HIV-1 transcriptional silencing caused by TRIM22 inhibition of Sp1 binding to the viral promoter
© Turrini et al. 2015
Received: 4 June 2015
Accepted: 1 December 2015
Published: 18 December 2015
Intracellular defense proteins, also referred to as restriction factors, are capable of interfering with different steps of the viral life cycle. Among these, we have shown that Tripartite motif 22 (TRIM22) suppresses basal as well as phorbol ester-induced HIV-1 long terminal repeat (LTR)-mediated transcription, independently of its E3 ubiquitin ligase activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) binding to the U3 region and Tat interaction with the TAR region of the HIV-1 LTR. As basal HIV-1 transcription is driven by the transcription factor specificity protein 1 (Sp1), we have investigated whether TRIM22 could interfere with Sp1-driven transcriptional activation of the HIV-1 LTR.
293T cells, devoid of endogenous TRIM22 expression, were transfected with a TRIM22-expressing plasmid together with reporter plasmids driven by the HIV-1 LTR promoter either containing or lacking Sp1 binding sites or with reporter plasmids driven by non-viral promoter sequences either containing or lacking the three Sp1 binding sites from the HIV-1 LTR. These reporter assays showed that TRIM22 efficiently inhibited Sp1-driven transcription. Knocking down TRIM22 expression in the CD4+ SupT1 T cell line increased the replication of Sp1-dependent HIV-1 variants. TRIM22 did not interact with Sp1, but prevented binding of Sp1 to the HIV-1 promoter, as demonstrated in protein-DNA pull down and chromatin immunoprecipitation assays.
TRIM22 acts as a suppressor of basal HIV-1 LTR-driven transcription by preventing Sp1 binding to the HIV-1 promoter.
Tripartite Motif (TRIM) proteins form a large family that encompasses several members with broad antiviral activities against both DNA and RNA viruses [1, 2]. TRIM22 has been previously shown to inhibit the replication of HIV-1 [3, 4], Influenza A virus , Hepatitis B and C viruses [6, 7] and encephalomyocarditis virus , although by different mechanisms. We have shown that TRIM22 inhibited both basal and PMA (phorbol, 12-myristate, 13-acetate) plus ionomycin-induced HIV-1 transcription, independently of its E3 ubiquitin-ligase activity. Furthermore, TRIM22 did not affect either NF-kB or Tat-activated HIV-1 transcription . As HIV-1 basal transcription is mainly driven by the transcription factor Sp1 that binds to the three binding sites present in the core enhancer of the U3 region in the HIV-1 LTR , we tested whether TRIM22 interfered with Sp1-dependent transcription of HIV-1. For this purpose, Luciferase (Luc) -based reporter constructs driven by a minimal HIV-1 LTR (HIV-1 LTR Luc) containing the TATA box, Tat-binding TAR sequences, and the three Sp1 sites (WT) were transfected in 293T cells, which are devoid of endogenous TRIM22 expression. To determine whether TRIM22 inhibition of Sp1-dependent transcription was also related to the hierarchical clustering of the three Sp1 binding sites, HIV-1 LTR Luc deletion/mutation variants that retained two (ΔSp1-III), one (ΔSp1-III + II) and no Sp1 sites (mSp1) were tested in the presence and absence of TRIM22 expression. As these reporters contain two tet operator (tetO) sites for the binding of the doxycycline-inducible transcriptional activator rtTA , an rtTA-expressing plasmid was co-transfected in 293T cells and transcription was activated by doxycycline added to the culture medium. The effect of TRIM22 was determined by co-transfecting 293T cells with either a TRIM22-expressing plasmid or the empty control plasmid pcDNA3.1(+).
To verify whether TRIM22 could inhibit Sp1-mediated transcription activation out of the context of the HIV-1 LTR, a reporter construct driven by two tetO sites coupled to the minimal cytomegalovirus (CMV) promoter and lacking any HIV-1 related promoter sequence (tetO-CMV configuration) was tested in the presence or absence of TRIM22-expressing plasmid. As shown in Fig. 1b, TRIM22 expression did not affect the Luc activity driven by tetO-CMV promoter. A similar promoter construct that included the three HIV-1 Sp1 binding sites (tetO-CMV-Sp1 configuration) was also tested. The presence of Sp1 binding sites increased the Luc activity ~ fourfold and, importantly, restored the inhibitory effect of TRIM22 on promoter-dependent transcription. All together, these findings demonstrate that TRIM22 expression inhibits Sp1-driven transcription from the HIV-1 LTR.
HIV-rtTA replicated more efficiently in TRIM22-KD cells than in CTRL-KD cells (Fig. 2b). In this virus, three Sp1 sites are present in the U3 promoter region, which explains why TRIM22 negatively influences viral replication. The tetO-CMV virus did not show any replication upon infection of CTRL-KD and TRIM22-KD SupT1 cells, which is likely due to the absence of NF-kB and Sp1 binding sites (Fig. 2c). The tetO-CMV-Sp1 virus replicated also very poorly in CTRL-KD cells (RT activity became detectable only from day 29 PI), but it replicated significantly better in the TRIM22-KD SupT1 cells (Fig. 2d). Altogether, these results demonstrate that TRIM22 interferes with HIV-1 replication that is dependent on Sp1 binding sites in the LTR.
Next, we performed chromatin IP (ChIP) on 293T cells transfected with an HIV-1 LTR-Luc plasmid in the presence or absence of a Flag-TRIM22 expression plasmid to further analyze the impact of TRIM22 on Sp1 binding to the viral promoter in the context of the complete HIV-1 LTR in vivo. IP was performed using anti-Sp1 and anti-Flag Abs to isolate Sp1-bound and TRIM22-bound DNA, respectively. The DNA released from the immune-complexes was analyzed by two independent quantitative real-time PCR that generated amplicons between −103/+14 (LTR promoter ) and +164/+244 (leader region ), respectively, relative to the transcription start site (+1). The LTR promoter PCR encompasses the three Sp1 binding sites whereas the leader region PCR is nearby two Sp1 binding sites. The rate of amplification was normalized on cross-linked non-IP chromatin (% of input). As expected, analysis of the Sp1-IP samples revealed reduced binding of the HIV-1 DNA in the presence of TRIM22, confirming that TRIM22 interferes with Sp1 binding to the HIV-1 LTR (Fig. 4c). In contrast, when the anti-Flag Ab was used to IP TRIM22 or when unrelated immunoglobulins (IgGs) were used as negative control, a similar low level of LTR fragments was detected in the presence and absence of TRIM22.
Overall, these results demonstrate that TRIM22 causes transcriptional repression of HIV-1 by interfering with the binding of Sp1 to the HIV-1 LTR promoter region. Consistent with the lack of DNA binding domains in TRIM proteins , we did not observe direct binding of TRIM22 to the HIV-1 LTR. These observations together with our inability to co-IP TRIM22 and Sp1 suggest an indirect effect of TRIM22 on Sp1 binding to the HIV-1 LTR, which may involve one or several other factors. This/these unidentified TRIM22 partner(s) could either promote a transcriptionally silenced heterochromatin configuration, as in the case of COUP-TF interacting protein 2 (CTIP2)  and c-Myc , or stimulate post-translational modifications of Sp1, as previously suggested for histone deacetylases (HDAC)  and p300 , thereby affecting its DNA-binding affinity. Furthermore, by preventing Sp1 binding to the promoter, TRIM22 might favor binding of Sp3, another Sp family member considered a repressor of transcription .
Sp1 is an ubiquitous factor and TRIM22 is expressed in different immune cells and induced by type I interferons . Therefore, TRIM22 has the potential to inhibit the transcription of several host genes driven by Sp1 and to suppress cellular or even tumor growth . Recent work has highlighted the peculiar configuration of the three adjacent Sp1 binding sites of the HIV-1 promoter as a guanine-rich sequence that can fold into a G-quadruplex (G4) structure  previously identified in eukaryotic oncogenes . Pharmacological stabilization of this structure promotes transcription silencing of both the HIV-1 LTR  and oncogenes . By preventing Sp1 binding, TRIM22 could favor the formation of the G4 structure in the HIV-1 LTR but also in oncogenes.
In conclusion, in the present study we demonstrate that TRIM22 acts as a negative regulator of HIV-1 replication via inhibition of basal Sp1-driven proviral transcription. We can thus speculate that TRIM22 favors either the establishment or maintenance of HIV-1 latency. TRIM22 could therefore represent a novel target for pharmacological interference in latently infected cells harboring replication-competent proviruses.
FT, AKR, SM and EV participated in the design of the study, study implementation, data analyses, and drafting of the report. ML, CVL, AD, AH and BB participated in study implementation, and drafting of the report. All authors read and approved the final manuscript.
FT performed experiments of the study as fulfillment of his Ph.D. degree of the International Ph.D. School of Molecular Medicine, Università Vita-Salute San Raffaele, Milan, Italy. We thank Antony Rodari (Service of Molecular Virology, Department of Molecular Biology, Université Libre de Bruxelles (ULB), Gosselies, Belgium), Annapaola Andolfo, Cinzia Magagnotti (ProMiFa, Protein Microsequencing Facility, San Raffaele Scientific Institute, Milano) for technical support and Guido Poli (San Raffaele Scientific Institute, Milano) for critical reading of the manuscript. CVL is Research Director of the Belgian Fund for Scientific Research (FRS-FNRS).
The authors declare that they have no competing interests.
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