA (16 nmol/L) and then treated with IFNβ (PBL Biomedical Laboratories). Blood-derived primary macrophages from adult rhesus macaques were isolated and cultured as previously described (8) with modifications noted in the figure legends where appropriate.

SIV infection of primary rhesus macrophages

Unless indicated otherwise, adherent macrophages were infected with SIV/17EFr (8) at a multiplicity of infection (MOI) of 0.05 for 2–6 h at 37°C. After infection, cells were washed with PBS five times and cultured in the appropriate medium. IFNβ was added once at the indicated concentration. Cell supernatants (1 ml) were collected at the indicated intervals and replaced with fresh medium without the further addition of IFNβ. Virus replication was evaluated by quantitating levels of the SIV capsid protein p27 (SIV p27 ELISA kit; PerkinElmer) in supernatants and SIV gag RNA (real-time RT-PCR) in cells. The real-time RT-PCR assay used to quantitate SIV gag RNA has been described elsewhere (28).

RNA extraction and analysis

Total RNA was extracted using an RNeasy Total RNA kit (Qiagen). At the indicated times after transfection with siRNA, total RNA and protein were isolated using a PARIS (Protein and RNA Isolation System) kit (Ambion). SIV RNA was quantitated using real-time RT-PCR (28). For quantitation of 18 S ribosomal RNA, the following primers and probe (300 nmol/L and 100 nmol/L, respectively) were used in a 50-µl reaction: 5'-AGTCCCTGCCCTTTGTACACA-3' (forward primer), 5'-GATCCGAGGGCCTCACTAAAC-3' (reverse primer), and 5'-CGCCCGTCGCTACTACCGATTGG-3' (probe). Sequences were amplified from plasmid standards or 0.5–1 µg of RNA (in triplicate) using a Chromo 4 Thermal Cycler (MJ Research) programmed at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, and 60°C for 1 min. The 18 S standard was generated from a human cDNA library.

Protein isolation and Western blot analysis

Whole cell lysates were prepared with ice-cold modified radioimmunoprecipitation assay (RIPA) buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM sodium fluoride) containing Protease Inhibitor Cocktail III (Calbiochem) and Phosphatase Inhibitor Cocktail II (Sigma-Aldrich). Cytoplasmic fractions and nuclear extracts from U937 cells (107) were prepared using a nuclear fractionation kit (Marligen Biosciences). Cells transfected with siRNA were lysed with Cell Disruption Buffer from the PARIS kit (Ambion) containing protease and phosphatase inhibitor mixtures and RNaseOUT (0.5 U/ml; Invitrogen Life Technologies). Protein (10–80 µg) was resolved by electrophoresis (10 or 12.5% polyacrylamide gel) and blotted onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% milk in TBS with 0.1% Tween 20 (TBST), membranes were probed with 1 µg/ml anti-CUGBP1 mAb (Upstate Biotechnology) or anti-C/EBPβ polyclonal Ab at 1/500 (Santa Cruz Biotechnology). Membranes were washed with TBST three times for 5–10 min with agitation, incubated for 1 h with appropriate secondary Abs conjugated with HRP (DakoCytomation), and analyzed by chemiluminescence (Pierce). As previously demonstrated, wild-type C/EBPβ and LIP migrate in SDS-PAGE to ~36 and ~19 kDa, respectively. To control for equal loading of whole cell lysates, blots were re-probed with anti-GAPDH mAb at 1/5,000 (Santa Cruz Biotechnology). To control for the quality of subcellular fractionations, anti-GAPDH or anti-Lamin A/C (1/200; Santa Cruz Biotechnology) sera were used to identify cytoplasmic and nuclear markers, respectively.

Ribonucleoprotein immunoprecipitation (RIP) assay

The assay used to detect complexes of CUGBP1 and C/EBPβ mRNA has been described elsewhere (29) and was used in this study with the following modifications. PMA-differentiated U937 cells (107) were fixed in 0.5% formaldehyde/PBS at room temperature for 15 min. Crosslinking reactions were quenched with 0.25 M glycine (pH 7.0) and incubated at room temperature for 5 min. Fixed cells were resuspended in modified RIPA buffer (50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, and 150 mM NaCl) containing 1x Protease Inhibitor Cocktail III and RNaseOUT (0.5 U/ml) and sonicated three times (50%; Branson 250, Branson Ultrasonics). Solubilized cell lysates (300 µl) were precleared for 1 h at 4°C with 30 µl of protein G-Dynabeads (Invitrogen Life Technologies), and immunoprecipitation reactions were performed using 2 µg of anti-CUGBP1 polyclonal Ab (4°C overnight; Santa Cruz Biotechnology) and protein G-Dynabeads (30 µl for 1 h at room temperature). Immune complexes were washed five to six times with high-stringency RIPA (50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 1 M NaCl) and resuspended in 100 µl of 50 mM Tris-HCl (pH 7.0), 5 mM EDTA, 10 mM DTT, and 1% SDS. After reverse crosslinking (1 h at 70°C), RNA was extracted with a TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions and used as a template for cDNA synthesis (SuperScript First-Strand; Invitrogen Life Technologies) and analyzed by PCR using the C/EBPβ-specific primers 5'-CCAGCCCCCTCACTAATAGC-3' (forward primer) and 5'-TACGCAGCAGCCAAGCAGTC-3' (reverse primer) in 50-µl reaction containing cDNA (2 µl; one-tenth of the total cDNA reaction volume), 200 µmol/L dNTP, 200 nmol/L each primer, 1 M GC-Melt (BD Clontech), 5% DMSO, 2.5 U of Taq DNA polymerase (New England Biolabs), and 1 µl of [{alpha}-32P]CTP (6000 Ci/mmol). After 30 cycles (94°C for 30 s, 65-55°C gradient for 30 s, and 72°C for 30 s), amplified products were resolved on a 5% polyacrylamide gel, visualized by autoradiography, and quantitated using a Typhoon 9210 PhosphorImager. Water was used as a negative control and U937 cDNA (2 µl) was used as a positive control for PCR. To control for input C/EBPβ mRNA, solubilized lysates (300 µl) were reverse crosslinked (70°C) and 1 µg of isolated RNA was used in RT-PCR.

Immunoprecipitation and in vitro kinase assay

PMA-differentiated U937 cells (107) were lysed with modified RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.25% sodium deoxycholate, 10 mM EGTA, and 150 mM NaCl with protease and phosphatase inhibitors as above. Lysates were precleared with Protein G-Dynabeads and immunoprecipitation reactions were performed at 4°C overnight with anti-CUGBP1 (1 µg) in a volume of 200 µl of lysis buffer, after which immunoprecipitates were incubated with Protein G-Dynabeads for 1 h at room temperature. Immune complexes were washed three times with lysis buffer, once with kinase buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 20 mM MgCl2, resuspended in 22 µl of kinase buffer with 20 µCi of [{gamma}-32P]ATP and incubated at 30°C for 30 min. Reactions were stopped with 1 ml of ice-cold lysis buffer and immune complexes were pelleted, resolved by SDS-PAGE, visualized by autoradiography, and quantitated using a Typhoon 9210 PhosphorImager.

Metabolic labeling

PMA-differentiated U937 cells (107) were labeled either with [35S]Met/Cys (ICN Radiochemicals) or [32P]orthophosphate (PerkinElmer). For 35S labeling, cells were incubated in methionine-free, cysteine-free RPMI 1640 (Sigma-Aldrich) containing 2% FBS, 2 mM L-glutamine, 10 mM HEPES, 0.5 mg/ml gentamicin, and 16 nmol/L PMA for 30 min before the addition of 0.2 mCi/ml [35S]Met/Cys and IFNβ for the indicated periods of time. For 32P labeling, cells were incubated in phosphate-free RPMI 1640 (Invitrogen Life Technologies) containing 2% dialyzed FBS, 2 mM L-glutamine, 10 mM HEPES, 0.5 mg/ml gentamicin, and 16 nmol/L PMA for 30 min before the addition of 0.5 mCi/ml [32P]orthophosphate for the indicated periods of time. Whole cell lysates, cytoplasmic fractions, and nuclear extracts were prepared and resolved by SDS-PAGE as described above.

siRNA depletion of CUGBP1

Primary macaque macrophages seeded into 6-well plates were treated with Lipofectamine alone or in combination with 100 pmol of either a CUGBP1-specific siRNA pool (Dharmacon) or a nonspecific siRNA pool (Dharmacon) once a day for 2 days using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s protocol. The efficiency of the CUGBP1 knockdown was evaluated by Western blotting. Macrophages transfected with CUGBP1 siRNA, nonspecific siRNA, or Lipofectamine alone were infected with SIV/17EFr at a MOI of 0.05 for 2 h at 37°C, washed, and treated with IFNβ for 24 h. Viral replication was measured by quantitating p27 and SIV RNA as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IFNβ induces LIP and suppresses SIV replication in primary macaque macrophages

Previous studies from our laboratory have demonstrated that LIP suppresses C/EBPβ-induced SIV LTR activity and that the increased expression of LIP in the brain parallels the increase in IFNβ during the suppression of acute SIV replication in the CNS as evidenced by the suppressed levels of SIV RNA (4, 12, 13). To examine the effect of IFNβ on LIP expression in vitro, whole cell lysates prepared from primary blood-derived macaque macrophages treated with IFNβ were processed for Western blot analysis as described in Materials and Methods. Our results demonstrate that IFNβ increased LIP expression in primary macaque macrophages as early as 12 h post-IFNβ treatment (data not shown) and that LIP expression was maintained for at least 96 h after treatment (Fig. 1A). This IFNβ-mediated effect on LIP expression is consistent with the IFNβ-mediated induction of LIP in uninfected U937 cells and HIV-infected alveolar macrophages (12, 17, 30). The expression of wild-type full-length C/EBPβ, also referred to as LAP (liver-enriched transcriptional activator protein) (22), fluctuated at various times following IFNβ treatment in some experiments, but no consistent pattern was observed (Fig. 1A; compare also to Fig. 6C). Importantly, the resulting ratios of LIP to LAP (>0.2) were inhibitory (22) in each IFNβ-treated sample for at least 96 h, which, as we have previously reported, leads to the suppression of SIV LTR activity (12). These results demonstrate for the first time that IFNβ induces LIP in primary macaque macrophages.


Figure 1
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  		FIGURE 1. Effect of IFNβ on LIP expression and SIV replication in primary macaque macrophages. A, Western blot analysis of C/EBPβ isoforms. Cell lysates from primary macaque macrophages treated with 100U/ml IFNβ were subjected to Western blot analysis for detection of the C/EBPβ isoforms LIP and LAP. Band intensities quantitated using the Kodak Image Station were used to calculate the ratio of LIP to LAP for each sample, which is indicated below each lane. Shown is a representative experiment, n = 2. B, p27 analysis of SIV particle production. Primary macaque macrophages were infected (or not) with SIV/17EFr (MOI of 0.05) for 6 h, washed extensively, and then treated with 100 U/ml recombinant human IFNβ for the indicated periods of time. For infected cultures IFNβ was added 2 days postinfection without washing the cells, when SIV p27 was detectable in culture supernatants. Supernatants were collected from infected cultures at 24, 48, 72, and 96 h post-IFNβ treatment for p27 analysis. C, Real time RT-PCR analysis of SIV RNA. At 24, 48, 72, and 96 h post-IFNβ treatment cells were harvested and examined for the production of full-length viral RNA by using real-time RT-PCR. Data reflect the number of copies of SIV gag RNA per 1 µg of total RNA normalized to infected, untreated controls. Mock-infected, p27, and SIV RNA levels were below the limit of detection. Data are representative of three independent experiments.

 

Figure 6
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  		FIGURE 6. siRNA knockdown of CUGBP1. Primary macrophages transfected with siRNA specific for CUGBP1 (CUGBP1), nonspecific siRNA (NS), or Lipofectamine 2000-only control (None) were infected with SIV for 2 h, washed to remove free virus, and immediately treated with 100 U/ml IFNβ for 24 h as described in Materials and Methods. A, Western blot analysis of CUGBP1. Band intensities were quantitated using the Kodak Image Station and normalized to GAPDH. The numbers below each lane represent the intensity of CUGBP1 normalized to the nonspecific-transfected control for each treatment. B, Impact of siRNA on SIV replication. Loss of CUGBP1 suppresses the anti-SIV activity of IFNβ. Viral replication (24 h post-IFNβ treatment) was assessed by quantitating p27 in supernatants and SIV gag mRNA in cells, which are depicted in this graph as a percentage of the infected, untreated control (100%; dashed line). C, Western blot analysis of LIP and LAP after 24 h of treatment with IFNβ. Band intensities quantitated using the Kodak Image Station were used to calculate the ratio of LIP to LAP for each sample, which is indicated below each lane. All data are representative of three independent experiments.

 
To examine the effect of IFNβ on SIV replication in vitro, primary macaque macrophages were infected with SIV as described in Materials and Methods. When SIV p27 was detected in culture supernatants 48 h postinfection, IFNβ was added (or not) to the cultures without washing the cells. Virus replication was monitored thereafter at the indicated times by quantitating p27 in supernatants and SIV RNA from harvested cells. Treatment with IFNβ suppressed ongoing virus production, which was reflected by the decreased levels of SIV p27 and full-length viral RNA as early as 24 h after treatment (Fig. 1, B and C). The balance of the p27 detected over time in the IFNβ-treated cultures is likely residual virus, which suggests that particle production is also impacted by IFNβ treatment such that no increase in p27 is observed over time (not even at 50% of the untreated cultures, which might be expected based on the impact of IFNβ on SIV RNA). This strongly suggests that multiple stages in the replication cycle are vulnerable to IFNβ treatment, consistent with several reports in the literature (31). In conjunction with our previous studies, these results demonstrate that the in vivo correlation between IFNβ, LIP, and the suppression of viral RNA during acute SIV infection in the brain (12, 13) can be modeled in vitro by using primary macaque macrophages or human U937 cells.

IFNβ induces the association of CUGBP1 with C/EBPβ mRNA

C/EBPβ is an important regulator of HIV/SIV replication in macrophages, and the ratio of its activating isoforms to inhibitory isoforms is subject to control by IFNβ. Because IFNβ suppresses SIV replication and induces LIP within 24 h of treatment (Fig. 1), we suspected that a previously unrecognized IFNβ-mediated pathway exists to induce this acute antiviral activity. Because a single RNA binding protein, CUGBP1, has been implicated to date in directing LIP expression (23, 24, 25, 26), we hypothesized that induction of the inhibitory C/EBPβ isoform by IFNβ in macrophages might be mediated through CUGBP1. Therefore, we examined the association of CUGBP1 with C/EBPβ mRNA in PMA-differentiated U937 cells treated with IFNβ by using a RIP assay (29). This method combines a highly reactive, reversible crosslinking reaction with a high-stringency immunoprecipitation protocol to identify specific RNAs complexed with a given protein. Briefly, U937 cells were crosslinked with formaldehyde and RNA/CUGBP1 complexes were immunoprecipitated, after which the crosslinks were reversed and the presence of C/EBPβ mRNA was detected by RT-PCR. The results from this RIP assay indicate that increased levels of C/EBPβ mRNA are complexed with CUGBP1 after treatment with IFNβ (Fig. 2A). The levels of C/EBPβ mRNA in these complexes increase as early as 6 h after the addition of IFNβ, peak after 12–24 h of IFNβ treatment, and subside thereafter (Fig. 2, A and B), suggesting that this is an acute downstream effector pathway for IFNβ that dissipates thereafter.


Figure 2
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  		FIGURE 2. IFNβ induces the formation of CUGBP1-C/EBPβ mRNA complexes. A, RIP assay performed on lysates from PMA-differentiated U937 macrophages treated with 100U/ml IFNβ for 6, 12, 24, and 48 h. Band intensities quantitated by using a Typhoon PhosphorImager were normalized to control for input C/EBPβ mRNA in each reaction. This graph depicts the fold induction of C/EBPβ mRNA complexed with CUGBP1 after treatment with IFNβ normalized to an untreated control at each time point (1.0). There is a statistically significant increase in the association of CUGBP1 protein with C/EBPβ mRNA in IFNβ-treated samples compared with untreated samples at all time points except for 48 h (*, p < 0.05; paired t test). Data are representative of four independent experiments. B, Representative autoradiograph (n = 4) of the RIP assay performed at 12 h post-IFNβ treatment. CUGBP1-specific Ab was used in conjunction with C/EBPβ-specific primers as described in Materials and Methods. Control lanes represent negative (–) and positive (+) controls for PCR and no Ab (No AB) is the control for the immunoprecipitation step as described in Materials and Methods. The control for input C/EBPβ mRNA in each treatment group is shown. There is an increase in the levels of C/EBPβ mRNA complexed with CUGBP1 after treatment with IFNβ. C, Western blot of CUGBP1 immunoprecipitates from U937 lysates (crosslinked or not) prepared at 12 h post-IFNβ treatment. That no difference is observed between total CUGBP1 immunoprecipitated (IP) with CUGBP1-specific Ab from non-crosslinked (left lane), crosslinked untreated (middle lane), and crosslinked IFNβ-treated (right lane) lysates demonstrates that equal amounts of CUGBP1 are present in each treatment group. Shown is a representative experiment, n = 4.

 
This transient RNA binding activity of CUGBP1 precedes and directs the increased expression of LIP, which is quite stable compared with wild-type C/EBPβ (32). Because type I IFN can also enhance protein stability (33, 34), it is conceivable that treatment with IFNβ contributes to accumulation of the LIP isoform. Thus, although not actively participating during sustained events mediating the suppression of SIV replication, CUGBP1 induces a protein whose activity correlates temporally with the suppression of acute (24 h) and sustained (96 h) virus replication. These findings demonstrate directly that IFNβ induces the formation of CUGBP1-C/EBPβ mRNA complexes. Furthermore, because increases in CUGBP1 levels are not observed (Figs. 2C and 3, A and B), we conclude that IFNβ most likely potentiates the RNA binding activity of constitutively expressed CUGBP1 protein.


Figure 3
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  		FIGURE 3. Western blot analysis of CUGBP1 protein in IFNβ-treated macrophages. Cell lysates from PMA-differentiated U937 cells treated for 3 to 48 h with 100 U/ml IFNβ (A) or primary macaque macrophages treated for 6 to 48 h with 100 U/ml IFNβ (B) were subjected to Western blot analysis for the detection of CUGBP1. Band intensities were quantitated using the Kodak Image Station and are shown below each lane as the ratio of CUGBP1 to GAPDH control. Results represent at least three independent experiments.

 
Activation of CUGBP1 by IFNβ

Accumulating reports indicate that C/EBPβ isoforms are overexposed in a variety of cancers (35, 36, 37, 38), fueling interest in the mechanism leading to the activation of CUGBP1. In this venue, recent studies have linked the activation of CUGBP1 to cytoplasmic translocation and phosphorylation (25, 39). To examine the subcellular distribution of CUGBP1 before and after IFNβ treatment, we performed Western blot analysis on subcellular fractions of differentiated U937 cells treated or not with IFNβ. The results of these studies demonstrated that IFNβ did not alter CUGBP1 expression in the cytoplasm or nucleus (Fig. 4, A and B). Further, we found no difference in the subcellular distribution of CUGBP1 when quantitating CUGBP1 immunoprecipitated from the nucleus and cytoplasm of untreated and IFNβ-treated [35S]Met/Cys-labeled U937 cells (data not shown). That these results differ from those obtained by others (39) suggests that the cellular localization of CUGBP1 is signal and cell type specific.


Figure 4
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  		FIGURE 4. Subcellular distribution of CUGBP1 after treatment with IFNβ. A, Western blot analysis of cytoplasmic and nuclear fractions prepared from PMA-differentiated U937 cells that had been treated or not treated with 100 U/ml IFNβ for 6–24 h. The efficiency of subcellular fractionation was determined by probing the representative blots for GAPDH (cytoplasmic) or Lamin A/C (nuclear). Band intensities were quantitated in each compartment using the Kodak Image Station. B, Graph of subcellular CUGBP1 levels that reflects the abundance of CUGBP1 in each compartment over time. Band intensities of cytoplasmic and nuclear fractions were normalized to appropriate controls, added, and graphed relative to 100%. No change in subcellular distribution of CUGBP1 was observed in an extended analysis for 5 days after IFNβ treatment (data not shown). Results are representative of three independent experiments.

 
Phosphorylation of CUGBP1 is thought to be critical for its activity, because the dephosphorylation of CUGBP1 abolishes the binding to C/EBPβ mRNA and consequently decreases the translation of LIP (25). Further, overexpression of CUGBP1 in the absence of cellular activation fails to stimulate LIP expression (39), suggesting that CUGBP1 binding to RNA requires posttranslational modification(s). To examine the phosphorylation state of CUGBP1 before and after IFNβ treatment, in vitro kinase assays were performed on CUGBP1 immunoprecipitated from differentiated U937 cells (Fig. 5A). Stimulation of cells with IFNβ increased the phosphorylation of CUGBP1; the phosphorylation was greatest 12–24 h after treatment with IFNβ (Fig. 5A, inset) and paralleled the increased formation of CUGBP1-C/EBPβ mRNA complexes (Fig. 2A). We also examined CUGBP1 phosphorylation in immunoprecipitates from differentiated cells that were metabolically labeled with [32P]orthophosphate in the presence or absence of IFNβ. Consistent with the results from the kinase assay, treatment with IFNβ resulted in increased incorporation of 32P into CUGBP1 (Fig. 5B). Collectively, these data support a model in which IFNβ-induced phosphorylation of CUGBP1 increases its binding to RNA. Given the rapid effect of IFNβ on CUGBP1 phosphorylation and the formation of CUGBP1-C/EBPβ mRNA complexes, it is reasonable to predict that IFNβ-mediated activation of CUGBP1 regulates the production of LIP and the subsequent suppression of SIV replication, which in our system occurs within 24 h after treatment with IFNβ.


Figure 5
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  		FIGURE 5. IFNβ signaling induces the phosphorylation of CUGBP1. A, Quantitative analysis of five independent in vitro kinase assays of CUGBP1 immunoprecipitates from PMA-differentiated U937 cells treated or not treated with 100 U/ml IFNβ for the indicated times as described in Materials and Methods. Bars represent a fold induction in the intensity of CUGBP1 phosphorylation after treatment with IFNβ over the untreated control (1.0) at the indicated times. There is a statistically significant difference in the phosphorylation of CUGBP1 in IFNβ-treated samples compared with untreated samples (*, p < 0.05; paired t test. The inset depicts the expression of CUGBP1 in [35S]Met/Cys-labeled U937 cells and the in vitro phosphorylation of CUGBP1 at 12 h post-IFNβ treatment. No statistically significant difference in CUGBP1 expression was observed in five experiments (p > 0.05; paired t test). B, Autoradiograph of analysis of CUGBP1 immunoprecipitates prepared from [32P]orthophosphate- and [35S]Met/Cys-labeled U937 cells that were treated or not treated with 100 U/ml IFNβ for the indicated times. There is a statistically significant increase in 32P labeling but not 35S labeling of CUGBP1 in IFNβ-treated samples compared with untreated samples (p < 0.05; paired t test). A basal level of phosphorylation of CUGBP1 is observed in the untreated cells, which is increased upon stimulation with IFNβ. Shown is a representative experiment, n = 3.

 
CUGBP1 is required for IFNβ-mediated inhibition of SIV replication

Because IFNβ inhibits SIV replication in macrophages and induces the activation of CUGBP1, which has been linked to the production of LIP that, in turn, suppresses SIV LTR activity, we reasoned that the depletion of CUGBP1 would suppress the antiviral activity of IFNβ. Transfection of a CUGBP1-specific siRNA pool into macrophages decreased the level of CUGBP1 protein by ~75% as compared with parallel nonspecific siRNA or Lipofectamine-only control transfections (Fig. 6A). When cells were depleted (~75%) of CUGBP1, IFNβ failed to induce LIP and inhibit SIV replication to the same extent as in control transfections (Fig. 6, B and C), confirming that CUGBP1 is an essential component of at least one antiviral effector pathway induced by IFNβ. Not surprisingly, IFNβ inhibition of SIV replication was not completely relieved by depleting CUGBP1 with siRNA, likely due to the incomplete knockdown of CUGBP1 as well as other antiviral mechanisms triggered by IFNβ.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Studies from our laboratory have consistently demonstrated that the suppression of acute SIV replication in the brain occurs without an observable decrease in viral DNA levels (12, 13), indicating that active virus replication is suppressed, in large part, at a transcriptional level. Moreover, we have reported compelling evidence to implicate IFNβ-induced LIP in this suppression and have argued that this classical innate immune response to virus infection provides an inducible conduit for HIV/SIV latency in the brain (12). In the present study, we demonstrate that IFNβ-mediated induction of LIP and suppression of SIV replication in primary macaque macrophages requires CUGBP1. By extension, these results strongly suggest that this CUGBP1-dependent mechanism mediates the suppression of acute SIV replication in the brain and the establishment of a latent CNS viral reservoir.

Because truncated, dominant-negative LIP is translated from the same mRNA as full-length C/EBPβ, it has been impossible to selectively knock down the expression of LIP without negatively impacting the expression of full-length C/EBPβ, which is an important transactivator of the SIV/HIV LTR in macrophages. Thus, we have previously been unable to demonstrate unequivocally that LIP is required for IFNβ-mediated suppression of SIV replication. What we have demonstrated, using a titration strategy in cotransfection experiments, is that increasing the expression of LIP relative to full-length C/EBPβ suppresses C/EBPβ-mediated transactivation of the SIV LTR (12). In the present study, we make use of the only experimental strategy to date able to knock down the expression of LIP but not of full-length C/EBPβ, namely, the knockdown of CUGBP1 expression. Although it is conceivable that CUGBP1 regulates the expression of other yet unidentified proteins, the results from these studies nonetheless strengthen the hypothesis that LIP is essential for IFNβ-mediated suppression of SIV replication.

The significant contribution of the IFN system, particularly type I IFN, to immune responses against virus infection is underscored by the number of strategies that have evolved by viruses to thwart the antiviral activities of IFNs (reviewed in Ref. 40). To effectively protect cells from a broad spectrum of viruses, it is not surprising that type I IFN exerts its antiviral activity through multiple mechanisms that include the RNA-dependent protein kinase (PKR)/eukaryotic initiation factor 2 {alpha} pathway, the oligoadenylate synthetase/RNase L pathway, and myxovirus resistance GTPase (40, 41). Several new host cell-associated proteins, such as TRIM5{alpha} and APOBEC3G (42, 43), have been recently identified that represent potent opposition to HIV infection, at least in some circumstances, after viral entry and before viral integration into the nucleus. The demonstration that IFNβ activates CUGBP1, an RNA-binding protein (RBP), provides a new dimension by which type I IFN mediates its antiviral activity. RBPs are involved in a variety of processes that are critical for appropriate protein expression, including posttranscriptional processing of RNAs, splicing, polyadenylation, nuclear export, and translational control (44). Recent work indicates that several host RBPs are usurped into the HIV replication cycle (45). For example, the DDX3 and DDX1 DEAD box proteins have been identified as critical cofactors for the Rev-mediated export of singly spliced and unspliced viral mRNAs (46), and heterogeneous nuclear ribonucleoproteins have been implicated in both the trafficking and the splicing of HIV RNAs (47, 48, 49, 50). In striking contrast, the activation of CUGBP1 by IFNβ represents a unique and powerful antiviral mechanism to complement another IFN-inducible RBP that has been shown to inhibit Rev-mediated HIV-1 expression (51). It follows from these observations that RBPs may represent important cellular factors that can be manipulated to alter viral replication in several therapeutically useful ways.

Although limited, existing reports have consistently linked the ability of CUGBP1 to bind C/EBPβ mRNA to phosphorylation (25, 52). Dephosphorylation of CUGBP1 was shown to abolish binding to C/EBPβ mRNA and, accordingly, decrease the translation of LIP (25). In apparent agreement, our data show that the formation of CUGBP1-C/EBPβ mRNA complexes is distinctly enhanced by IFNβ treatment and correlates temporally with IFNβ-induced phosphorylation of CUGBP1. Despite the presence of multiple phosphorylation sites, little is known about the kinase(s) responsible for the phosphorylation of CUGBP1. Previous studies, however, have demonstrated that myotonin protein kinase and cyclin-dependent kinase 4 can phosphorylate CUGBP1 in some tissues (52, 53). It will be interesting to determine whether common pathways exist within complex independent signaling pathways to mediate the phosphorylation of CUGBP1 or whether CUGBP1 represents a common target for a variety of kinase pathways.

Although CUGBP1 can be activated to bind C/EBPβ mRNA in a number of cell types and in response to signals that mobilize pathways leading to increased expression and increased cytoplasmic localization of CUGBP1 (39, 54), neither event occurred in our studies of primary macaque macrophages or differentiated human U937 cells exposed to IFNβ. This observation suggests that the critical level of CUGBP1 protein required to achieve the appropriate balance of C/EBPβ to LIP in response to IFNβ already exists in macrophages. However, the same may not be true in other cell types in response to other stimuli. Moreover, specific ratios of LAP to LIP may differ between cell types. For example, because C/EBPβ is the predominant C/EBP family member up-regulated upon monocyte-macrophage differentiation (55), there may be an unforgiving ratio of these proteins that is inconsistent with survival or phenotype identity in macrophages. In this regard, CUGBP1 is also induced upon monocyte differentiation (J. M. Dudaronek, unpublished observations) and may be required to support macrophage-specific responses and functions.

The ability to recognize a diverse assortment of microbial pathogens is critical for the surveillance role played by cells of the innate immune system, including macrophages, as well as the ability to initiate an appropriate immune response (56). Downstream events in this process include the production of proinflammatory cytokines, a hallmark of macrophage activation. C/EBP binding sites are integral to the production of and response to many proinflammatory mediators including cytokines such as TNF-{alpha}, IL-1β, and IL-6 (57, 58, 59, 60), which are produced during acute SIV infection of the brain (61, 62). In fact, the expression of these cytokines is entirely in phase with virus replication: high levels during active virus replication and low/undetectable levels during viral latency (62). Because LIP is a repressor of C/EBP sites whether in the context of viral LTRs or promoters such as TNF-{alpha} (21, 58), an attractive model emerges in which IFNβ, through CUGBP1 and subsequently LIP, interrupts the self-perpetuating cycle of macrophage activation and virus replication during acute SIV infection in the brain. Although a safe and effective response in an immune-privileged site, this process also promotes the establishment of a long-lived viral reservoir in the brain (12, 13) that, unfortunately, is preferable to the escalated inflammatory state characteristic of SIV CNS disease and HIV-associated dementia.


   Acknowledgments
 
We thank Lucio Gama and Brandon T. Bullock for technical assistance as well as the rest of the Retrovirus Laboratory for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants MH70306, NS47984, and NS07392 (to J.E.C.). Back

2 Address correspondence and reprint requests to Dr. Janice E. Clements, Johns Hopkins School of Medicine, Department of Molecular and Comparative Pathobiology, 733 North Broadway, Building 820, Baltimore, MD 21205. E-mail address: jclement{at}jhmi.edu Back

3 Abbreviations used in this paper: LTR, long terminal repeat; CUGBP1, CUG-repeat RNA-binding protein 1; LAP, liver-enriched transcriptional activator protein; LIP, liver-enriched transcriptional inhibitory protein; RBP, RNA-binding protein; RIP, ribonucleoprotein immunoprecipitation; RIPA, radioimmunoprecipitation assay; siRNA, small interfering RNA. Back

Received for publication May 3, 2007. Accepted for publication September 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Griffin, D. E.. 2003. Immune responses to RNA-virus infections of the CNS. Nat. Rev. Immunol. 3: 493-502. [Medline]
  2. Honda, Y., L. Rogers, K. Nakata, B. Y. Zhao, R. Pine, Y. Nakai, K. Kurosu, W. N. Rom, M. Weiden. 1998. Type I interferon induces inhibitory 16-kD CCAAT/enhancer binding protein (C/EBP)β, repressing the HIV-1 long terminal repeat in macrophages: pulmonary tuberculosis alters C/EBP expression, enhancing HIV-1 replication. J. Exp. Med. 188: 1255-1265. [Abstract/Free Full Text]
  3. Kornbluth, R. S., P. S. Oh, J. R. Munis, P. H. Cleveland, D. D. Richman. 1990. The role of interferons in the control of HIV replication in macrophages. Clin. Immunol. Immunopathol. 54: 200-219. [Medline]
  4. Barber, S. A., D. S. Herbst, B. T. Bullock, L. Gama, J. E. Clements. 2004. Innate immune responses and control of acute simian immunodeficiency virus replication in the central nervous system. J. Neurovirol. 10: (Suppl. 1):15-20. [Medline]
  5. Williams, K. C., S. Corey, S. V. Westmoreland, D. Pauley, H. Knight, C. deBakker, X. Alvarez, A. A. Lackner. 2001. Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS. J. Exp. Med. 193: 905-915. [Abstract/Free Full Text]
  6. Davis, L. E., B. L. Hjelle, V. E. Miller, D. L. Palmer, A. L. Llewellyn, T. L. Merlin, S. A. Young, R. G. Mills, W. Wachsman, C. A. Wiley. 1992. Early viral brain invasion in iatrogenic human immunodeficiency virus infection. Neurology 42: 1736-1739. [Abstract/Free Full Text]
  7. An, S. F., M. Groves, F. Gray, F. Scaravilli. 1999. Early entry and widespread cellular involvement of HIV-1 DNA in brains of HIV-1 positive asymptomatic individuals. J. Neuropathol. Exp. Neurol. 58: 1156-1162. [Medline]
  8. Flaherty, M. T., D. A. Hauer, J. L. Mankowski, M. C. Zink, J. E. Clements. 1997. Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus. J. Virol. 71: 5790-5798. [Abstract]
  9. Mankowski, J. L., M. T. Flaherty, J. P. Spelman, D. A. Hauer, P. J. Didier, A. M. Amedee, M. Murphey-Corb, L. M. Kirstein, A. Munoz, J. E. Clements, M. C. Zink. 1997. Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence. J. Virol. 71: 6055-6060. [Abstract]
  10. Zink, M. C., A. M. Amedee, J. L. Mankowski, L. Craig, P. Didier, D. L. Carter, A. Munoz, M. Murphey-Corb, J. E. Clements. 1997. Pathogenesis of SIV encephalitis: selection and replication of neurovirulent SIV. Am. J. Pathol. 151: 793-803. [Abstract]
  11. Clements, J. E., M. Li, L. Gama, B. Bullock, L. M. Carruth, J. L. Mankowski, M. C. Zink. 2005. The central nervous system is a viral reservoir in simian immunodeficiency virus, infected macaques on combined antiretroviral therapy: a model for human immunodeficiency virus patients on highly active antiretroviral therapy. J. Neurovirol. 11: 180-189. [Medline]
  12. Barber, S. A., L. Gama, J. M. Dudaronek, T. Voelker, P. M. Tarwater, J. E. Clements. 2006. Mechanism for the establishment of transcriptional HIV latency in the brain in a simian immunodeficiency virus-macaque model. J. Infect. Dis. 193: 963-970. [Medline]
  13. Clements, J. E., T. Babas, J. L. Mankowski, K. Suryanarayana, M. Piatak, Jr, P. M. Tarwater, J. D. Lifson, M. C. Zink. 2002. The central nervous system as a reservoir for simian immunodeficiency virus (SIV): steady-state levels of SIV DNA in brain from acute through asymptomatic infection. J. Infect. Dis. 186: 905-913. [Medline]
  14. Gessani, S., P. Puddu, B. Varano, P. Borghi, L. Conti, L. Fantuzzi, G. Gherardi, F. Belardelli. 1994. Role of endogenous interferon-β in the restriction of HIV replication in human monocyte/macrophages. J. Leukocyte Biol. 56: 358-361. [Abstract]
  15. Gessani, S., P. Puddu, B. Varano, P. Borghi, L. Conti, L. Fantuzzi, M. Papacchini, F. Belardelli. 1994. Role of interferons in the restriction of HIV replication in human monocytes/macrophages. Res. Immunol. 145: 659-662; discussion 662–653. [Medline]
  16. Gessani, S., P. Puddu, B. Varano, P. Borghi, L. Conti, L. Fantuzzi, F. Belardelli. 1994. Induction of β interferon by human immunodeficiency virus type 1 and its gp120 protein in human monocytes-macrophages: role of β interferon in restriction of virus replication. J. Virol. 68: 1983-1986. [Abstract/Free Full Text]
  17. Weiden, M., N. Tanaka, Y. Qiao, B. Y. Zhao, Y. Honda, K. Nakata, A. Canova, D. E. Levy, W. N. Rom, R. Pine. 2000. Differentiation of monocytes to macrophages switches the Mycobacterium tuberculosis effect on HIV-1 replication from stimulation to inhibition: modulation of interferon response and CCAAT/enhancer binding protein β expression. J. Immunol. 165: 2028-2039. [Abstract/Free Full Text]
  18. Ossipow, V., P. Descombes, U. Schibler. 1993. CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials. Proc. Natl. Acad. Sci. USA 90: 8219-8223. [Abstract/Free Full Text]
  19. Henderson, A. J., X. Zou, K. L. Calame. 1995. C/EBP proteins activate transcription from the human immunodeficiency virus type 1 long terminal repeat in macrophages/monocytes. J. Virol. 69: 5337-5344. [Abstract]
  20. Henderson, A. J., R. I. Connor, K. L. Calame. 1996. C/EBP activators are required for HIV-1 replication and proviral induction in monocytic cell lines. Immunity 5: 91-101. [Medline]
  21. Henderson, A. J., K. L. Calame. 1997. CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+ T cells. Proc. Natl. Acad. Sci. USA 94: 8714-8719. [Abstract/Free Full Text]
  22. Descombes, P., U. Schibler. 1991. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67: 569-579. [Medline]
  23. Timchenko, N. A., A. L. Welm, X. Lu, L. T. Timchenko. 1999. CUG repeat binding protein (CUGBP1) interacts with the 5' region of C/EBPβ mRNA and regulates translation of C/EBPβ isoforms. Nucleic Acids Res. 27: 4517-4525. [Abstract/Free Full Text]
  24. Timchenko, N. A., G. L. Wang, L. T. Timchenko. 2005. RNA CUG-binding protein 1 increases translation of 20-kDa isoform of CCAAT/enhancer-binding protein β by interacting with the {alpha} and β subunits of eukaryotic initiation translation factor 2. J. Biol. Chem. 280: 20549-20557. [Abstract/Free Full Text]
  25. Baldwin, B. R., N. A. Timchenko, C. A. Zahnow. 2004. Epidermal growth factor receptor stimulation activates the RNA binding protein CUG-BP1 and increases expression of C/EBPβ-LIP in mammary epithelial cells. Mol. Cell. Biol. 24: 3682-3691. [Abstract/Free Full Text]
  26. Timchenko, N. A., Z. J. Cai, A. L. Welm, S. Reddy, T. Ashizawa, L. T. Timchenko. 2001. RNA CUG repeats sequester CUGBP1 and alter protein levels and activity of CUGBP1. J. Biol. Chem. 276: 7820-7826. [Abstract/Free Full Text]
  27. Welm, A. L., S. L. Mackey, L. T. Timchenko, G. J. Darlington, N. A. Timchenko. 2000. Translational induction of liver-enriched transcriptional inhibitory protein during acute phase response leads to repression of CCAAT/enhancer binding protein {alpha} mRNA. J. Biol. Chem. 275: 27406-27413. [Abstract/Free Full Text]
  28. Zink, M. C., K. Suryanarayana, J. L. Mankowski, A. Shen, M. Piatak, Jr, J. P. Spelman, D. L. Carter, R. J. Adams, J. D. Lifson, J. E. Clements. 1999. High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis. J. Virol. 73: 10480-10488. [Abstract/Free Full Text]
  29. Niranjanakumari, S., E. Lasda, R. Brazas, M. A. Garcia-Blanco. 2002. Reversible crosslinking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26: 182-190. [Medline]
  30. Hoshino, Y., K. Nakata, S. Hoshino, Y. Honda, D. B. Tse, T. Shioda, W. N. Rom, M. Weiden. 2002. Maximal HIV-1 replication in alveolar macrophages during tuberculosis requires both lymphocyte contact and cytokines. J. Exp. Med. 195: 495-505. [Abstract/Free Full Text]
  31. Korth, M. J., M. D. Taylor, M. G. Katze. 1998. Interferon inhibits the replication of HIV-1, SIV, and SHIV chimeric viruses by distinct mechanisms. Virology 247: 265-273. [Medline]
  32. Sears, R. C., L. Sealy. 1994. Multiple forms of C/EBP β bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat. Mol. Cell. Biol. 14: 4855-4871. [Abstract/Free Full Text]
  33. Silverman, R. H.. 1997. 2–5A-Dependent RNase L: a regulated endoribonuclease in the interferon system. G. D’ Alessio, Jr, and J. F. Riordan, Jr, eds. Ribonucleases: Structure and Functions 515-551. Academic Press, New York, NY.
  34. de Veer, M. J., M. Holko, M. Frevel, E. Walker, S. Der, J. M. Paranjape, R. H. Silverman, B. R. Williams. 2001. Functional classification of interferon-stimulated genes identified using microarrays. J. Leukocyte Biol. 69: 912-920. [Abstract/Free Full Text]
  35. Sebastian, T., P. F. Johnson. 2006. Stop and go: anti-proliferative and mitogenic functions of the transcription factor C/EBPβ. Cell Cycle 5: 953-957. [Medline]
  36. Sebastian, T., R. Malik, S. Thomas, J. Sage, P. F. Johnson. 2005. C/EBPβ cooperates with RB:E2F to implement Ras(V12)-induced cellular senescence. EMBO J. 24: 3301-3312. [Medline]
  37. Zahnow, C. A., R. D. Cardiff, R. Laucirica, D. Medina, J. M. Rosen. 2001. A role for CCAAT/enhancer binding protein β-liver-enriched inhibitory protein in mammary epithelial cell proliferation. Cancer Res. 61: 261-269. [Abstract/Free Full Text]
  38. Zahnow, C. A., P. Younes, R. Laucirica, J. M. Rosen. 1997. Overexpression of C/EBPβ-LIP, a naturally occurring, dominant-negative transcription factor, in human breast cancer. J. Natl. Cancer Inst. 89: 1887-1891. [Abstract/Free Full Text]
  39. Bae, E. J., S. G. Kim. 2005. Enhanced CCAAT/enhancer-binding protein β-liver-enriched inhibitory protein production by Oltipraz, which accompanies CUG repeat-binding protein-1 (CUGBP1) RNA-binding protein activation, leads to inhibition of preadipocyte differentiation. Mol. Pharmacol. 68: 660-669. [Abstract/Free Full Text]
  40. Samuel, C. E.. 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14: 778-809. , table of contents. [Abstract/Free Full Text]
  41. Peng, G., K. J. Lei, W. Jin, T. Greenwell-Wild, S. M. Wahl. 2006. Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti-HIV-1 activity. J. Exp. Med. 203: 41-46. [Abstract/Free Full Text]
  42. Sheehy, A. M., N. C. Gaddis, J. D. Choi, M. H. Malim. 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418: 646-650. [Medline]
  43. Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, J. Sodroski. 2004. The cytoplasmic body component TRIM5{alpha} restricts HIV-1 infection in Old World monkeys. Nature 427: 848-853. [Medline]
  44. Musunuru, K.. 2003. Cell-specific RNA-binding proteins in human disease. Trends Cardiovasc. Med. 13: 188-195. [Medline]
  45. Krishnan, V., S. L. Zeichner. 2004. Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication. Retrovirology 1: 42[Medline]
  46. Dayton, A. I.. 2004. Within you, without you: HIV-1 rev and RNA export. Retrovirology 1: 35[Medline]
  47. Jacquenet, S., A. Mereau, P. S. Bilodeau, L. Damier, C. M. Stoltzfus, C. Branlant. 2001. A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H. J. Biol. Chem. 276: 40464-40475. [Abstract/Free Full Text]
  48. Marchand, V., A. Mereau, S. Jacquenet, D. Thomas, A. Mougin, R. Gattoni, J. Stevenin, C. Branlant. 2002. A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. J. Mol. Biol. 323: 629-652. [Medline]
  49. Mouland, A. J., H. Xu, H. Cui, W. Krueger, T. P. Munro, M. Prasol, J. Mercier, D. Rekosh, R. Smith, E. Barbarese, et al 2001. RNA trafficking signals in human immunodeficiency virus type 1. Mol. Cell. Biol. 21: 2133-2143. [Abstract/Free Full Text]
  50. Valente, S. T., S. P. Goff. 2006. Inhibition of HIV-1 gene expression by a fragment of hnRNP U. Mol. Cell. 23: 597-605. [Medline]
  51. Constantoulakis, P., M. Campbell, B. K. Felber, G. Nasioulas, E. Afonina, G. N. Pavlakis. 1993. Inhibition of rev-mediated HIV-1 expression by an RNA binding protein encoded by the interferon-inducible 9–27 gene. Science 259: 1314-1318. [Abstract/Free Full Text]
  52. Timchenko, L. T., E. Salisbury, G. L. Wang, H. Nguyen, J. H. Albrecht, J. W. Hershey, N. A. Timchenko. 2006. Age-specific CUGBP1-eIF2 complex increases translation of CCAAT/enhancer-binding protein β in old liver. J. Biol. Chem. 281: 32806-32819. [Abstract/Free Full Text]
  53. Roberts, R., N. A. Timchenko, J. W. Miller, S. Reddy, C. T. Caskey, M. S. Swanson, L. T. Timchenko. 1997. Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice. Proc. Natl. Acad. Sci. USA 94: 13221-13226. [Abstract/Free Full Text]
  54. Tanaka, N., Y. Hoshino, J. Gold, S. Hoshino, F. Martiniuk, T. Kurata, R. Pine, D. Levy, W. N. Rom, M. Weiden. 2005. Interleukin-10 induces inhibitory C/EBPβ through STAT-3 and represses HIV-1 transcription in macrophages. Am. J. Respir. Cell Mol. Biol. 33: 406-411. [Abstract/Free Full Text]
  55. Natsuka, S., S. Akira, Y. Nishio, S. Hashimoto, T. Sugita, H. Isshiki, T. Kishimoto. 1992. Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. Blood 79: 460-466. [Abstract/Free Full Text]
  56. Kaisho, T., S. Akira. 2006. Toll-like receptor function and signaling. J. Allergy Clin. Immunol. 117: 979-987; quiz 988. [Medline]
  57. Wedel, A., G. Sulski, H. W. Ziegler-Heitbrock. 1996. CCAAT/enhancer binding protein is involved in the expression of the tumour necrosis factor gene in human monocytes. Cytokine 8: 335-341. [Medline]
  58. Pope, R. M., A. Leutz, S. A. Ness. 1994. C/EBP β regulation of the tumor necrosis factor {alpha} gene. J. Clin. Invest. 94: 1449-1455. [Medline]
  59. Shirakawa, F., K. Saito, C. A. Bonagura, D. L. Galson, M. J. Fenton, A. C. Webb, P. E. Auron. 1993. The human prointerleukin 1 β gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction. Mol. Cell. Biol. 13: 1332-1344. [Abstract/Free Full Text]
  60. Akira, S., H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, T. Kishimoto. 1990. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9: 1897-1906. [Medline]
  61. Orandle, M. S., A. G. MacLean, V. G. Sasseville, X. Alvarez, A. A. Lackner. 2002. Enhanced expression of proinflammatory cytokines in the central nervous system is associated with neuroinvasion by simian immunodeficiency virus and the development of encephalitis. J. Virol. 76: 5797-5802. [Abstract/Free Full Text]
  62. Mankowski, J. L., S. E. Queen, J. E. Clements, M. C. Zink. 2004. Cerebrospinal fluid markers that predict SIV CNS disease. J. Neuroimmunol. 157: 66-70. [Medline]



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