Basic Science: Concise Communication
Expression levels of the innate response gene RIG-I and its regulators RNF125 and TRIM25 in HIV-1-infected adult and pediatric individuals
Britto, Alan M.A.a; Amoedo, Nívea D.b; Pezzuto, Paulaa; Afonso, Adriana O.a; Martínez, Ana M.B.c; Silveira, Jussarac; Sion, Fernando S.d; Machado, Elizabeth S.e; Soares, Marcelo A.a,f; Giannini, Ana L.M.a
aDepartamento de Genética, Instituto de Biologia
bInstituto de Bioquímica Médica, UFRJ, Rio de Janeiro
cFaculdade de Medicina, UFRG, Rio Grande
dHospital Universitário Gaffrée e Guinle, UNIRIO
eInstituto de Puericultura e Pediatria Martagão Gesteira, UFRJ
fPrograma de Genética, INCA, Rio de Janeiro, Brazil.
Correspondence to Ana Lúcia M. Giannini, CCS-Departamento de Genética, Instituto de Biologia, UFRJ, Rua Rodolpho P. Rocco, s/n, Bl. A, room A2-066, Ilha do Fundão, CEP 21941-617, Rio de Janeiro, RJ, Brazil. E-mail: firstname.lastname@example.org
Received 14 September, 2012
Revised 26 March, 2013
Accepted 3 April, 2013
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Objective: TLRs (Toll-like receptors) and RLRs (RIG-I-like receptors) mediate innate immune responses by detecting microorganism invasion. RIG-I activation results in the production of interferon (IFN) type 1 and IFN responsive genes (ISGs). As the ubiquitin ligases RNF125 and TRIM25 are involved in regulating RIG-I function, our aim was to assess whether the levels of these three genes vary between healthy and HIV-infected individuals and whether these levels are related to disease progression.
Design: Gene expression analyses for RIG-I, RNF125, and TRIM25 were performed for HIV-infected adults and the children's peripheral blood mononuclear cells (PBMCs).
Methods: Reverse transcription-quantitative PCRs (RT-qPCRs) were performed in order to quantify the expression levels of RIG-I, RNF125 and TRIM25 from PBMCs purified from control or HIV-infected individuals.
Results: Controls express higher levels of the three genes when compared to HIV-infected patients. These expressions are clearly distinct between healthy and progressors, and are reproduced in adults and children. In controls, RNF125 is the highest expressed gene, whereas in progressors, RIG-I is either the highest expressed gene or is expressed similarly to RNF125 and TRIM25.
Conclusion: A pattern of expression of RIG-I, RNF125, and TRIM25 genes in HIV patients is evident. The high expression of RNF125 in healthy individuals reflects the importance of keeping RIG-I function off, inhibiting unnecessary IFN production. Consistent with this assumption, RNF125 levels are lower in HIV patients and importantly, the RNF125/RIG-I ratio is lower in patients who progress to AIDS. Our results might help to predict disease progression and unveil the role of poorly characterized host genes during HIV infection.
Over 34 million people are infected by HIV worldwide  and resistant strains have emerged since the development of the HAART therapy . Recent focus on host proteins, which interfere with infection outcome, led to the discovery of host restriction factors [3–7] and viral sensors (Toll-like receptor, TLR and RIG-I-like receptor, RLR) [8–11]. Recently, it has been shown that genomic HIV RNA can be detected by RIG-I , a RNA helicase. Upon RNA recognition, the caspase recruitment domains (CARD domains) present in this protein interact with similar ones in MAVS (mitochondrial antiviral signaling protein) at the mitochondria  activating the transcription of interferon (IFN) via nuclear factor-κB (NF-κB) and IRF3 (interferon regulatory factor 3). RIG-I CARD domains undergo posttranslational modifications [13–16], including ubiquitination , which influence its capacity to regulate IFN production. RNF125 and TRIM25 are ubiquitin ligases that can mediate the latter process [18,19]. TRIM25 belongs to the tripartite motif (TRIM) family and its function activates RIG-I, hence increasing IFN production [19,20]. Several TRIM proteins participate in HIV infection ; TRIM5α interferes with viral uncoating  and is differentially expressed in HIV-positive patients as is TRIM22 [23–25].
RNF125 targets RIG-I for destruction in the proteasome , is involved in T-cell activation, and inhibits HIV transcription . TRIM25 and RNF125 genes are regulated by IFN [18,23] providing regulatory mechanisms for the production of this cytokine, whose fine balance is essential to achieve a protective rather than an immunopathogenic effect in HIV-positive patients [27,28].
Data suggest that high levels of expression of IFN responsive genes (ISGs) are related to viral replication inhibition in vitro; however, in-vivo data are scarce and controversial . Higher levels of mRNA for RIG-I and RNF125 were found in hepatic tissues from hepatitis C virus (HCV)-positive patients . However, the simultaneous analysis of RIG-I expression levels and those of its positive (TRIM25) and negative (RNF125) regulators have not been investigated in an HIV setting. This information could be useful to predict progression to AIDS. Here, we performed qPCR analysis of RIG-I, RNF125, and TRIM25 in HIV-positive adults and children. Our results suggest that the levels of these three genes vary according to disease progression.
Participants came from the University Hospitals at Federal Universities of Rio Grande (FURG) and Rio de Janeiro (UNIRIO), Brazil. Children were from the Pediatrics Institute, Federal University of Rio de Janeiro (UFRJ). This study was approved by Institutional Review Boards of the three institutions.
Thirty-four adults (14 HIV-negative controls and 20 HIV-positive patients from which 10 were nonprogressors and 10 progressors as defined elsewhere ) were included in the study. The children's group had eight HIV-negative controls and 12 HIV-positive children – (four nonprogressors and eight progressors as defined in the literature ) and samples were retrieved from a previous study .
Sample preparation and quantitative PCR
RNA was extracted by TRIzol (Life Technologies, Grand Island, New York, USA) from Ficoll-paque isolated peripheral blood mononuclear cells (PBMCs) and cDNA was synthesized using SuperScript II (Promega, Madison, Wisconsin, USA) and random primers (Life Technologies) following the manufecturer's instructions. Taqman primers, probes, and equipment (ABI 7500) were from Life Technologies. Results are shown as the ratio: target gene/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expressions (2-ΔCt). Mann–Whitney U-tests and Spearman's rho test were used for statistical data and correlations analysis, respectively.
’In-vitro’ interferon treatment, HIV infection, and western blots
Jurkat cells were treated for different times with IFN-α (100 ng/ml) or IFN-γ (50 ng/ml; data shown as 2-ΔΔCt). For HIV infections, 106 PBMCs were spinoculated at 1200 g for 2 h with 50 ng of NL4.3-Luc (normalized by p24 ELISA; Zeptometrix Kit-Retrotek). Frozen PBMCs were used to perform the western blots. RIG-I and α-tubulin antibodies were from Abcam (Cambridge, Massachusetts, USA).
Participants’ clinical data
Adult patients were followed up for at least 3 years with quarterly quantification of HIV viral load and CD4+ T-cells. Ages, CD4+ T-cell counts, and HIV viral load averaged 40.4 years ± 10.7; 582.1/μl ± 343.53, and 4.33 log ± 1.31, respectively. For the children cohort, ages, CD4+ T-cell counts, and viral load averaged 7.16 years ± 4.59; 19.75% ± 9.8, and 4.09 log ± 1.22, respectively (Supplementary Table 1, http://links.lww.com/QAD/A348).
Expression of RIG-I, RNF125, and TRIM25
The genes studied here are involved in antiviral signaling, they respond to IFN, and their protein products directly interact, so we assessed their expression levels in HIV-negative and HIV-positive individuals to verify whether these reflected HIV infection status and could be linked to disease progression.
HIV-negative controls expressed higher levels of the three genes and, in these participants, RNF125 was the highest expressed gene (78% of the adults and 75% of the children), reaching twice the levels of TRIM25 in more than 62% of these (Fig. 1a–e). This expression was four and 2.5 times higher in HIV-negative compared to HIV-positive adults and children, respectively. In both cohorts, progressors and nonprogressors expressed similar levels of RNF125, indicating that RNF125 expression does not reflect disease progression (Supplementary Figs 1 and 2, http://links.lww.com/QAD/A348). In contrast, RIG-I was the highest expressed gene in progressor adults (70%) and children (50%; Supplementary Figs 1 and 2, http://links.lww.com/QAD/A348). In adult progressors, the ratio RNF125/RIG-I is significantly lower than this ratio in HIV-negative controls (Fig. 1f). This pattern of higher expression of RIG-I in HIV-negative controls compared to HIV-positive participants was confirmed at protein levels for some of the samples which were still available (Fig. 2a and b).
In order to elucidate whether the expression patterns observed could be seen at the beginning of the infection, in-vitro HIV infections using PBMCs isolated from two HIV-negative blood donors were performed, followed by qPCRs. As seen in Fig. 2c and d, RNF125 and RIG-I doubled in the first 48 h; however, no major increases were detected for TRIM25 and the two other ISGs APOBE3G and 3F after HIV exposure.
The results suggest that the genes for RIG-I regulators and RIG-I itself are expressed differently in progressors compared to HIV-negative individuals, following a specific pattern that is independent of age, mode of transmission, or antiretroviral therapy (ART) [samples were collected at a time when only two of the 19 adults (F7 and F12) were subjected to therapy]. However, this pattern is only observed in an established chronic disease, suggesting the participation of other players of the immune system.
Although a negative correlation exists between the levels of RIG-I and CD4+ T-cell counts, no significant correlation was found for any of the genes expressions alone and HIV viral load. These results show that RIG-I is not the sole determinant of viremia (Supplementary Table 2, http://links.lww.com/QAD/A348).
Levels of RIG-I and TRIM25 were correlated, but this was not found for RIG-I and RNF125. As the three genes are regulated by IFN, their expressions should all be correlated, unless they respond differently to this cytokine. To clarify this issue, Jurkat cells were treated with IFN-α or IFN-γ and q-PCR was performed. RIG-I and TRIM25 expressions were detected 2 h after IFN-α treatment, whereas that of RNF125 doubled only at 48 h (Fig. 2e). RIG-I expression in response to IFN-α was more intense (eight-fold increase after 24 h), whereas TRIM25 mRNA levels triplicated at this time point. Hence, RIG-I and TRIM25 genes are more sensitive to IFN-α in vitro than the RNF125 gene. Of these three ISGs, only RIG-I responded to IFN-γ (Fig. 2f).
Our hypothesis was that the levels of innate immunity genes would reflect HIV infection and disease progression. Hence, levels of RIG-I (a viral sensor) and two of its regulators, RNF125 and TRIM25 (ubiquitin ligases with opposing functions) were quantified by qPCR. Our study is the first to demonstrate the potential relevance of RIG-I and the ubiquitin ligases RNF125 and TRIM25 together in an HIV scenario using HIV-positive adults and children.
We concluded that none of the gene levels per se can be used to predict disease progression. However, when the expressions of the three are analyzed together, a disease progression-dependent pattern was detected. RNF125 was the highest expressed gene in HIV-negative controls, whereas RIG-I was the highest expressed gene in progressors and the ratio RNF125/RIG-I was higher in the former group (Fig. 1f). We speculate that in a chronically infected individual, with detectable viral load, the presence of the virus maintains the innate response activated via RIG-I, resulting in IFN production, which on its turn, maintains RIG-I and TRIM25 transcription ongoing, explaining the positive correlation found for RIG-I and TRIM25 mRNAs. High levels of RIG-I would keep IFN production on, resulting in CD4+ T-cell apoptosis . In fact, a negative correlation was found between CD4+ cell counts and RIG-I levels, but none was found between RIG-I's expression and viral load, suggesting the participation of other host restriction factors in the control of viremia. This is also corroborated by the suggested role of RNF125 in inhibition of HIV transcription .
Although RNF125 has been described to respond to type I IFNs , in our hands this induction was approximately four times smaller than that observed for RIG-I, leading us to conclude that RNF125 expression is regulated by factors other than IFN. This conclusion is also corroborated by the higher levels of RNF125 found in controls, whose levels of circulating IFN should not be high in the absence of infection. We believe that RNF125 protein has a major role in maintaining the innate immunity signaling off (by targeting RIG-I for destruction). In fact, when RNF125 is exogenously expressed, endogenous levels of RIG-I decrease . RNF125 also has a short half-life , so cells would have to continually transcribe and translate its gene to maintain its functional cellular levels. The identification and characterization of the RNF125's promoter would help to elucidate its regulation.
Although it would have been interesting to sort the different cell types from the patients’ PBMCs to evaluate how each one contributes to the expression of genes studied here, the fact that Jurkat cells (immortalized CD4+T-cell) analysis leads to similar expression pattern found for PBMCs from controls suggests that T-lymphocytes are the main contributors to the results found here (data not shown). This is also supported by the expression data published in BioGPS site, in which the expression of the RNF125 and RIG-I genes was higher in CD8+ and CD4+ cells. Two features detected during HIV infection are the depletion of the CD4+ T-cell population and the expansion of the CD8+ T-cell one. As both CD8+ and CD4+ cells express the highest levels of both RIG-I and RNF125, the sorting of these cell types from patients might not have been clarifying here [34,35].
In a HCV-positive scenario, sustained nonvirological responders (NVRs) express more RIG-I and less RNF125 (lower RNF125/RIG-I ratio) compared to sustained virological responders (SVRs) [29,36]. Similarly, we found that in an HIV scenario, progressors have a lower RNF125/RIG-I ratio when compared to nonprogressors and controls (Fig. 1f). Hence, these ratios can be useful to predict both NVR and progression in HCV and HIV scenarios, respectively.
Although at present we are unable to explain the higher expression of the three genes in PBMCs from controls, this has been also found for IRF1 in a HCV scenario . Moreover, TLR3, TLR7, RIG-I, and IFN-α mRNA levels are significantly downregulated in patients with chronic HCV infection when compared with healthy controls . Also, there has been controversy concerning ISG expression during HIV infection [38–40]. The different virus biology and escape routes might account for these differences [41–45]. Nevertheless, our results support the findings of others and start to unveil the important role of yet uncharacterized players in HIV infections.
The authors would like to thank Dr Amilcar Tanuri and his group for the help and facilities to set the qRT-PCR.
A.M.A.B., N.D.A., and A.O.A. contributed to the purifications of PBMCs, RNA, cDNA, and the performance and analysis of the qRT-PCR reactions. P.P. performed the IFN treatment of Jurkat cells, prepared their cDNAs and performed the western blots. A.M.B.M. and J.S. organized the clinical data from adults enrolled in the Dr Miguel Riet Corrêa Junior University Hospital-Federal University of Rio Grande (FURG). F.S.S. and E.S.M. were responsible for the clinical data collection from the adult patients from the Gaffrée and Guinle University Hospital-Federal University of the Rio de Janeiro State (UNIRIO) and the pediatric individuals from the Martagão Gesteira Institute of Pediatrics-Federal University of Rio de Janeiro (UFRJ). A.L.M.G. and M.A.S. were responsible for the design of the experiments and preparing the article.
This work was funded by CNPq and FAPERJ agencies, Brazil.
Conflicts of interest
The authors declare that no conflicts of interest exist.
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HIV infection; innate immunity; ubiquitin ligase RNF125; ubiquitin ligase TRIM25; viral sensor RIG-I
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