
==== Front
Mem Inst Oswaldo Cruz
Mem Inst Oswaldo Cruz
mioc
Memórias do Instituto Oswaldo Cruz
0074-0276
1678-8060
Instituto Oswaldo Cruz, Ministério da Saúde

10.1590/0074-02760240071
01123
Research Article
HIV-1 controllers exhibit an enhanced antiretroviral innate state characterised by overexpression of p21 and MCPIP1 and silencing of ERVK-6 RNA expression
https://orcid.org/0000-0001-6681-0987
de Azevedo Suwellen Sardinha Dias conducted the experiments analysed the data together with MR-A, ED and GB wrote the first draft 1 +
Ribeiro-Alves Marcelo wrote the first draft 2
Côrtes Fernanda Heloise participated in sample processing provided intellectual input for interpretation of the results 1
Delatorre Edson collaborated in the mRNA gene expression analysis provided intellectual input for interpretation of the results 3
Hoagland Brenda conducted patient recruitment and follow-up 2
Villela Larissa M 2
Grinsztejn Beatriz conducted patient recruitment and follow-up 2
Veloso Valdilea Gonçalvez conducted patient recruitment and follow-up 2
Morgado Mariza G provided intellectual input for interpretation of the results 1
Souza Thiago Moreno L conceived and designed the study supervised the experiments 4 5
Bello Gonzalo conceived and designed the study supervised the experiments participated in sample processing provided intellectual input wrote the first draft 1 6
1 Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de AIDS & Imunologia Molecular, Rio de Janeiro, RJ, Brasil
2 Fundação Oswaldo Cruz-Fiocruz, Instituto Nacional de Infectologia Evandro Chagas, Laboratório de Pesquisa Clínica em DST/AIDS, Rio de Janeiro, RJ, Brasil
3 Universidade Federal do Espírito Santo, Centro de Ciências da Saúde, Departamento de Patologia, Laboratório de Genômica e Ecologia Viral, Vitória, ES, Brasil
4 Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Imunofarmacologia, Rio de Janeiro, RJ, Brasil
5 Fundação Oswaldo Cruz-Fiocruz, Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças de Populações Negligenciadas, Rio de Janeiro, RJ, Brasil
6 Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Arbovírus e Vírus Hemorrágicos, Rio de Janeiro, RJ, Brasil
+ Corresponding author: suwellendias@gmail.com
he authors declare no conflict of interest.

All authors have read and agreed to the published version of the manuscript.

16 9 2024
2024
119 e24007101 4 2024
12 7 2024
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License
BACKGROUND

Human immunodeficiency virus (HIV)-1 infection can activate the expression of human endogenous retroviruses (HERVs), particularly HERV-K (HML-2). HIV controllers (HICs) are rare people living with HIV (PLWHs) who naturally control HIV-1 replication and overexpress some cellular restriction factors that negatively regulate the LTR-driven transcription of HIV-1 proviruses.

OBJECTIVES

To understand the ability of HICs to control the expression of endogenous retroviruses.

METHODS

We measured endogenous retrovirus type K6 (ERVK-6) RNA expression in peripheral blood mononuclear cells (PBMCs) of HICs (n = 23), antiretroviral (ART)-suppressed subjects (n = 8), and HIV-1-negative (NEG) individuals (n = 10) and correlated the transcript expression of ERVK-6 with multiple HIV-1 cellular restriction factors.

FINDINGS

Our study revealed that ERVK-6 RNA expression in PBMCs from HICs was significantly downregulated compared with that in both the ART and NEG control groups. Moreover, we detected that ERVK-6 RNA levels in PBMCs across all groups were negatively correlated with the expression levels of p21 and MCPIP1, two cellular restriction factors that limit the activation of macrophages and T cells by downregulating the activity of NF-kB.

MAIN CONCLUSIONS

These findings support the hypothesis that HICs activate innate antiviral mechanisms that may simultaneously downregulate the transcription of both exogenous (HIV-1) and endogenous (ERVK-6) retroviruses. Future studies with larger cohorts should be performed to confirm this hypothesis and to explore the role of p21 and MCPIP1 in regulating HERV-K expression in physiological and pathological conditions.

Key words:

HIV-1
ERVK-6
elite controllers
viremic controllers
restriction factors
FAPERJE-26/110.123/2014 E-26/202.335/2019 E-26/202.896/2018 CNPq401220/2016-8 304883/2020-4 FAPERJ (Grant no. E-26/110.123/2014), CNPq (Grant no. 401220/2016-8).SSDA is financed by a postdoctoral fellowship from the “Pós-Doutorado Nota 10 (PDR 10)” by FAPERJ (Grant no. E-26/202.335/2019); GB is funded by CNPq (Grant no. 304883/2020-4) and FAPERJ (Grant no. E-26/202.896/2018)
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pmcHuman endogenous retroviruses (HERVs) are remnants of ancient viral infections integrated into the human genome throughout evolutionary history. These genetic elements are estimated to comprise nearly 8% of the human genome and are passed from generation to generation. 1 Although most HERVs are now functionally inactive due to mutations and deletions, some have been implicated in disease processes, such as autoimmune and neurodegenerative disorders, several types of cancer, and immune dysregulation and coagulopathy in critical coronavirus disease 2019 (COVID-19). 2 - 9

HERV-K represents the latest classified family, encompassing the most recently integrated HERV groups within the human genome. 10 HERV-Ks can be further divided into ten subfamilies (HML 1 - 10), with some retaining their structural integrity and ability to generate viral proteins such as HERV-K (HML-2). 11 Interestingly, previous studies revealed that human immunodeficiency virus (HIV)-1 can activate HERV-K (HML-2) mRNA and protein expression in vitro. 12 , 13 , 14 , 15 Moreover, untreated people living with HIV (PLWHs) display higher levels of HERV-K RNA in peripheral blood mononuclear cells (PBMCs) 4 , 13 , 16 or plasma 17 , 18 , 19 than antiretroviral therapy (ART)-treated individuals and/or HIV-uninfected healthy subjects. One study demonstrated that HIV-1 Tat plays an important role in activating the expression of HERV-K (HML-2). 20 HIV-1 infection may also enhance HERV-K (HML-2) transcription in both infected and uninfected cells, 4 probably by indirect mechanisms influencing immune activation and inflammation 21 and/or the cellular epigenetic environment. 22

These findings suggest that LTR-directed transcription of both HERV-K and HIV-1 proviruses could be regulated by common viral and cellular transcription factors. We previously observed that the expression levels of two multifunctional cellular proteins, cyclin-dependent kinase (CDK) inhibitor 1A (CDKN1A/p21) and monocyte chemotactic protein-induced protein 1 (MCPIP1), were significantly elevated in a group of rare PLWHs who naturally control plasma viremia without ART, called HIV controllers (HICs), compared with ART-suppressed and HIV-1-negative individuals. 23 Interestingly, p21 and MCPIP1 are critical to maintaining immune-system homeostasis and contribute to limiting the activation of macrophages and T cells by downregulating NF-kB activity, 24 , 25 , 26 , 27 , 28 , 29 a transcription factor that stimulates the LTR-driven activation of both HIV-1 and HERV-K proviruses. Thus, HICs overexpress cellular restriction factors that may negatively regulate the transcription of HERV-K. However, the precise relationship between the natural control of HIV replication and the expression of HERVs remains unclear.

To test the hypothesis that cellular restriction factors overexpressed in HIC may also negatively regulate HERVs, we performed an exploratory analysis focused on the transcriptional activity of endogenous retrovirus type K6 (ERVK-6) proviral locus that belongs to the HERV-K (HML-2) subfamily and has been reported to be activated in cells persistently infected by HIV. 14 We measured the ERVK-6 RNA expression levels (envelope protein fragment, also called HERV-K [HML-2] or HERV-K_7p22.1) in PBMCs of HICs (n = 23), HIV-1 ART-suppressed subjects (n = 10) and HIV-1-negative individuals (n = 8), and we also correlated the transcript expression levels of ERVK-6 with multiple HIV-restriction factors.

SUBJECTS AND METHODS

Study subjects - We analysed a cohort of 23 HICs followed up at the Instituto Nacional de Infectologia Evandro Chagas (INI) in Rio de Janeiro, Brazil and provided written informed consent documents approved by the INI Institutional Review Board (Addendum 049/2010) and the Brazilian National Human Research Ethics Committee (CONEP 14430/2011). The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 1983.

All HICs maintained an RNA VL of < 2000 copies/mL without antiretroviral therapy for at least five years and were subdivided into two subgroups: elite controllers (EC, n = 13) in whom most (≥ 70%) plasma VL determinations were below the limit of detection (LOD) and viremic controllers (VC, n = 10) in whom most (≥ 70%) VL determinations were > LOD and < 2000 copies/mL. The limit of detection of plasma VL determinations varied over the follow-up period according to the Brazilian Ministry of Health guidelines, with methodologies being updated over time to improve sensitivity: Nuclisens HIV-1 RNA QT assay (Organon Teknika, Durham, NC, USA, limit of detection: 80 copies/mL) from 1999 to 2007; the Versant HIV-1 3.0 RNA assay (bDNA 3.0, Siemens, Tarrytown, New York, NY, USA, limit of detection: 50 copies/mL) from 2007 to 2013; and the Abbott RealTime HIV-1 assay (Abbott Laboratories, Wiesbaden, Germany, limit of detection: 40 copies/mL) from 2013 to present. Previous studies detailed these subjects’ virological and immunological characteristics. 23 , 30 , 31 ART-suppressed subjects (ART, n = 8) and healthy HIV-1-uninfected subjects (NEG, n = 10) were used as controls.

mRNA gene-expression analysis - Total RNA was extracted from 1 × 107 PBMCs using an RNeasy mini kit (Qiagen, North Rhine-Westphalia, Hilden, Germany) in which buffer RLT was supplemented with β-mercaptoethanol and displaced on-column DNase treatment using a Qiagen RNase-Free DNase Set (Qiagen, North Rhine-Westphalia, Hilden, Germany) according to the manufacturer’s instructions. Total RNA yield and quality were determined using NanoDrop® 8000 spectrophotometer and Agilent® (Santa Clara, CA, USA) 2100 Bioanalyzer. Only samples with an RNA integrity number (RIN) greater than 8.0 were used for further analysis. Purified RNA (1 μg) was reverse transcribed to cDNA using an RT2 First Strand Kit (Qiagen, North Rhine-Westphalia, Hilden, Germany). The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix (Qiagen, North Rhine-Westphalia, Hilden, Germany), and the mixture was added to a customised RT2 RNA PCR Array (Qiagen, North Rhine-Westphalia, Hilden, Germany) to measure the mRNA expression of ERVK-6 envelope protein, also called HERV-K (HML-2) or HERV-K_7p22.1 (Gene RefSeq #PPH60565A-200/NM_001007236), 13 cellular restriction factors (CDKN1A/p21, ZC3H12A/MCPIP1, APOBEC3G, IFITM1, IFITM2, IFITM3, SAMHD1, Mx1, Mx2, SERINC3, SERINC5, SLFN11, and Tetherin/Bst2), and three housekeeping genes (GAPDH, β-actin, and RNase-P), according to the manufacturer’s instructions. Values of the crossing point at the maximum of the second derivative of the four-parameter fitted sigmoid curve second derivative, Cp, were determined for each sample. The efficiency of each amplification reaction was calculated as the ratio between the fluorescence of the cycle of quantification and the fluorescence of the cycle immediately preceding that. Genes used in the normalisation among samples were selected by the geNorm method. 32 Data were expressed as fold changes in mRNA abundance calculated as the normalised gene expression in any test sample divided by the mean normalised gene expression in the control HIV-1 NEG group.

Data analyses - The comparisons of mean log(base 2)-fold changes (log-FC) in mRNA abundance were performed by either nonparametric t tests or one-way analysis of variance (ANOVA) permutation tests (B = 1000 permutations), followed by pairwise comparisons with Holm‒Bonferroni adjustment, 33 for two or more groups, respectively. The Pearson coefficient was used for correlation analyses. Finally, a multivariate principal component analysis (PCA) was performed for the log-transformed expression data to visualise the distribution of sample individuals according to either their group or their mRNA ERV-K 6 expression levels (divided into four different quartiles) in two-dimensional (2D) space. The proportion of explained variation was calculated by adding the successive proportions of variation explained to obtain the running total. The contributions (in percentage) of the variables to the principal components were calculated as (var. cos2 × 100)/(total cos2 of the component), where cos2 indicates square cosine or squared coordinates. Accordingly, the contributions (in percentage) of individuals to the principal components were calculated as (ind. cos2 × 100)/(total cos2 of the component). Ellipses of the quantiles with 66% of the normal distribution adjusted for the individuals in the different interest groups are presented in these new dimensional spaces. A p value ≤ 0.05 was used as the significance level in the analysis. All analyses were performed using R software version 4.1.2 34 and the packages ‘base’ for descriptive and correlation analyses and ‘FactoMineR’ 35 and ‘factoextra’ 36 for PCA and its graphic representation.

Availability of data - The datasets during and/or analysed during the current study are available from the corresponding author upon reasonable request.

RESULTS

Epidemiological, clinical and virological characteristics of the studied individuals - Thirty-one HIV-1-positive (23 HICs and eight ART-suppressed) and 10 HIV-negative individuals were included in this cross-sectional study (Table). The participants had a median age of 46 years [interquartile range (IQR): 40-52 years old]. Most HIV-positive (58%) and HIV-negative (60%) individuals were females, and all individuals displayed CD4+ T-cell counts above 500 cells/μL. The HICs were subdivided into EC and VC subgroups based on the history of viral load measurements since the positive diagnosis for HIV-1. The ECs had a median follow-up time of nine years and showed undetectable (< 40-80 copies/mL) viral load at most (≥ 70%) measurements, while the VCs had a median of 10 years of follow-up and showed low-level viremia (81-2000 copies/mL) at most (≥ 70%) visits. The control group of ART-suppressed subjects had a median of 13 years (IQR: 9-22.5) since HIV diagnosis and almost nine years (IQR: 7.25-11) on the use of ART.

TABLE Main epidemiological, clinical, and virological characteristics of individuals

Characteristics	HICs (n = 23)	ART (n = 8)	NEG (n = 10)	
ECs (n = 13)	VCs (n = 10)	
Sex, no. (%)					
Female	10 (77)	4 (40)	4 (50)	6 (60)	
Male	3 (23)	6 (60)	4 (50)	4 (40)	
Age (years)*	45 (39-60)	45 (42-49)	47 (38-53)	47 (36-51)	
Time since HIV-1 diagnosis (years) *	9 (6-15)	10 (7-15)	13 (9-23)	-	
Time since ART started (years)*	NA	NA	9 (7-11)	NA	
CD4+ T-cell (cells/μL)*	1027 (835-1255)	664 (607-1018)	889 (678-1097)	1043 (784-1581)	
CD4/CD8 ratio*	1.33 (1.24-1.61)	0.90 (0.62-1.07)	1.06 (0.73-1.5)	1.69 (1.62-2.00)	
Median viremia* (RNA copies/mL)	< 50	719 (512-2242)	< 40	-	
ART: antiretroviral therapy; ECs: elite controllers; HICs: human immunodeficiency virus controllers; NEG: negative; VCs: viremic controllers. *At the study point. Interquartile ranges (IQR) are shown in parentheses.

ERVK-6 mRNA levels are downregulated in HICs - We first evaluated the ERVK-6 transcript levels in the HIC and ART groups compared to the NEG group. We found a significant (p < 0.05) downregulation of ERVK-6 mRNA in the HIC (0.88 - mean fold-change), VC (0.84 - mean fold-change), and EC subgroups (0.86 - mean fold-change), while no significant differences in ERVK-6 expression levels were observed between the NEG and ART groups (0.94 - mean fold-change, p = 0.33) (Fig. 1). In comparison to the ART group, we found a significant downregulation of ERVK-6 mRNA in VCs (0.89-mean fold-change, p = 0.02) and a lower, but not significant, expression in ECs (0.94-mean fold-change, p = 0.70) and HICs (0.92-mean fold-change, p = 0.08) (Fig. 1). No significant differences in ERVK-6 expression levels were observed between the EC and VC subgroups (p = 0.08) (Fig. 1).

Fig. 1: the endogenous retrovirus type K6 (ERVK-6) mRNA levels are downregulated in human immunodeficiency virus controllers (HICs). Boxplots represent the interquartile and sample median (central solid black line) of the relative changes [fold-change values relative to the mean of human immunodeficiency virus (HIV)-1-uninfected (negative - NEG) subjects] of ERVK-6 in HIC (A) and viremic controllers (VCs) and elite controllers (ECs) subgroups (B) compared with NEG and antiretroviral therapy (ART)-suppressed subjects’ (ART) ERVK-6 expression. p-values < 0.05 were considered statistically significant.

ERVK-6 mRNA and cellular restriction factor levels are correlated - We next assessed whether reduced ERVK-6 transcripts in HICs could be associated with mRNA expression levels of 13 cellular restriction factors that act against HIV-1. Pearson’s correlation of all groups combined showed that the mRNA expression of ERVK-6 was negatively correlated with both p21 (r = −0.48; p = 0.0013) and MCPIP1 (r = −0.35; p = 0.0248) (Fig. 2A-B), positively correlated with SERINC3 (r = 0.58; p < 0.0001), SERINC5 (r = 0.60; p < 0.0001), APOBEC3G (r = 0.43; p = 0.005), IFITM2 (r = 0.43; p = 0.004), SAMHD1 (r = 0.43; p = 0.005), and SLFN11 (r = 0.40; p = 0.009) (Fig. 2C-H), and was not significantly correlated (p > 0.05) with the other five restriction factors (Mx1, Mx2, IFITM1, IFITM3, and Tetherin) analysed [Supplementary data (Figure)].

Fig. 2: correlation between mRNA levels of endogenous retrovirus type K6 (ERVK-6) and some restriction factors (RFs): p21 (A); MCPIP1 (B); SERINC3 (C); SERINC5 (D); APOBEC3G (E); IFITM2 (F); SAMHD1 (G), and SLFN11 (H) in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus controllers (HICs) [viremic controllers (VCs) and elite controllers (ECs)] and control groups [negative (NEG) and antiretroviral therapy (ART)]. The points’ colours indicate the patient group, according to the legend. Correlation coefficients (Pearson’s ρ) are shown in each graph’s upper right or left corner. p-values < 0.05 were considered statistically significant.

We also evaluated the correlation coefficient between restriction factors significantly correlated with ERVK-6 mRNA expression levels. We detected a significant negative correlation between p21 and SERINC3 (r = −0.53; p = 0.0004) and APOBEC3G (r = −0.40; p = 0.009) (Fig. 3A). Significant positive correlations were observed between SERINC3 and APOBEC3G (r = 0.49; p = 0.0013) and between SERINC5 and SLFN11 (r = 0.37; p = 0.018), SAMHD1 (r = 0.45; p = 0.003), and IFITM2 (r = 0.34; p = 0.028). Finally, we also observed positive correlations between SLFN11 and APOBEC3G (r = 0.41; p = 0.008) (Fig. 3A).

Fig. 3: (A) Correlogram representing the matrices of Pearson’s rank-order correlation coefficient (ρ) between restriction factors (RFs) and endogenous retrovirus type K6 (ERVK-6) mRNA expression levels. (B-C) Principal component analysis showing the four principal components of the dataset. In Fig. 3B, each circle represents one sample, and the circle’s colour indicates the mRNA ERVK-6 expression levels (divided into four different quartiles) according to the legend in the right corner. In Fig. 3C, the circles representing each sample are coloured according to groups of the study [negative (NEG), antiretroviral therapy (ART), elite controllers (ECs), and viremic controllers (VCs)], as shown in the legend in the right corner.

Multivariate PCA comprising p21, MCPIP1, SERINC3, and SERINC5 variables is shown in Fig. 3B-C, according to either mRNA ERVK-6 expression levels divided into four different quartiles (Fig. 3C) or the study group (Fig. 3D). Our results revealed that 80.6% of the total variance in response to the four restriction factors was expressed by two principal components. The first component (Dim1) represented 47.2%, while the second (Dim2) represented 33.4% of the total variance. The variables p21 and MCPIP1 contributed most to the first component (Dim1), while SERINC5 and SERINC3 contributed most to the second component (Dim2).

In general, we observed a strong association between the ERVK-6 expression quartiles and the HIV-1 control groups (X2 = 32.293, df = 9, p = 0.0001772). This association was even more evident between the NEG control group and the quartiles with the highest ERVK-6 expression (i.e., seven in the fourth quartile, two in the third quartile, one in the second quartile, and none in the first quartile) (X2 = 16.056, df = 3, p = 0.001104). The opposite situation, also highly associated, was observed between the VC group and ERVK-6 expression (seven in the first quartile, one in the second quartile, two in the third quartile, and zero in the fourth quartile) (X2 = 13.641, df = 3, p = 0.003437). This association was confirmed when examining the HIC group (ECs + VCs), i.e., 10 in the first quartile, 8 in the second quartile, four in the third quartile, and only one in the fourth quartile (X2 = 17.413, df = 3, p = 0.0005812).

The separation between the HICs (ECs + VCs) and the control (NEG + ART) groups occurred almost perfectly along the first component (Dim1), with the first group to the right of the origin (first and fourth quadrants) and the second to the left of the origin (second and third quadrants), except for two ECs and one VC individual, in the third and second quadrants, respectively; two of them had low ERVK-6 expression (second quartile), and another had very low ERVK-6 expression (first quartile). The same observation regarding the first component (Dim1) was made for the levels of ERVK-6 expression, with the individuals with the lowest expression (first and second quartile) being further to the right of the origin (first and fourth quadrants) and those with the highest expression to the left of the origin (second and third quadrants). Notably, the individuals with the highest ERVK-6 expression (fourth quartile) were concentrated in the second quadrant of the PCA, coinciding with the predominance of healthy control individuals (NEG), while the individuals with the lowest ERVK-6 expression (first quartile) were concentrated in the fourth quadrant of the PCA, coinciding with the predominance of VCs.

DISCUSSION

Previously published studies revealed that HERV-K (HML-2) RNA expression in PBMCs from untreated viremic PLWHs was higher than that in ART-treated PLWHs and/or HIV-uninfected healthy subjects. 4 , 13 , 16 However, none of these studies analysed HERV-K (HML-2) RNA expression in HICs. Our study reveals that ERVK-6 (HML-2) RNA expression in PBMCs from HICs, particularly the VC subgroup, was significantly downregulated compared with both ART-treated PLWHs and/or HIV-uninfected healthy control groups.

Our analyses revealed that ERVK-6 RNA level in our group of ART-treated PLWHs with undetectable viremia was comparable to those detected in HIV-uninfected healthy, consistent with the notion that successful ART may reduce the expression level of HERVs in HIV-infected individuals. 18 Other studies, by contrast, described the persistently elevated expression of some HERVs, including HERV-K (HML-2), in PBMCs of HIV patients under ART compared to healthy controls. 4 , 37 The contradictory results could be explained by varying levels of residual replication and duration of therapy among ART-treated PLWHs, or by the influence of factors other than HIV replication on the expression of HERVs. Indeed, we would expect HICs to exhibit slightly higher expression levels than both control groups in our study because HICs usually displayed higher residual viremia than ART-treated PLWHs. 38 , 39 Thus, the downregulation of ERVK-6 RNA levels observed in our group of HICs cannot be attributed solely to the extremely low viral load.

We hypothesise that HICs may activate some antiviral mechanisms that control the expression of both exogenous and endogenous retroviruses. Consistent with this notion, we detected that ERVK-6 RNA levels in PBMCs were negatively correlated with the expression levels of two cellular restriction factors, namely, p21 and MCPIP1. These proteins can block HIV-1 replication in macrophages and CD4+ T cells 40 , 41 , 42 , 43 , 44 and further limit aberrant immune activation. 24 , 45 , 46 Interestingly, p21 and MCPIP1 limit the activation of macrophages and T cells by downregulating the activity of NF-κB, 24 , 25 , 26 , 27 , 28 , 29 a transcription factor that stimulates the LTR-driven transcription of HIV-1 and HERV-K proviruses. 47 , 48 In a previous study, we showed that MCPIP1 and p21 mRNA and protein expression levels were upregulated in PBMCs from our HIC cohort. 23 Therefore, we suggest that HICs activate a homeostatic anti-inflammatory response that comprises antiviral factors, such as p21 and MCPIP1, to prevent excessive immune activation driven by residual HIV-1 replication. This negative homeostatic response inhibits the NF-κB pathway, which may in turn reduce the efficiency of the LTR-driven transcription of both HIV-1 and HERV-K proviruses.

Interestingly, we detected that ERVK-6 RNA expression in our cohort was positively correlated with two cellular restriction factors, SERINC3 and SERINC5, that may upregulate HERV-K (HML-2) RNA levels by activating the NF-κB pathway. SERINC3/5 were initially identified as cell restriction factors that can potently suppress HIV-1 infectivity by incorporating into budding viral particles and impairing subsequent virion fusion and infection of new target cells. 49 , 50 A recent study revealed that SERINC3/5 exhibited additional antiviral activities by forming a signalling complex with the mitochondrial antiviral signalling protein (MAVS) and TRAF6 at the mitochondria that increased the phosphorylation and activation of IRF3 and IκBα, thus cooperatively enhancing the NF-κB inflammatory pathway and type I IFN (IFN-I) production. 51 Moreover, we detected a significant negative correlation between the expression of p21 and SERINC3, suggesting that these restriction factors may display antagonist transcriptional regulation and may be part of the mechanism that controls the repression and activation of HERVs.

Our findings also revealed that the expression of some ISGs analysed here (APOBEC3G, SLFN11, and SAMDH1) was positively correlated with the expression of both ERVK-6 and SERINC3/5. Cytosolic HERV-K dsRNA/cDNA may activate different nucleic acid sensors involved in innate immunity, such as retinoic acid-inducible gene-I-like receptors (RIG-I and MDA5) 52 , 53 , 54 and cGMP-AMP synthase (cGAS). 55 Activation of these innate immune sensors leads to phosphorylation and activation of TANK-binding kinase 1 (TBK1) and IκB kinase-ϵ (IKKϵ) kinases, which in turn activate the transcription factors IRF3 and NF-κB, ultimately leading to upregulation of type I IFNs, ISGs, and other proinflammatory cytokines. Interestingly, SAMHD1 and SLFN11 belong to a subset of ISGs that may be directly induced by IRF3, 56 , 57 and APOBEC3G may be positively modulated by the NF-κB pathway. 58 We propose that increased levels of SERINC3/5 and cytosolic HERV-K RNA/DNA may directly upregulate some ISGs by triggering the activation of IRF-3 and NF-κB, independent of the engagement of the type I IFN-JAK-STAT pathway.

Previous studies described that HICs have more robust cellular and antibody responses against HERV-K than ART-suppressed, viremic no controllers and uninfected subjects. 59 , 60 The generation and maintenance of strong HERV-specific cellular and antibody responses in HICs seem inconsistent with the very low ERVK-6 mRNA expression detected here in PBMCs. Some findings, however, may explain these apparently conflicting results. Previous studies conducted in our and other cohorts of HICs showed that HIV-1 continues to replicate and evolve despite undetectable or extremely low levels of viremia, 30 , 61 - 66 and HIV-1 replication in HICs seems to occur mainly in the lymph nodes. 66 Thus, HERV-K expression in HICs may be mostly restricted to HIV-infected cells residing in lymph nodes, and the expression of both HERV-K and HIV-1 antigens at those sites may stimulate the continuous generation of HERV-specific cellular and antibody responses. In contrast, most PBMCs and HIV-infected cells in the periphery display an antiviral transcriptional signature that limits the activation of both exogenous and endogenous retroviruses.

Persistent overexpression of IFN-α/β and ISGs is a hallmark of chronic HIV-1 infection and is associated with detrimental immune activation, bystander CD4+ T-cell apoptosis, and progressive disease. 67 , 68 Some data suggest that augmented HERV expression may play an active role in exacerbating and perpetuating chronic inflammation in some autoimmune diseases via type I IFN- and MAVS-positive signalling feedback loops. 69 On the other hand, HERV-K (HML-2) knockdown significantly downregulated genes containing interferon-stimulated response elements (ISREs) in their promoters in basal and IFN-γ-challenged macrophages and had implications for the paracrine activation of nearby cells following macrophage activation. 21 Moreover, repression of endogenous retrovirus in vivo alleviates tissue aging and, to some extent, organismal aging by attenuating innate immune responses. 54 Thus, repression of HERV-K expression in HICs may contribute to attenuating the activation of innate immune responses, preventing chronic overexpression of IFN-α/β/ISGs and preserving immune homeostasis in those subjects.

Previous studies from our cohort and other groups showed that HICs displayed elevated levels of some key plasma inflammatory markers, such as sCD14, IP-10, IL-18, and D-dimer, and higher CD8+ T-cell activation concerning HIV-negative individuals. 31 , 70 - 76 Moreover, VCs displayed higher levels of immune activation and residual inflammation than ECs. The proinflammatory profile observed in HICs, however, is different from the aberrant inflammation observed in untreated no controllers. 77 , 78 Indeed, gene expression analysis of PBMCs and purified T cells from HICs showed that these individuals, compared with ART-treated individuals, have lower expression of several inflammatory genes, such as IL1A/B, IL-6, CXCL5, CXCL13, and CXCL1, but maintain upregulation of genes associated with cytotoxicity and the T-cell response. 79 We suggest that the low levels of ERVK-6 detected here in HICs may be linked to the upregulation of an anti-inflammatory response that limits aberrant inflammation, and such homeostatic mechanisms would be more critical for VCs due to the higher levels of chronic HIV-1 antigenic stimulation compared with ECs.

A recent study, made available in preprint format, that compared the (retro)transcriptome of ECs, PLWH-on-ART, viremic progressors and healthy controls supports a quite different scenario. 80 According to this study, ECs display elevated levels of specific ISGs and transposable elements (TEs) located upstream of ISGs for control groups. The authors propose a model in which ECs upregulate TEs serving as promoters or enhancers for ISGs that inhibit HIV replication. This is markedly different from our model of natural control, but several factors make direct comparisons between studies challenging. First, different HERV proviruses may respond differently to HIV infection. 12 , 37 , 81 , 82 Second, the referenced study focused on analysing HERVs expression using previously published RNAseq data from activated CD4+ T-cells, differing greatly from our study of ex vivo (unstimulated) PBMCs. Third, the preprint study described different (retro)transcriptomic profiles among different ECs, reinforcing the heterogeneity of resistance mechanisms among ECs cohorts.

Our study has two main limitations. The first limitation is the small sample size. Our study was exploratory and included a relatively small number of HICs, so future studies with larger cohorts of ECs and VCs are needed to confirm the findings described here. The second limitation is that we do not provide a comprehensive overview of the expression profile of all HERV-K (HML-2) loci. Our exploratory analysis was centred on the transcriptional activity of ERVK-6 because this proviral locus was shown to be activated in persistently HIV-infected cells. 14 However, different HERV-K (HML-2) loci could be differentially regulated by either HIV-1 infection or proinflammatory stimuli, and the ERVK-6 provirus represents a small percentage (< 0.2%) of the total HERV-K HML-2 expression under physiological conditions. 12 , 21 Thus, future investigations exploring the expression of different HERV-K (HML-2) loci are essential to understanding the ability of HICs to downregulate the expression of this human endogenous retrovirus group-K subgroup.

In summary, our findings revealed that ERVK-6 mRNA levels in PBMCs of HICs are lower than those seen in aviremic ART-treated subjects and HIV-negative individuals. We found that ERVK-6 mRNA levels were negatively correlated with the expression of some cellular restriction factors (p21 and MCPIP1) and positively correlated with the expression of others (SERINC3/5, APOBEC3G, IFITM2, SAMHD1, and SLFN11). We suggest that reduced expression of ERVK-6 in HICs may result from the upregulation of a distinctive antiviral and anti-inflammatory homoeostatic response that contributes both to control HIV-1 replication and to limit chronic immune activation and inflammation driven by persistent HIV-1 antigenic stimulation. Larger confirmatory studies should be conducted to validate the conclusions of this hypothesis-generating study.

ACKNOWLEDGEMENTS

To the participants in the study, as well as all the INI and LabAIDS technical staff involved in the clinical follow-up and blood collection from of the participants. We also thank the Plataforma de PCR em Tempo Real e Digital - RJ (RPT09A) - FIOCRUZ and Plataforma de Sequenciamento de Ácidos Nucléicos de Nova Geração - RJ (RPT01J) -FIOCRUZ.

Financial support: FAPERJ (Grant no. E-26/110.123/2014), CNPq (Grant no. 401220/2016-8).SSDA is financed by a postdoctoral fellowship from the “Pós-Doutorado Nota 10 (PDR 10)” by FAPERJ (Grant no. E-26/202.335/2019); GB is funded by CNPq (Grant no. 304883/2020-4) and FAPERJ (Grant no. E-26/202.896/2018).
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REFERENCES

1 Jern P Coffin JM Effects of retroviruses on host genome function Annu Rev Genet 2008 42 1 709 732 18694346
2 Agoni L Guha C Lenz J Detection of human endogenous retrovirus K (HERV-K) transcripts in human prostate cancer cell lines Front Oncol 2013 3 180 180 23847768
3 Cegolon L Salata C Weiderpass E Vineis P Palù G Mastrangelo G Human endogenous retroviruses and cancer prevention evidence and prospects BMC Cancer 2013 13 1 4 4 23282240
4 Bhardwaj N Maldarelli F Mellors J Coffin JM HIV-1 Infection leads to increased transcription of human endogenous retrovirus HERV-K (HML-2) proviruses in vivo but not to increased virion production J Virol 2014 88 19 11108 11120 25056891
5 Argaw-Denboba A Balestrieri E Serafino A Cipriani C Bucci I Sorrentino R HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features J Exp Clin Cancer Res 2017 36 1 20 20 28125999
6 Douville R Liu J Rothstein J Nath A Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis Ann Neurol 2011 69 1 141 151 21280084
7 Li W Lee MH Henderson L Tyagi R Bachani M Steiner J Human endogenous retrovirus-K contributes to motor neuron disease Sci Transl Med 2015 7 307 307ra153 307ra153
8 Balada E Vilardell-Tarrés M Ordi-Ros J Implication of human endogenous retroviruses in the development of autoimmune diseases Int Rev Immunol 2010 29 4 351 370 20635879
9 Temerozo JR Fintelman-Rodrigues N Dos Santos MC Hottz ED Sacramento CQ Da Silva APD Human endogenous retrovirus K in the respiratory tract is associated with COVID-19 physiopathology Microbiome 2022 10 1 65 65 35459226
10 Costas J Evolutionary dynamics of the human endogenous retrovirus family HERV-K inferred from full-length proviral genomes J Mol Evol 2001 53 3 237 243 11523010
11 Subramanian RP Wildschutte JH Russo C Coffin JM Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses Retrovirology 2011 8 1 90 90 22067224
12 Young GR Terry SN Manganaro L Cuesta-Dominguez A Deikus G Bernal-Rubio D HIV-1 infection of primary CD4 + T cells regulates the expression of specific human endogenous retrovirus HERV-K (HML-2) elements J Virol 2018 92 1 e01507 e01517 29046457
13 Contreras-Galindo R López P Vélez R Yamamura Y HIV-1 infection increases the expression of human endogenous retroviruses type K (HERV-K) in vitro AIDS Res Human Retroviruses 2007 23 1 116 122 17263641
14 Vincendeau M Göttesdorfer I Schreml JMH Wetie AGN Mayer J Greenwood AD Modulation of human endogenous retrovirus (HERV) transcription during persistent and de novo HIV-1 infection Retrovirology 2015 12 1 27 27 25886562
15 Jones RB Garrison KE Mujib S Mihajlovic V Aidarus N Hunter DV HERV-K-specific T cells eliminate diverse HIV-1/2 and SIV primary isolates J Clin Invest 2012 122 12 4473 4489 23143309
16 Ormsby CE SenGupta D Tandon R Deeks SG Martin JN Jones RB Human endogenous retrovirus expression is inversely associated with chronic immune activation in HIV-1 infection PLoS One 2012 7 8 e41021 22879884
17 Contreras-Galindo R Kaplan MH Markovitz DM Lorenzo E Yamamura Y Detection of HERV-K(HML-2) viral RNA in plasma of HIV type 1-infected individuals AIDS Res Hum Retroviruses 2006 22 10 979 984 17067267
18 Contreras-Galindo R Almodóvar-Camacho S González-Ramírez S Lorenzo E Yamamura Y Comparative longitudinal studies of HERV-K and HIV-1 RNA titers in HIV-1-infected patients receiving successful versus unsuccessful highly active antiretroviral therapy AIDS Res Hum Retroviruses 2007 23 9 1083 1086 17919102
19 Garrison KE Jones RB Meiklejohn DA Anwar N Ndhlovu LC Chapman JM T cell responses to human endogenous retroviruses in HIV-1 infection PLoS Pathog 2007 3 11 e165 17997601
20 Gonzalez-Hernández MJ Swanson MD Contreras-Galindo R Cookinham S King SR Noel RJ Expression of human endogenous retrovirus type K (HML-2) is activated by the tat protein of HIV-1 J Virol 2012 86 15 7790 7805 22593154
21 Russ E Mikhalkevich N Iordanskiy S Expression of human endogenous retrovirus group K (HERV-K) HML-2 correlates with immune activation of macrophages and type I interferon response Microbiol Spectr 2023 11 2 e04438 e04422 36861980
22 Hurst T Magiorkinis G Epigenetic control of human endogenous retrovirus expression focus on regulation of long-terminal repeats (LTRs) Viruses 2017 9 6 130 130 28561791
23 De Azevedo SSD Ribeiro-Alves M Côrtes FH Delatorre E Spangenberg L Naya H Increased expression of CDKN1A/p21 in HIV-1 controllers is correlated with upregulation of ZC3H12A/MCPIP1 Retrovirology 2020 17 1 18 18 32615986
24 Fu M Blackshear PJ RNA-binding proteins in immune regulation a focus on CCCH zinc finger proteins Nat Rev Immunol 2017 17 2 130 143 27990022
25 Sun X Feng W Guo Y Wang Q Dong C Zhang M MCPIP1 attenuates the innate immune response to influenza A virus by suppressing RIG-I expression in lung epithelial cells J Med Virol 2018 90 2 204 211 28892164
26 Chen X Zhao Q Xie Q Xing Y Chen Z MCPIP1 negatively regulate cellular antiviral innate immune responses through DUB and disruption of TRAF3-TBK1-IKKe complex Biochem Biophy Res Commun 2018 503 2 830 836
27 Lloberas J Celada A p21waf1/CIP1.a CDK inhibitor and a negative feedback system that controls macrophage activation Eur J Immunol 2009 39 3 691 694
28 Scatizzi JC Mavers M Hutcheson J Young B Shi B Pope RM The CDK domain of p21 is a suppressor of IL-1ß-mediated inflammation in activated macrophages Eur J Immunol 2009 39 3 820 825 19189309
29 Trakala M Arias CF García MI Moreno-Ortiz MC Tsilingiri K Fernández PJ Regulation of macrophage activation and septic shock susceptibility via p21(WAF1/CIP1) Eur J Immunol 2009 39 3 810 819 19224635
30 De Azevedo SSD Caetano DG Côrtes FH Teixeira SLM Dos Santos Silva K.Hoagland B Highly divergent patterns of genetic diversity and evolution in proviral quasispecies from HIV controllers Retrovirology 2017 14 1 29 29 28464889
31 Côrtes FH De Paula HHS Bello G Ribeiro-Alves M De Azevedo SSD Caetano DG Plasmatic levels of IL-18, IP-10, and activated CD8+ T cells are potential biomarkers to identify HIV-1 elite controllers with a true functional cure profile Front Immunol 2018 9 1576 1576 30050532
32 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biol 2002 3 7 research0034 research0034 12184808
33 Solari A Salmaso L Pesarin F Basso D Permutation tests for stochastic ordering and ANOVA. Theory and applications with R. Lecture Notes in Statistics. 194 Springer 2009 New York
34 R Core Team R: a language and environment for statistical computing. Vienna, 2017 Austria R Foundation for Statistical Computing
35 Lê S Josse J Husson F FactoMineR an R package for multivariate analysis J Stat Soft 2008 25 1 1 18
36 Kassambara A Mundt F factoextra: extract and visualize the results of multivariate data analyses 2016 https://CRAN.R-project.org/package=factoextra
37 Mantovani F Kitsou K Paraskevis D Lagiou P Magiorkinis G The interaction of human immunodeficiency virus-1 and human endogenous retroviruses in patients (primary cell cultures) and cell line models Microbiol Spectr 2023 11 6 e01379 e01323 37811936
38 Hatano H Delwart EL Norris PJ Lee TH Dunn-Williams J Hunt PW Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy J Virol 2009 83 1 329 335 18945778
39 Landay A Golub ET Desai S Zhang J Winkelman V Anastos K HIV RNA levels in plasma and cervical-vaginal lavage fluid in elite controllers and HAART recipients AIDS 2014 28 5 739 743 24326356
40 Bergamaschi A David A Le Rouzic E Nisole S Barré-Sinoussi F Pancino G The CDK Inhibitor p21Cip1/WAF1 is induced by Fc R activation and restricts the replication of human immunodeficiency virus type 1 and related primate lentiviruses in human macrophages J Virol 2009 83 23 12253 12265 19759136
41 Valle-Casuso JC Allouch A David A Lenzi GM Studdard L Barré-Sinoussi F et al p21 restricts HIV-1 in monocyte-derived dendritic cells through the reduction of deoxynucleoside triphosphate biosynthesis and regulation of SAMHD1 antiviral activity Virol 2017 91 23 e01324 e01317
42 Liu S Qiu C Miao R Zhou J Lee A Liu B MCPIP1 restricts HIV infection and is rapidly degraded in activated CD4+ T cells Proc Natl Acad Sci USA 2013 110 47 19083 19088 24191027
43 Allouch A David A Amie SM Lahouassa H Chartier L Margottin-Goguet F et al p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway Proc Natl Acad Sci USA 2013 110 42 E3997 E4006
44 Lin RJ Chien HL Lin SY Chang BL Yu HP Tang WC MCPIP1 ribonuclease exhibits broad-spectrum antiviral effects through viral RNA binding and degradation Nucleic Acids Res 2013 41 5 3314 3326 23355615
45 Jura J Skalniak L Koj A Monocyte chemotactic protein-1-induced protein-1 (MCPIP1) is a novel multifunctional modulator of inflammatory reactions Biochim Biophys Acta 2012 1823 10 1905 1913 22771441
46 Uehata T Iwasaki H Vandenbon A Matsushita K Hernandez-Cuellar E Kuniyoshi K Malt1-induced cleavage of regnase-1 in CD4+ helper T cells regulates immune activation Cell 2013 153 5 1036 1049 23706741
47 Chan JK Greene WC Dynamic roles for NF- B in HTLV-I and HIV-1 retroviral pathogenesis Immunol Rev 2012 246 1 286 310 22435562
48 Manghera M Douville RN Endogenous retrovirus-K promoter a landing strip for inflammatory transcription factors? Retrovirology 2013 10 1 16 16 23394165
49 Rosa A Chande A Ziglio S De Sanctis V Bertorelli R Goh SL HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation Nature 2015 526 7572 212 217 26416734
50 Usami Y Wu Y Göttlinger HG SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef Nature 2015 526 7572 218 223 26416733
51 Zeng C Waheed AA Li T Yu J Zheng YM Yount JS SERINC proteins potentiate antiviral type I IFN production and proinflammatory signaling pathways Sci Signal 2021 14 700
52 Di Giorgio E Xodo LE Endogenous retroviruses (ERVs) Does RLR (RIG-I-like receptors)-MAVS pathway directly control senescence and aging as a consequence of ERV de-repression? Front Immunol 2022 13 917998 917998 35757716
53 Min X Zheng M Yu Y Wu J Kuang Q Hu Z Ultraviolet light induces HERV expression to activate RIG-I signalling pathway in keratinocytes Exp Dermatol 2022 31 8 1165 1176 35332586
54 Mikhalkevich N O'Carroll IP.Tkavc R.Lund K.Sukumar G.Dalgard CL Response of human macrophages to gamma radiation is mediated via expression of endogenous retroviruses Johnson WE, editor PLoS Pathog 2021 17 2 e1009305 33556144
55 Liu X Liu Z Wu Z Ren J Fan Y Sun L Resurrection of endogenous retroviruses during aging reinforces senescence Cell 2023
56 Li M Kao E Gao X Sandig H Limmer K Pavon-Eternod M Codon-usage-based inhibition of HIV protein synthesis by human schlafen 11 Nature 2012 491 7422 125 128 23000900
57 Yang S Zhan Y Zhou Y Jiang Y Zheng X Yu L Interferon regulatory factor 3 is a key regulation factor for inducing the expression of SAMHD1 in antiviral innate immunity Sci Rep 2016 6 1 29665 29665 27411355
58 Pauli EK Schmolke M Hofmann H Ehrhardt C Flory E Münk C High level expression of the anti-retroviral protein APOBEC3G is induced by influenza A virus but does not confer antiviral activity Retrovirology 2009 6 1 38 38 19371434
59 De Mulder M SenGupta D Deeks SG Martin JN Pilcher CD Hecht FM Anti-HERV-K (HML-2) capsid antibody responses in HIV elite controllers Retrovirology 2017 14 1 41 41 28830571
60 SenGupta D Tandon R Vieira RGS Ndhlovu LC Lown-Hecht R Ormsby CE Strong human endogenous retrovirus-specific T cell responses are associated with control of HIV-1 in chronic infection J Virol 2011 85 14 6977 6985 21525339
61 Bailey JR Williams TM Siliciano RF Blankson JN Maintenance of viral suppression in HIV-1-infected HLA-B*57+ elite suppressors despite CTL escape mutations J Exp Med 2006 203 5 1357 1369 16682496
62 Mens H Kearney M Wiegand A Shao W Schønning K Gerstoft J HIV-1 continues to replicate and evolve in patients with natural control of HIV infection J Virol 2010 84 24 12971 12981 20926564
63 Salgado M Brennan TP O'Connell KA.Bailey JR.Ray SC.Siliciano RF Evolution of the HIV-1 nefgene in HLA-B*57 positive elite suppressors Retrovirology 2010 7 1 94 94 21059238
64 O'Connell KA.Brennan TP.Bailey JR.Ray SC.Siliciano RF.Blankson JN Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus J Virol 2010 84 14 7018 7028 20444904
65 Pernas M Tarancón-Diez L Rodríguez-Gallego E Gómez J Prado JG Casado C Factors leading to the loss of natural elite control of HIV-1 infection J Virol 2018 92 5 e01805 e01817 29212942
66 Boritz EA Darko S Swaszek L Wolf G Wells D Wu X Multiple origins of virus persistence during natural control of HIV infection Cell 2016 166 4 1004 1015 27453467
67 Utay NS Douek DC Interferons and HIV infection the good, the bad, and the ugly Pathog Immun 2016 1 1 107 116 27500281
68 Doyle T Goujon C Malim MH HIV-1 and interferons who's interfering with whom? Nat Rev Microbiol 2015 13 7 403 413 25915633
69 Russ E Iordanskiy S Endogenous retroviruses as modulators of innate immunity Pathogens 2023 12 2 162 162 36839434
70 Côrtes FH Passaes CPB Bello G Teixeira SLM Vorsatz C Babic D HIV Controllers with different viral load cutoff levels have distinct virologic and immunologic profiles J Acquir Immune Defic Syndr 2015 68 4 377 385 25564106
71 Caetano DG Ribeiro-Alves M Hottz ED Vilela LM Cardoso SW Hoagland B Increased biomarkers of cardiovascular risk in HIV-1 viremic controllers and low persistent inflammation in elite controllers and art-suppressed individuals Sci Rep 2022 12 1 6569 6569 35449171
72 Hunt PW Brenchley J Sinclair E McCune JM Roland M Page-Shafer K Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy J Infect Dis 2008 197 1 126 133 18171295
73 Krishnan S Wilson EMP Sheikh V Rupert A Mendoza D Yang J Evidence for innate immune system activation in HIV type 1-infected elite controllers J Infect Dis 2014 209 6 931 939 24185941
74 Pereyra F Lo J Triant VA Wei J Buzon MJ Fitch KV Increased coronary atherosclerosis and immune activation in HIV-1 elite controllers AIDS 2012 26 18 2409 2412 23032411
75 Noel N Boufassa F Lécuroux C Saez-Cirion A Bourgeois C Dunyach-Remy C Elevated IP10 levels are associated with immune activation and low CD4+ T-cell counts in HIV controller patients AIDS 2014 28 4 467 476 24378753
76 Platten M Jung N Trapp S Flossdorf P Meyer-Olson D Zur Wiesch JS Cytokine and chemokine signature in elite versus viremic controllers infected with HIV AIDS Res Human Retroviruses 2016 32 6 579 587 26751176
77 Neuhaus J Jacobs DR Jr Baker JV Calmy A Duprez D La Rosa A Markers of inflammation, coagulation, and renal function are elevated in adults with HIV infection J Infect Dis 2010 201 12 1788 1795 20446848
78 Deeks SG HIV infection, inflammation, immunosenescence, and aging Annu Rev Med 2011 62 1 141 155 21090961
79 Hocini H Bonnabau H Lacabaratz C Lefebvre C Tisserand P Foucat E HIV controllers have low inflammation associated with a strong HIV-specific immune response in blood J Virol 2019 93 10 e01690 e01618 30814287
80 Singh M Leddy SM Iñiguez LP Bendall ML Nixon DF Feschotte C Transposable elements may enhance antiviral resistance in HIV-1 elite controllers bioRxiv 2023 http://biorxiv.org/lookup/doi/10.1101/2023.12.11.571123
81 Grandi N Pisano MP Scognamiglio S Pessiu E Tramontano E Comprehensive analysis of HERV transcriptome in HIV+ cells absence of HML2 activation and general downregulation of individual HERV loci Viruses 2020 12 4 481 481 32340287
82 Gonzalez-Hernandez MJ Cavalcoli JD Sartor MA Contreras-Galindo R Meng F Dai M Regulation of the human endogenous retrovirus K (HML-2) transcriptome by the HIV-1 tat protein J Virol 2014 88 16 8924 8935 24872592
