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Open Access

Peer-reviewed

Research Article

Identification of viral protein R of human immunodeficiency virus-1 (HIV) and interleukin-6 as risk factors for malignancies in HIV-infected individuals: A cohort study

  • Akihiro Matsunaga,

    Roles Formal analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft

    Affiliation Department of Intractable Diseases, Research Institute, National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan

    ⨯
  • Naokatsu Ando,

    Roles Investigation, Resources

    Affiliation AIDS Clinical Center, Hospital, National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan

    ⨯
  • Yuko Yamagata,

    Roles Investigation, Methodology

    Affiliations Department of Intractable Diseases, Research Institute, National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan, RIKEN SPring-8 Center, Koto, Sayo, Hyogo, Japan

    ⨯
  • Mari Shimura,

    Roles Writing – review & editing

    Affiliations Department of Intractable Diseases, Research Institute, National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan, RIKEN SPring-8 Center, Koto, Sayo, Hyogo, Japan

    ⨯
  • Hiroyuki Gatanaga,

    Roles Project administration, Resources, Writing – review & editing

    Affiliation AIDS Clinical Center, Hospital, National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan

    ⨯
  • Shinichi Oka,

    Roles Funding acquisition, Project administration, Resources

    Affiliation AIDS Clinical Center, Hospital, National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan

    ⨯
  • Yukihito Ishizaka

    Roles Funding acquisition, Project administration, Supervision, Writing – original draft

    zakay@ri.ncgm.go.jp

    Affiliation Department of Intractable Diseases, Research Institute, National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan

    ⨯

Abstract

Background

Despite effective antiretroviral therapy, patients with human immunodeficiency virus type-1 (HIV) suffer from a high frequency of malignancies, but related risk factors remain elusive. Here, we focused on blood-circulating viral protein R (Vpr) of HIV, which induces proinflammatory cytokine production and genotoxicity by exogenous functions.

Methods and findings

A total 404 blood samples of HIV patients comprising of 126 patients with malignancies (tumor group) and 278 patients without malignancies (non-tumor group), each of 96 samples was first selected by one-to-one propensity score matching. By a detergent-free enzyme-linked immunosorbent assays (detection limit, 3.9 ng/mL), we detected Vpr at a higher frequency in the matched tumor group (56.3%) than in the matched non-tumor group (39.6%) (P = 0.030), although there was no different distribution of Vpr levels (P = 0.372). We also detected anti-Vpr immunoglobulin (IgG), less frequently in the tumor group compared with the tumor group (22.9% for tumor group vs. 44.8% for non-tumor group, P = 0.002), and the proportion of patients positive for Vpr but negative of anti-Vpr IgG was significantly higher in the tumor group than in the non-tumor group (38.6% vs. 15.6%, respectively, P < 0.001). Additionally, Interleukin-6 (IL-6), the levels of which were high in HIV-1 infected patients (P < 0.001) compared to non-HIV-infected individuals, was significantly higher in advanced cases of tumors (P < 0.001), and IL-6 level was correlated with Vpr in the non-tumor group (P = 0.010). Finally, multivariate logistic regression analysis suggested a positive link of Vpr with tumor occurrence in HIV patients (P = 0.002).

Conclusion

Vpr and IL-6 could be risk factors of HIV-1 associated malignancies, and it would be importance to monitor these molecules for well managing people living with HIV-1.

Citation: Matsunaga A, Ando N, Yamagata Y, Shimura M, Gatanaga H, Oka S, et al. (2024) Identification of viral protein R of human immunodeficiency virus-1 (HIV) and interleukin-6 as risk factors for malignancies in HIV-infected individuals: A cohort study. PLoS ONE 19(1): e0296502. https://doi.org/10.1371/journal.pone.0296502

Editor: Roberto F. Speck, University Hospital Zurich, SWITZERLAND

Received: May 31, 2023; Accepted: December 14, 2023; Published: January 2, 2024

Copyright: © 2024 Matsunaga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Data are available from doi: 10.6084/m9.figshare.24580450.

Funding: This study was partly supported by a Health and Labor Science Research Grant (19HB1003), a grant from the Research Program on HIV/AIDS (17fk0410208h0002) from the Japan Agency for Medical Research and Development (AMED) and grants from National Center for Global Health and Medicine Intramural Research (29A1003 and 19A1018).

Competing interests: The authors have declared that no competing interests exist.

Introduction

Before the advent of antiretroviral therapy (ART) initiated in 1996, human immunodeficiency virus type-1 (HIV)-infected people were susceptive to a high incidence of numerous malignancies, especially AIDS-defining malignancies (ADMs) such as non-Hodgkin lymphoma (NHL), Kaposi sarcoma (KS), and invasive cervical cancer [1]. In the post-ART era, the risk of opportunistic infection and ADMs have been dramatically decreased with remarkable improvement of their mortality and quality of life [2,3]. However, lines of evidence revealed that ART did not eliminate the risk of a part of ADMs and non-AIDS defining malignancies (NADMs) that include cancers of the stomach, colon, pancreatic, anal, lung, liver, skin and prostate and Hodgkin lymphoma (HL). Strikingly, the incidence of these NADMs among people living with HIV infection (PLWH) is 1.69-fold higher compared with the general population [4–6]. Malignancies in HIV patients are characterized by more advanced tumor progression and poor prognosis, causing approximately 50% of mortality among PLWH [7,8]. It is crucially important to develop predictive and preventive methods, but both modes of NADMs and their risk factors remain elusive.

As candidate risks for NADMs, it has been proposed that the co-infection of tumor viruses, immunocompromised state, chronic inflammation (inflammatory cytokines), and viral proteins with transforming activities [9,10] are mutually involved in the tumor development. The chronic overproduction of inflammatory cytokines in response to inflammation, infection, or tissue damage can promote tumor growth and metastasis by stimulating cell proliferation, survival, angiogenesis, and invasion [11,12]. Additionally, it is particularly important to note that viral proteins such as transactivator of transcription (Tat), negative factor (Nef), glycoprotein 120 (gp120), p17, and viral protein R (Vpr) are present in patients’ blood as soluble forms and function as transforming factors [13–17]. These observations suggest that PLWH is exposed to multiple cancer risks even under ART; however, it is unclear how each molecule is involved in carcinogenesis.

Vpr is a highly conserved viral accessory protein that is packaged in the virion [18,19] and delivered into the nucleus of infected cells upon viral entry. By forced expression, Vpr shows pleiotropic functions, which include the enhancement of viral replication by transactivation of the viral promoter [20,21], the suppression of host immune responses [22,23], the activation of the oxidative stress pathway [24], DNA damage response with cell cycle arrest at the G2 phase [25–30], suppression of the repair of DNA double-strand breaks [31–33], and inhibition of telomerase activity [34]. Of note, extracellular Vpr is biologically active and induces apoptosis in astrocytes and neurons [35–37], mitochondrial oxidative stress [38,39], and DNA damage response [40,41]. We reported that extracellular Vpr has multiple functions active for induction of interleukin-6 (IL-6) production via Toll-like receptor 4 (TLR4) and MyD88 [42,43], and of retrotransposition [44]. To explore the biological relevance of Vpr in vivo, we developed an enzyme-linked immunosorbent assay system for detecting Vpr (Vpr-ELISA) and reported that Vpr is present in approximately 20% of HIV patients [45], even in well-controlled PLWH with undetectable HIV RNA under ART control. Because Vpr present in blood was active in inducing retrotransposition [44], and it has been reported that Vpr activates the production of tumor necrosis factor alpha (TNFα) and Interleukin-1 beta (IL-1β) [37,46], it is plausible that Vpr itself and/or inflammatory cytokines cooperatively function as cancer risks in PLWH [47].

To prove this possibility, we measured levels of Vpr in sera of 404 PLWH with or without malignancies and analyzed the relationship between Vpr and inflammatory cytokines in relation to tumor development.

Materials and methods

Ethical approval

This study was performed following the national regulations and institutional policies and was approved by the ethics committees of the National Center for Global Health and Medicine (#NCGM-G-003183-00) on Mar. 8, 2019 in accordance with the Helsinki Declaration of the World Medical Association. All subjects were provided based on written informed consent.

Study participants

We obtained 404 serum samples of HIV patients from the National Center for Global Health and Medicine (NCGM) Biobank in Japan, which were collected from the period Mar. 30, 2010 to Apr. 2, 2021. Among them, 126 samples belonged to HIV patients with malignancies (tumor group), and 278 samples belonged to HIV patients without any malignancies (non-tumor group) (Table 1). The non-tumor group had no evidence of tumor development for at least one year after the blood collection. We accessed and retrieved clinical data at the time of the sampling and the tumor-free status for at least one year from Sep. 4, 2019 to Apr. 21, 2023.

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Table 1. Demographic and clinical characteristics of both the full cohort and the matched cohort.

https://doi.org/10.1371/journal.pone.0296502.t001

The tumor group consisted of 51 patients with ADMs and 75 with NADMs. The ADM patients included 28 cases with NHL, 22 with KS, and 1 with cervical cancer, while the NADM patients included 12 cases with colon cancer, 8 with anal cancer, 7 with stomach cancer, 5 with pancreatic cancer, 4 with liver cancer, 4 with oral cancer, 3 with prostate cancer, 3 with lung cancer, 3 with skin cancer, 7 with HL, and 19 with other cancers. The serum samples were aliquoted, frozen, and stored at –80°C. The demographic and clinical characteristics of all participants were obtained using routine clinical tests and included age, sex, blood CD4+ and CD8+ T-cell counts (cells/μl), plasma HIV RNA, hepatitis B virus (HBV) antigen, and hepatitis C virus (HCV) antibody. The tumor stages, local and advanced, were determined by professional physicians. The advanced stage means that the tumor has spread to the tissues around primary lesion or the other organs of the body.

Vpr-ELISA

The Vpr-ELISA was performed as previously reported [45]. Briefly, microtiter plates pre-coated with polyclonal antibodies against recombinant Vpr (rVpr) were blocked with a buffer comprising 1% BlockAce (Megmilk Snow Brand, Tokyo, Japan) diluted in phosphate-buffered saline, pH 7.4 (PBS). Then, 10 μL serum or standard rVpr was incubated at 37°C for 1 h with 40 μL of 0.4% BlockAce/PBS. After washing with PBS, 50 μL of 5 ng/μL B34 anti-Vpr monoclonal antibody was applied and incubated at 37°C for 1 h, followed by incubation with a horseradish peroxidase (HRP)-conjugated anti-penta-His antibody (Thermo Fisher Scientific, Waltham, MA). 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added for development, and the signal was measured after 30 min at a wavelength of 450 nm using a microplate reader (Bio-Rad, Hercules, CA). By optimizing the incubation temperature (37.0°C) and pH condition (pH 7.40) of the solutions, we tried improving the signal-to-background ratio and successfully increased the detection limit to 3.9 from 39 ng/mL compared with the previous study. All samples were measured in triplicate.

Anti-Vpr IgG ELISA

According to the reported procedures [48], rVpr was produced by a wheat germ cell-free protein expression system (CellFree Sciences, Ehime, Japan) and by using strep-tagII (IBA Lifesciences, Göttingen, Germany) and His-tag (Qiagen, Hilden, Germany) affinity purification methods. Microtiter plates (Thermo Fisher Scientific) were coated with 50 μL of 1 ng/μL rVpr at 4°C overnight. Then, 100 μL of 1:100 diluted serum was applied and incubated at 37°C for 1 h. After washing with PBS, the plates were incubated with 1:10,000-diluted anti-human IgG antibody conjugated to horseradish peroxidase (GeneTex, Irvine, CA) and developed with TMB substrate solution (Nacalai Tesque, Kyoto, Japan). Absorbance was measured at a wavelength of 450 nm (OD450) using a microplate reader (Bio-Rad, Irvine, CA). HIV-uninfected volunteer samples were used as the negative control, whereas those of infected patients with high levels of anti-Vpr antibodies were used as the positive control. All measurements were made in triplicate. The positive cut-off value was set to the mean plus three times standard deviation of the negative control.

Inflammatory cytokine ELISA

Inflammatory cytokines, IL-6, TNFα and IL-1β, were measured using the AlphaLISA immune assay kit (PerkinElmer, Waltham, MA) according to the manufacturer’s kit protocols. Briefly, a 384-well plate was filled with 2μl of serum sample, 4μl of 50 μl/mL AlphaLISA acceptor bead-conjugated antibody, and 4μl of 5 nM biotinylated antibody against a cytokine in AlphaLISA immunoassay buffer, then incubated for 1 h at 23°C. Without a washing step, 10 μL of 80 μg/mL streptavidin-coated donor beads were added. After incubation for 30 min at 23°C in the dark, the plate was read on a Nivo microplate reader (PerkinElmer) using the AlphaLISA measurement protocol (excitation wavelength of 680 nm and emission detection to 615 nm). The concentration of cytokines was calculated from the slope of the standard curve of an analyte standard provided by the supplier (PerkinElmer). Lowest limits of quantification (LLOQ) by the AlphaLISA were 1.3 pg/mL of IL-6, 2.2 pg/mL of TNFα, and 0.65 pg/mL of IL-1β, respectively. If the measurement value were below the LLOQ, we used the half values of LLOQ, 0.65 pg/mL for IL-6, 1.1 pg/mL for TNFα, and 0.325 pg/mL for IL-1b, for statistical analyses.

Statistical analysis

To avoid confounding differences due to baseline varieties in comparing between the non-tumor group and the tumor group, we performed a propensity score matching by SPSS software (version 26; IBM, Chicago, IL). The propensity scores were generated with the prospective characteristics, including age, sex at birth, CD4+ T-cell count, CD8+ T-cell count, HIV viremia, HBV viremia and HCV viremia by fitting a logistic regression model. The matching was performed using 1:1 ratio without replacement by caliper-matching on the estimated propensity score. The caliper was calculated by 0.25 × (standard deviation of propensity score). Other statistical analyses were performed with Prism software (version 9; GraphPad Software, La Jolla, CA). The statistical significance of differences between the two groups was tested using the Wilcoxon matched pairs signed rank test for the matched pair group analysis and the Mann–Whitney U nonparametric test. The differences between categorical variables were analyzed using Fisher’s exact test. Statistical significance was set at P < 0.05. To analyze the distribution of Vpr concentration in the tumor and non-tumor groups, we used 1.95 ng/mL for the samples, the concentrations of which were less than 3.9 ng/mL, the detection limit of the ELISA. To compare three groups, a nonparametric Kruskal-Wallis test was performed, and then the post hoc Dunn’s multiple comparisons test was used to identify which specific groups were significantly different from each other. To extract predictor variables with correlation with the outcome variable (IL-6 or Vpr), the multivariate linear regression analyses were performed with forced entry method. Binary logistic regression model was used to identify risk factors associated with tumor occurrence in HIV patients as the reference (odds ratio = 1). Receiver operating characteristic (ROC) curve was plotted to evaluate the performance of risk factors and the area under curve (AUC) was measured to represent the degree of separability.

Results

Characteristics of the enrolled HIV patients

The cohort analyzed included 404 HIV patients: 126 patients with malignancies (tumor group) and 278 patients without malignancies (non-tumor group). There was no difference in gender or age distribution between these two groups (P > 0.999 and 0.289, respectively; Table 1). Of note, there was a higher frequency of HIV viremia in the tumor group compared with the non-tumor group (37.3% vs. 9.0%, P < 0.001; Table 1), which was associated with a significant difference in CD4+ T-cell counts (median [interquartile range], 369 [158.3–543.5] cells/μL for the tumor group vs. 549 [398.5–717.5] cells/μL for the non-tumor group, P < 0.001; Table 1) and CD4/CD8 ratios (0.502 [0.201–0929] vs. 0.770 [0.561–1.144], respectively, P < 0.001; Table 1). To reduce these biases of confounding factors with significant differences, we extracted 96 patients with malignancies (matched tumor group) and 96 patients without malignancies (matched non-tumor group) by one-to-one propensity score matching. There was no difference in demographic and clinical characteristics between these two matched groups (Table 1).

Higher detection frequency of Vpr in the tumor group

In 96 patients of the matched tumor group, we detected 54 patients positive for circulating Vpr (56.3%), whereas 38 of 96 patients were positive in the matched non-tumor group (39.6%). Data implied that Vpr is present at significantly higher frequency in the tumor group than in the non-tumor group (P = 0.0300; Fig 1A, left panel). However, there was no remarkable difference in the concentrations of Vpr between the two groups (P = 0.372; Fig 1A, right panel). The clinical characteristics were also similar in the Vpr-positive and -negative cases (Table 2). Additionally, both Vpr detection frequency and concentration distribution were not different in the aviremic (HIV RNA level < detection limit 20 copy/mL) and viremic patients among both the non-tumor group and the tumor-group (P = 0.672 and P = 0.769 for the non-tumor group and P = 0.716 and P = 0.309 for the tumor group; S1A Fig). The incidence of viral blips in ART-controlled patients, in which the viral load temporarily increased to a maximum of 500 copies/mL, but quickly decreased to <50 copies/mL, was 5.5–8.3% and not different between two groups (P = 0.855 for the tumor group and P = 0.744 for the non-tumor group; Table 2). Data also indicated that circulating Vpr is not directly linked with the HIV present in the peripheral blood. Moreover, the detection frequency of Vpr was not different in NADM and ADMs (P = 0.469; S1B Fig and S1 Table) or the virus-related (VR) and the virus-unrelated (VUR) tumors (P = 0.597 for VR and VUR; S1C Fig).

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Fig 1. Inverse detection frequencies of circulating Vpr and anti-Vpr IgG in HIV patients with tumors.

(A) Incidence of Vpr and distribution of Vpr concentrations in HIV patients. Left panel, stacked bar chart showing the proportion of Vpr-positive samples in the matched tumor group (T) and the matched non-tumor group (NT). Black bar, Vpr positivity. White bar, Vpr negativity. Right panel. violin plot showing the distribution of Vpr concentrations (ng/mL). Dotted line is the detection limit of Vpr (3.8 ng/mL). (B) Incidence of anti-Vpr IgG in HIV patients. Left panel, bar chart showing the proportion of anti-Vpr IgG-positive samples in HIV patients. Black bar, anti-Vpr IgG positivity. White bar, anti-Vpr IgG negativity. Right panel, violin plot showing the distribution of anti-Vpr IgG titers (OD value). Dotted line shows the value of the negative control (OD = 0.38). (C) Combined positive proportion of Vpr and anti-Vpr IgG. The proportion of Vpr and anti-Vpr IgG-double positive (Vpr+, anti-Vpr IgG+; dark ray bar), only Vpr-positive (Vpr+, anti-Vpr IgG-; gray bar), only anti-Vpr IgG-positive (Vpr-, anti-Vpr IgG+; light gray bar), and Vpr and anti-Vpr IgG-double negative (Vpr-, anti-Vpr IgG-, white bar) patients was depicted. The percentage of each group was shown with a white or black number. Fisher’s exact test (for 2×2 contingency table) or chi-square test (for 2×4 contingency table) was used to compare the number of patients between the T and NT groups. (D) Relative frequency of cases positive for Vpr in representative tumors Representative doughnut chart depicting the relative frequency of Vpr-positive (black) and -negative (white) patients with NHL, HL, pancreatic cancer, stomach cancer, liver cancer, and skin cancer. The percentage of Vpr-positive and -negative patients was shown in white or black numbers, respectively. (E) The proportion of combined detection of Vpr and anti-Vpr IgG in each primary lesion of tumor. The percentage of each group was shown with a white or black number.

https://doi.org/10.1371/journal.pone.0296502.g001

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Table 2. Relationships between circulating Vpr and clinical characteristics.

https://doi.org/10.1371/journal.pone.0296502.t002

Lower detection frequency of anti-Vpr IgG in the tumor group

Given that some HIV-infected patients had seroconversion against Vpr [49–52], we next measured anti-Vpr IgG. Data revealed that in the tumor group, 22 patients (22.9%) were positive for anti-Vpr IgG, whereas 43 patients (44.8%) were positive in the non-tumor group, suggesting that the detection frequency of anti-Vpr IgG was significantly lower in the matched tumor group than in the matched non-tumor group (P = 0.0022; Fig 1B left panel), although the distribution of the anti-Vpr IgG concentration was significantly higher in the matched non-tumor group (P < 0.0001; Fig 1B, right panel). Curiously, the detection frequency of anti-Vpr IgG level was higher in the viremic patients of the tumor group (61.8% vs. 29.3%, P = 0.0017; Table 3) who had a lower CD4+ T-cell count (246 [121–404] vs. 409 [173–602], P = 0.0123; Table 3) and a lower CD4/CD8 ratio (0.322 [0.149–0.609] vs. 0.596 [0.286–1.062], P = 0.0021; Table 3). The anti-Vpr IgG level was not correlated with RNA level of HIV-1 (P = 0.700; S2A Fig), and the relationship between anti-Vpr IgG and HIV viremia remains unknown. No positive correlation of Vpr concentrations and anti-Vpr IgG level was observed both in non-tumor and tumor-groups (P = 0.812 for the non-tumor group, and P = 0.615 for the tumor group; S2B Fig).

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Table 3. Relationships between anti-Vpr IgG and clinical characteristics.

https://doi.org/10.1371/journal.pone.0296502.t003

We next analyzed the correlation of Vpr and anti-Vpr IgG in relation to tumor occurrence. As shown in Fig 1C, the detection frequency of Vpr in the anti-Vpr IgG-negative patients was significantly higher in the tumor group compared with the non-tumor group (38.6% vs. 15.6%, respectively, P < 0.001). In striking contrast, the detection frequency of anti-Vpr IgG in the Vpr-negative patients was significantly higher in the non-tumor group compared with the tumor group (20.8%% vs. 5.2%, respectively; Fig 1C). Data suggest that anti-Vpr IgG functions as a negative factor for tumorigenesis counteracting Vpr activity.

Quite interestingly, Vpr was detected at high frequency in particular types of tumors such as NHL (60.7% patients for Vpr positive), HL (57.1%), stomach (71.4%), and pancreatic cancers (80.0%) whereas it was not detected in live and skin cancers (0.0%; Fig 1D). The proportion of the patients who were positive for Vpr but negative of anti-Vpr IgG-negative was especially high in the stomach (57.1%), oral (50.0%), and pancreas cancers (60.0%) (Fig 1E), implicating the possible involvement of Vpr in certain types of tumor development.

Positive link of Vpr and IL-6 under non-tumor conditions

We measured IL-6, TNFα, and IL-1β, which are risk factors for cancer development [53] and are also involved in HIV-1 pathogenesis [54]. We found that concentrations of both IL-6 and TNFα were significantly high in the matched non-tumor and the matched tumor groups than healthy people (H) (P = 0.0289 and P < 0.001 for IL-6, and P = 0.001 and P < 0.001 for TNFα, respectively; Fig 2A), whereas IL-1β was not (P = 0.7156 and P > 0.9999; Fig 2A). Although the TNFα level was not different in the tumor and the non-tumor groups (P > 0.9999), the IL-6 level was significantly high in the tumor group (P < 0.001; Fig 2A, left panel). Further analysis revealed that IL-6 level was significantly higher in the non-tumor group of patients who were positive for Vpr (P = 0.0283; Fig 2B, left panel), but not in the tumor group (P = 0.680; Fig 2B, right panel). This observation was unique for IL-6, because there was no significant difference of TNFα in patients with or without Vpr among non-tumor and the tumor groups (P = 0.406 and P = 0.681, respectively; Fig 2C). To identify factors that correlate with IL-6, we performed the linear regression analyses in the non-tumor group and the tumor group and observed that Vpr concentration was the single molecule that correlated with IL-6 in the non-tumor group (S2 Table). We plotted the concentrations of Vpr and IL-6 and found that these molecules are well correlated in the non-tumor group (r = 0.266, P = 0.0095; Fig 2D, left panel). In the tumor group, however, there was no positive correlation between these molecules (r = 0.218, P = 0.0861; Fig 2D, right panel). Well consistent with the report that serum IL-6 is a risk factor for tumor progression [55], we observed that IL-6 concentrations were significantly high in the patients with advanced tumors (P < 0.001; Fig 2E), but it was not linked with Vpr (P = 0.655 for local tumor, and P = 0.944 for systemic tumor; Fig 2F).

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Fig 2. Circulating Vpr and inflammatory cytokines.

(A) Detection of cytokines. Concentrations of IL-6, TNFα and IL-1β detected in the patients of the matched tumor and the matched non-tumor groups were plotted (pg/mL). H, non-HIV infected healthy individuals; NT, HIV patients with non-tumor; T, HIV patients with tumor. Dotted line is the lower quantification limit (LLOQ) of IL-6 (1.3 pg/mL), of TNFα (2.2 pg/mL) and of IL-1b (0.65 pg/mL). Samples below LLOQ were plotted at half the detection limit (0.65 ng/mL for IL6, 1.1 pg/mL for TNFα, and 0.325 pg/mL for IL-1β). Horizontal bar represents the median value. *P < 0.05, **P < 0.001. (B) High concentrations of IL-6 in the Vpr-positive patients in the tumor group. Concentrations of IL-6 in the Vpr-negative (Vpr-) and Vpr-positive (Vpr+) groups in NT and T patients were plotted. Dotted line is the LLOQ of IL-6 (1.3 pg/mL). (C) TNFα concentration in the Vpr-negative (Vpr-) and Vpr-positive (Vpr+) groups in NT and T patients. Dotted line is the LLOQ of TNFα (2.2 pg/mL). (D) Correlation between Vpr and IL-6 concentrations in two groups. A log-log regression analysis was performed to assess the correlation. The correlation coefficient (r) and the p-value (P) are shown in graph. The solid line represents the best-fit line. In the T group, concentrations of IL-6 tended to be high in the Vpr-positive patients, but their statistical correlation was not observed. (E) IL-6 concentration in both local and advanced cases of HIV patients with malignancies. (F) IL-6 concentration in Vpr- and Vpr+ groups of local and advanced malignancies.

https://doi.org/10.1371/journal.pone.0296502.g002

Vpr as a novel risk factor for tumor in HIV patients

To analyze the risk factors for tumor occurrence in HIV patients, we finally performed multiple logistic regression analysis. The result showed that Vpr was positively related with tumor occurrence (odds ratio [85% confidence interval] = 2.38 [1.38–4.18]; P = 0.002; Fig 3A) whereas anti-Vpr IgG was negatively related with that (0.330 [0.169–0.616]; P < 0.001; Fig 3A). Beside Vpr, age, HIV viremia, HCV viremia, CRP, and IL-6 were also related with tumor occurrence. Furthermore, the classification between the non-tumor and the tumor groups could be more correctly predicted by including information of Vpr and anti-Vpr IgG into age, CD4, CD8, HIV viremia, HBV, viremia, HCV viremia, IL-6, and TNFα. (AUC = 0.853, and AUC = 0.826, respectively; Fig 3B). These data suggested that Vpr was a novel risk factor for tumorigenesis in HIV patients.

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Fig 3. Risk factors associated to tumor occurrence in HIV patients.

(A) Forest plot of odds ratio by multivariate logistic regression analysis. (B) ROC curves. Dashed line represents the performance on risk factors, age, CD4, CD8, HIV, HBV, HCV, CRP, IL-6 and TNFα, without Vpr and anti-Vpr IgG. Solid line represents the performance on risk factors including Vpr and anti-Vpr IgG. AUC, area under the curve.

https://doi.org/10.1371/journal.pone.0296502.g003

Discussion

Here we found that the circulating Vpr is present at a significantly high frequency in the tumor group compared with the non-tumor group (Fig 1A and Table 2). It is particularly interesting that Vpr was highly positive in NHL, HL, pancreatic, and stomach cancers (Fig 1D) and that the detection frequency of anti-Vpr IgG was low in the patients with gastric and pancreatic cancers (Fig 1E). Lines of evidence indicate that serum Vpr is biologically active [31], and extracellular Vpr causes mitochondrial stress, DNA damage, and retrotransposition at the ten ng/mL range [39,41,44]. Intriguingly, the median concentration of Vpr in HIV-positive patients was 152.5 ng/mL, implicating that approximately 750 μg of Vpr (0.15 μg/mL x 5000 mL total blood) can be continuously produced in vivo and the concentration in the vicinity of the infected cells would be high enough for inducing cellular damage. Because pancreatic acinar cells are the target of HIV infection via C-C chemokine receptor 5, both intracellular and secreted Vpr can induce mitochondrial stress and DNA damage on not only the infected acinar cells but also adjacent cells as a bystander effect [56,57]. If anti-Vpr IgG is not present, a high concentration of Vpr possibly induces more advanced pancreatitis and genotoxic insults leading to cancer development.

It has been proposed that even under ART control, chronic inflammation is continuously observed in HIV patients and plays a risk factor for malignancies [47,58]. Because Vpr is responsive for the induction of multiple inflammatory cytokines such as IL-1β [59], IL-6 [42,43], and TNF-α [46], we here measured these cytokines and investigated the possible link with Vpr. As shown in Fig 2A, the levels of IL-6 and TNFα were higher in HIV-positive patients compared with non-HIV infected healthy individuals, consistent with previous reports [60,61]. Although it has been reported that higher levels of IL-6 in plasma were associated with older age, higher HIV RNA, lower nadir CD4+ T-cell count and others during HIV infection [62], our current analysis revealed that IL-6 was correlated only with Vpr especially in the non-tumor group but not with age, HIV viremia and CD4+ T-cell count (P < 0.001; Fig 2D, left panel and S2 Table). The level of IL-6 was significantly higher in the patients with advanced tumors than local tumors (Fig 2E), but was not statistically correlated with the concentration of Vpr, or the presence of Vpr (Fig 2F). Data suggest that under non-tumor conditions, IL-6 concentration is directly influenced by Vpr, but once tumors are developed, IL-6 is produced by multiple factors alongside Vpr, for example, inflammation or tissue damage that associated with tumor development [63]. Because chronic overproduction of IL-6 can stimulate cell proliferation and survival [64], it is likely that Vpr and IL-6 are cooperatively involved in cellular transformation during tumorigenesis.

As to the emergence of viral proteins in patients’ blood, two possibilities have been postulated. First, there is evidence for residual viral replication due to the low concentrations of antiretroviral drugs in sanctuary sites are present throughout the body, including the lymph nodes, central nervous system, tissue macrophages, adipose tissue, and the gut-associated lymphoid tissue [65–70]. It has also been reported that temporal elevation of viral production, “viral blip”, is induced by transient inflammation in the sanctuary sites [71,72]. In our study, the proportion of viral blips in the Vpr-positive patients was negligible both in the tumor group (5.2%, 6 of 115 patients) and the non-tumor group (6.1%, 4 of 66 patients) (Table 3) and none of factors including HIV viremia were predicted with Vpr emergence status (S3 Table), further implying that the production of Vpr was not due to the viral blip. Second, reservoir cells with a defective provirus genome survive and secrete viral proteins for long periods. Under ART control, the vast majority (approximately 95%) of HIV proviruses, which are integrated into the CD4+ T-cell genome, are defective with large internal deletions, premature stop codons, and G-to-A hypermutations [73]. Even though these proviruses are unable to produce replication-competent viruses [74], they can generate fragments of HIV transcripts and produce encoded viral proteins [75]. Toward complete cure of HIV, various trials that include shock-and kill strategy and block-and-lock strategy have been recently designed and tried. In the shock-and kill strategy, it is expected that the reactivated virus from the cell reservoir is eliminated by the immune system or cytopathic effects [76,77]. However, cells with large internal deletion of the provirus genome would escape from HIV-specific cytotoxic T lymphocytes [73], implying that the block-and-lock strategy, in which transcription from the provirus genome is permanently shut off by inducing a deep and irreversible latency is more hopeful [78]. In any challenge, it is important to evaluate the efficacy of the system, and Vpr-ELISA would be useful for monitoring.

As potent Vpr inhibitors, several small molecules have been reported. They include vipirinin, fumagillin, quercetin, and VTD227, which inhibited Vpr-induced cell cycle arrest (G2/M arrest) and viral infection of macrophages [79–82]. They also include damnacanthal, isopimarane diterpenoids, icrasane quassinoids, bis-iridoid and iridoid glycosides, which inhibited Vpr-induced cell death [83–86]. Another inhibitor is mifepristone, which inhibited viral replication via extracellular Vpr and glucocorticoid receptor (GR) mediated HIV-1 LTR transactivation [87,88]. The effect of these Vpr inhibitors on the Vpr-induced IL-6 production has not been reported yet. Regarding the IL-6 promoter activation, glucocorticoids have both antagonistic and agonistic functions through five putative GR binding sites [89]. If the Vpr–GR complex could induce the IL-6 production via TLR4/MyD88 signaling [42], mifepristone might be a potential drug for preventing tumorigenesis caused by Vpr in HIV patients. The nuclear localization of Vpr–GR and poly (ADP-ribose) polymerase-1 complex was inhibited by anti-Vpr monoclonal antibody [90]. We previously reported that extracellular Vpr-induced retrotransposition activity was inhibited by anti-Vpr monoclonal antibody [44], implicating that anti-Vpr antibody drugs might be also useful for preventing Vpr-induced tumorigenesis in HIV patients.

Here we focused on Vpr, but other viral proteins such as Tat, Nef, gp120, and p17 have been reported to be present in patients’ blood under ART [13–17]. Of these viral proteins, Tat is involved in the transcriptional activation of host cell genes such as oncogenic c-MYC and activation-induced cytidine deaminase in B-cells, leading to B-cell malignancies [91,92]. HIV-1 p17, a structural matrix protein, promotes angiogenesis and lymphangiogenesis [93,94]. Its variant stimulates B-cell proliferation and is enriched in patients with NHL [95]. Vpr may co-operates with Tat or p17 for the development of B-cell malignancies because Vpr can bind directly to Tat or p17 and function in viral gene transcription and maturation [96,97]. It would be important to monitor these viral proteins by combining with Vpr and IL-6, since the current study revealed that these two molecules are candidate risk factors for tumorigenesis.

Our study had some limitations. In Vpr-ELISA, samples are subjected to analysis without use of any detergents [45], indicating that only soluble Vpr present as a free-form in blood is detected. This may raise the possibility that Vpr, which is masked by anti-Vpr IgG, might not be measured accurately. However, the detection frequency of Vpr in the non-tumor group was 39.6% (Fig 1A), well consistent with our previous report that 6 of 15 samples of patient serum were active in inducing retrotransposition [44]. Data imply that the frequency of biologically active Vpr was successfully detected by Vpr-ELISA. Second, we here measured Vpr in a single sample of each patient, but it would be necessary to examine several samples that are collected at different time points. It is especially important to know whether Vpr is continuously detected in the same patients, or not. By analyzing a larger cohort of HIV patients at different time-points, more reliable data would help us understand the roles of Vpr in tumor development and detecting malignancies at an early stage.

Supporting information

S1 Checklist. STROBE statement–checklist of items that should be included in reports of cohort studies.

https://doi.org/10.1371/journal.pone.0296502.s001

(DOCX)

S1 Fig. Circulating Vpr under HIV viremic conditions and oncovirus-related malignancies.

(A) Stacked bar chart showing the proportion of Vpr-positive samples in HIV patients with aviremia (Av) and viremia (V). Black bar, Vpr positivity. White bar, Vpr negativity. Violin plot showing the distribution of Vpr concentrations (ng/mL). Dotted line is the detection limit of Vpr (3.8 ng/mL). (B) The detection frequency of Vpr in patients with AIDS-defining malignancies (ADM) and non-AIDS defining malignancies (NADM). (C) The detection frequency of Vpr in patients with virus-related (VR) and virus-unrelated malignancies (VUR).

https://doi.org/10.1371/journal.pone.0296502.s002

(PDF)

S2 Fig. Correlation of anti-Vpr IgG level, HIV RNA level and Vpr levels.

(A) Relationship between anti-Vpr IgG level and HIV RNA in 72 HIV viremic patients. The horizontal dotted line is the detection limit of HIV RNA (20 copies/mL), and the vertical dotted line is the detection limit of anti-Vpr IgG (optical density at 450nm; OD450 = 0.38). (B) Relationship between anti-Vpr IgG and Vpr levels in the non-tumor and tumor groups. The horizontal dotted line is the detection limit of Vpr (3.9 pg/mL), and the vertical dotted line is the detection limit of anti-Vpr IgG (OD = 0.38). A log-log regression analysis was performed to assess the correlation. The correlation coefficient (r) and the p-value (P) are shown in graph. The solid line represents the best-fit line.

https://doi.org/10.1371/journal.pone.0296502.s003

(PDF)

S1 Table. Clinical characteristics of HIV patients with ADM and NADM.

https://doi.org/10.1371/journal.pone.0296502.s004

(PDF)

S2 Table. Multiple linear regression analysis of association between factors and IL-6 concentration.

https://doi.org/10.1371/journal.pone.0296502.s005

(PDF)

S3 Table. Multiple logistic regression analysis of association between factors and Vpr expression.

https://doi.org/10.1371/journal.pone.0296502.s006

(PDF)

Acknowledgments

The authors would like to thank Ms. Hitomi Igarashi for technical assistance, and NCGM Biobank for providing the study materials and clinical information.

References

  1. 1. Rihana N, Nanjappa S, Sullivan C, Velez AP, Tienchai N, Greene JN. Malignancy Trends in HIV-Infected Patients Over the Past 10 Years in a Single-Center Retrospective Observational Study in the United States. Cancer Control. 2018;25(1):1073274818797955. pmid:30185062
  2. 2. Lewden C, May T, Rosenthal E, Burty C, Bonnet F, Costagliola D, et al. Changes in causes of death among adults infected by HIV between 2000 and 2005: The "Mortalite 2000 and 2005" surveys (ANRS EN19 and Mortavic). J Acquir Immune Defic Syndr. 2008;48(5):590–8. pmid:18645512
  3. 3. Smith CJ, Ryom L, Weber R, Morlat P, Pradier C, Reiss P, et al. Trends in underlying causes of death in people with HIV from 1999 to 2011 (D:A:D): a multicohort collaboration. Lancet. 2014;384(9939):241–8. pmid:25042234
  4. 4. Yuan T, Hu Y, Zhou X, Yang L, Wang H, Li L, et al. Incidence and mortality of non-AIDS-defining cancers among people living with HIV: A systematic review and meta-analysis. EClinicalMedicine. 2022;52:101613. Epub 20220811. pmid:35990580
  5. 5. Shiels MS, Pfeiffer RM, Gail MH, Hall HI, Li J, Chaturvedi AK, et al. Cancer burden in the HIV-infected population in the United States. J Natl Cancer Inst. 2011;103(9):753–62. Epub 20110411. pmid:21483021
  6. 6. Hernandez-Ramirez RU, Shiels MS, Dubrow R, Engels EA. Cancer risk in HIV-infected people in the USA from 1996 to 2012: a population-based, registry-linkage study. Lancet HIV. 2017;4(11):e495–e504. Epub 20170810. pmid:28803888
  7. 7. Nishijima T, Inaba Y, Kawasaki Y, Tsukada K, Teruya K, Kikuchi Y, et al. Mortality and causes of death in people living with HIV in the era of combination antiretroviral therapy compared with the general population in Japan. AIDS. 2020;34(6):913–21. pmid:32039993
  8. 8. Yarchoan R, Ramaswami R, Lurain K. HIV-associated malignancies at 40: much accomplished but much to do. Glob Health Med. 2021;3(4):184–6. pmid:34532598
  9. 9. Isaguliants M, Bayurova E, Avdoshina D, Kondrashova A, Chiodi F, Palefsky JM. Oncogenic Effects of HIV-1 Proteins, Mechanisms Behind. Cancers (Basel). 2021;13(2). Epub 20210115. pmid:33467638
  10. 10. Boulware DR, Hullsiek KH, Puronen CE, Rupert A, Baker JV, French MA, et al. Higher levels of CRP, D-dimer, IL-6, and hyaluronic acid before initiation of antiretroviral therapy (ART) are associated with increased risk of AIDS or death. J Infect Dis. 2011;203(11):1637–46. pmid:21592994
  11. 11. Hirano T. IL-6 in inflammation, autoimmunity and cancer. Int Immunol. 2021;33(3):127–48. pmid:33337480
  12. 12. Ataie-Kachoie P, Pourgholami MH, Richardson DR, Morris DL. Gene of the month: Interleukin 6 (IL-6). J Clin Pathol. 2014;67(11):932–7. Epub 20140716. pmid:25031389
  13. 13. Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F. Tat protein of HIV-1 stimulates growth of cells derived from Kaposi’s sarcoma lesions of AIDS patients. Nature. 1990;345(6270):84–6. pmid:2184372
  14. 14. Ferdin J, Goricar K, Dolzan V, Plemenitas A, Martin JN, Peterlin BM, et al. Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA. PLoS One. 2018;13(1):e0191613. Epub 20180124. pmid:29364927
  15. 15. Popovic M, Tenner-Racz K, Pelser C, Stellbrink HJ, van Lunzen J, Lewis G, et al. Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therapy. Proc Natl Acad Sci U S A. 2005;102(41):14807–12. Epub 20050930. pmid:16199516
  16. 16. Carroll VA, Lafferty MK, Marchionni L, Bryant JL, Gallo RC, Garzino-Demo A. Expression of HIV-1 matrix protein p17 and association with B-cell lymphoma in HIV-1 transgenic mice. Proc Natl Acad Sci U S A. 2016;113(46):13168–73. Epub 20161031. pmid:27799525
  17. 17. Hoshino S, Sun B, Konishi M, Shimura M, Segawa T, Hagiwara Y, et al. Vpr in plasma of HIV type 1-positive patients is correlated with the HIV type 1 RNA titers. AIDS Res Hum Retroviruses. 2007;23(3):391–7. pmid:17411372
  18. 18. Yuan X, Matsuda Z, Matsuda M, Essex M, Lee TH. Human immunodeficiency virus vpr gene encodes a virion-associated protein. AIDS Res Hum Retroviruses. 1990;6(11):1265–71. pmid:2150318
  19. 19. Guenzel CA, Herate C, Benichou S. HIV-1 Vpr-a still "enigmatic multitasker". Front Microbiol. 2014;5:127. Epub 20140331. pmid:24744753
  20. 20. Deshmane SL, Amini S, Sen S, Khalili K, Sawaya BE. Regulation of the HIV-1 promoter by HIF-1alpha and Vpr proteins. Virol J. 2011;8:477. Epub 20111024. pmid:22023789
  21. 21. Duette G, Pereyra Gerber P, Rubione J, Perez PS, Landay AL, Crowe SM, et al. Induction of HIF-1alpha by HIV-1 Infection in CD4(+) T Cells Promotes Viral Replication and Drives Extracellular Vesicle-Mediated Inflammation. mBio. 2018;9(5). Epub 20180911. pmid:30206166
  22. 22. Majumder B, Janket ML, Schafer EA, Schaubert K, Huang XL, Kan-Mitchell J, et al. Human immunodeficiency virus type 1 Vpr impairs dendritic cell maturation and T-cell activation: implications for viral immune escape. J Virol. 2005;79(13):7990–8003. pmid:15956545
  23. 23. Khan H, Sumner RP, Rasaiyaah J, Tan CP, Rodriguez-Plata MT, Van Tulleken C, et al. HIV-1 Vpr antagonizes innate immune activation by targeting karyopherin-mediated NF-kappaB/IRF3 nuclear transport. Elife. 2020;9. Epub 20201210. pmid:33300875
  24. 24. Deshmane SL, Mukerjee R, Fan S, Del Valle L, Michiels C, Sweet T, et al. Activation of the oxidative stress pathway by HIV-1 Vpr leads to induction of hypoxia-inducible factor 1alpha expression. J Biol Chem. 2009;284(17):11364–73. Epub 20090209. pmid:19204000
  25. 25. DeHart JL, Zimmerman ES, Ardon O, Monteiro-Filho CM, Arganaraz ER, Planelles V. HIV-1 Vpr activates the G2 checkpoint through manipulation of the ubiquitin proteasome system. Virol J. 2007;4:57. Epub pmid:17559673.
  26. 26. Hrecka K, Gierszewska M, Srivastava S, Kozaczkiewicz L, Swanson SK, Florens L, et al. Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle. Proc Natl Acad Sci U S A. 2007;104(28):11778–83. Epub 20070703. pmid:17609381
  27. 27. Transy C, Margottin-Goguet F. HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle. 2009;8(16):2489–90. pmid:19667756
  28. 28. Zhang F, Bieniasz PD. HIV-1 Vpr induces cell cycle arrest and enhances viral gene expression by depleting CCDC137. Elife. 2020;9. Epub 20200615. pmid:32538781
  29. 29. Li D, Lopez A, Sandoval C, Nichols Doyle R, Fregoso OI. HIV Vpr Modulates the Host DNA Damage Response at Two Independent Steps to Damage DNA and Repress Double-Strand DNA Break Repair. mBio. 2020;11(4). Epub 20200804. pmid:32753492
  30. 30. Bregnard C, Benkirane M, Laguette N. DNA damage repair machinery and HIV escape from innate immune sensing. Front Microbiol. 2014;5:176. Epub 20140422. pmid:24795708
  31. 31. Levy DN, Refaeli Y, MacGregor RR, Weiner DB. Serum Vpr regulates productive infection and latency of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1994;91(23):10873–7. pmid:7971975
  32. 32. Belzile JP, Duisit G, Rougeau N, Mercier J, Finzi A, Cohen EA. HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase. PLoS Pathog. 2007;3(7):e85. pmid:17630831
  33. 33. Zimmerman ES, Sherman MP, Blackett JL, Neidleman JA, Kreis C, Mundt P, et al. Human immunodeficiency virus type 1 Vpr induces DNA replication stress in vitro and in vivo. J Virol. 2006;80(21):10407–18. Epub 20060906. pmid:16956949
  34. 34. Wang X, Singh S, Jung HY, Yang G, Jun S, Sastry KJ, et al. HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex. J Biol Chem. 2013;288(22):15474–80. Epub 20130423. pmid:23612978
  35. 35. Piller SC, Jans P, Gage PW, Jans DA. Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: implications for AIDS pathology. Proc Natl Acad Sci U S A. 1998;95(8):4595–600. pmid:9539783
  36. 36. Ferrucci A, Nonnemacher MR, Wigdahl B. Extracellular HIV-1 viral protein R affects astrocytic glyceraldehyde 3-phosphate dehydrogenase activity and neuronal survival. J Neurovirol. 2013;19(3):239–53. Epub 20130601. pmid:23728617
  37. 37. Guha D, Nagilla P, Redinger C, Srinivasan A, Schatten GP, Ayyavoo V. Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells. J Neuroinflammation. 2012;9:138. Epub 20120622. pmid:22727020
  38. 38. Ferrucci A, Nonnemacher MR, Cohen EA, Wigdahl B. Extracellular human immunodeficiency virus type 1 viral protein R causes reductions in astrocytic ATP and glutathione levels compromising the antioxidant reservoir. Virus Res. 2012;167(2):358–69. Epub 20120609. pmid:22691542
  39. 39. Arunagiri C, Macreadie I, Hewish D, Azad A. A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4+ lymphocytes. Apoptosis. 1997;2(1):69–76. pmid:14646566
  40. 40. Roshal M, Kim B, Zhu Y, Nghiem P, Planelles V. Activation of the ATR-mediated DNA damage response by the HIV-1 viral protein R. J Biol Chem. 2003;278(28):25879–86. Epub 20030508. pmid:12738771
  41. 41. Nakai-Murakami C, Shimura M, Kinomoto M, Takizawa Y, Tokunaga K, Taguchi T, et al. HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination. Oncogene. 2007;26(4):477–86. Epub 20060918. pmid:16983346
  42. 42. Hoshino S, Konishi M, Mori M, Shimura M, Nishitani C, Kuroki Y, et al. HIV-1 Vpr induces TLR4/MyD88-mediated IL-6 production and reactivates viral production from latency. J Leukoc Biol. 2010;87(6):1133–43. Epub 20100209. pmid:20145198
  43. 43. Lv L, Wang Q, Xu Y, Tsao LC, Nakagawa T, Guo H, et al. Vpr Targets TET2 for Degradation by CRL4(VprBP) E3 Ligase to Sustain IL-6 Expression and Enhance HIV-1 Replication. Mol Cell. 2018;70(5):961–70 e5. Epub 20180607. pmid:29883611
  44. 44. Iijima K, Okudaira N, Tamura M, Doi A, Saito Y, Shimura M, et al. Viral protein R of human immunodeficiency virus type-1 induces retrotransposition of long interspersed element-1. Retrovirology. 2013;10:83. Epub 20130805. pmid:23915234
  45. 45. Matsunaga A, Oka M, Iijima K, Shimura M, Gatanaga H, Oka S, et al. Short Communication: A Quantitative System for Monitoring Blood-Circulating Viral Protein R of Human Immunodeficiency Virus-1 Detected a Possible Link with Pathogenic Indices. AIDS Res Hum Retroviruses. 2019;35(7):660–3. Epub 20190506. pmid:30938169
  46. 46. Roesch F, Richard L, Rua R, Porrot F, Casartelli N, Schwartz O. Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells. J Virol. 2015;89(23):12118–30. Epub 20150923. pmid:26401039
  47. 47. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G. Inflammation and cancer: how hot is the link? Biochem Pharmacol. 2006;72(11):1605–21. Epub 20060804. pmid:16889756
  48. 48. Tachiwana H, Shimura M, Nakai-Murakami C, Tokunaga K, Takizawa Y, Sata T, et al. HIV-1 Vpr induces DNA double-strand breaks. Cancer Res. 2006;66(2):627–31. pmid:16423988
  49. 49. Richardson MW, Mirchandani J, Duong J, Grimaldo S, Kocieda V, Hendel H, et al. Antibodies to Tat and Vpr in the GRIV cohort: differential association with maintenance of long-term non-progression status in HIV-1 infection. Biomed Pharmacother. 2003;57(1):4–14. pmid:12642031
  50. 50. Wong-Staal F, Chanda PK, Ghrayeb J. Human immunodeficiency virus: the eighth gene. AIDS Res Hum Retroviruses. 1987;3(1):33–9. pmid:3476127
  51. 51. Reiss P, Lange JM, de Ronde A, de Wolf F, Dekker J, Danner SA, et al. Antibody response to viral proteins U (vpu) and R (vpr) in HIV-1-infected individuals. J Acquir Immune Defic Syndr (1988). 1990;3(2):115–22. pmid:2136912
  52. 52. Reiss P, Lange JM, de Ronde A, de Wolf F, Dekker J, Debouck C, et al. Speed of progression to AIDS and degree of antibody response to accessory gene products of HIV-1. J Med Virol. 1990;30(3):163–8. pmid:2341832
  53. 53. Dunlop RJ, Campbell CW. Cytokines and advanced cancer. J Pain Symptom Manage. 2000;20(3):214–32. pmid:11018340
  54. 54. Okay G, Koc MM, Guler EM, Yabaci A, Kocyigit A, Akkoyunlu Y. The Effect of Antiretroviral Therapy on IL-6, IL-1beta, TNF-alpha, IFN-gamma Levels and their Relationship with HIV-RNA and CD4+ T Cells in HIV Patients. Curr HIV Res. 2020;18(5):354–61. pmid:32652911
  55. 55. Kumari N, Dwarakanath BS, Das A, Bhatt AN. Role of interleukin-6 in cancer progression and therapeutic resistance. Tumour Biol. 2016;37(9):11553–72. Epub 20160603. pmid:27260630
  56. 56. New-Aaron M, Ganesan M, Dagur RS, Kharbanda KK, Poluektova LY, Osna NA. Pancreatogenic Diabetes: Triggering Effects of Alcohol and HIV. Biology (Basel). 2021;10(2). Epub 20210203. pmid:33546230
  57. 57. Whitcomb DC, Pogue-Geile K. Pancreatitis as a risk for pancreatic cancer. Gastroenterol Clin North Am. 2002;31(2):663–78. pmid:12134623
  58. 58. Zicari S, Sessa L, Cotugno N, Ruggiero A, Morrocchi E, Concato C, et al. Immune Activation, Inflammation, and Non-AIDS Co-Morbidities in HIV-Infected Patients under Long-Term ART. Viruses. 2019;11(3). Epub 20190227. pmid:30818749
  59. 59. Mamik MK, Hui E, Branton WG, McKenzie BA, Chisholm J, Cohen EA, et al. HIV-1 Viral Protein R Activates NLRP3 Inflammasome in Microglia: implications for HIV-1 Associated Neuroinflammation. J Neuroimmune Pharmacol. 2017;12(2):233–48. Epub pmid:27726055.
  60. 60. Nakajima K, Martinez-Maza O, Hirano T, Breen EC, Nishanian PG, Salazar-Gonzalez JF, et al. Induction of IL-6 (B cell stimulatory factor-2/IFN-beta 2) production by HIV. J Immunol. 1989;142(2):531–6. pmid:2783441
  61. 61. De Pablo-Bernal RS, Ruiz-Mateos E, Rosado I, Dominguez-Molina B, Alvarez-Rios AI, Carrillo-Vico A, et al. TNF-alpha levels in HIV-infected patients after long-term suppressive cART persist as high as in elderly, HIV-uninfected subjects. J Antimicrob Chemother. 2014;69(11):3041–6. Epub 20140709. pmid:25011654
  62. 62. Borges AH O’Connor JL, Phillips AN, Ronsholt FF, Pett S, Vjecha MJ, et al. Factors Associated With Plasma IL-6 Levels During HIV Infection. J Infect Dis. 2015;212(4):585–95. Epub 20150226. pmid:25722296
  63. 63. Jones SA, Jenkins BJ. Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer. Nat Rev Immunol. 2018;18(12):773–89. pmid:30254251
  64. 64. Taniguchi K, Karin M. IL-6 and related cytokines as the critical lynchpins between inflammation and cancer. Semin Immunol. 2014;26(1):54–74. Epub 20140216. pmid:24552665
  65. 65. Fukazawa Y, Lum R, Okoye AA, Park H, Matsuda K, Bae JY, et al. B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers. Nat Med. 2015;21(2):132–9. Epub 20150119. pmid:25599132
  66. 66. Solas C, Lafeuillade A, Halfon P, Chadapaud S, Hittinger G, Lacarelle B. Discrepancies between protease inhibitor concentrations and viral load in reservoirs and sanctuary sites in human immunodeficiency virus-infected patients. Antimicrob Agents Chemother. 2003;47(1):238–43. pmid:12499197
  67. 67. Chun TW, Nickle DC, Justement JS, Meyers JH, Roby G, Hallahan CW, et al. Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. J Infect Dis. 2008;197(5):714–20. pmid:18260759
  68. 68. Horiike M, Iwami S, Kodama M, Sato A, Watanabe Y, Yasui M, et al. Lymph nodes harbor viral reservoirs that cause rebound of plasma viremia in SIV-infected macaques upon cessation of combined antiretroviral therapy. Virology. 2012;423(2):107–18. Epub 20111221. pmid:22196013
  69. 69. Letendre S, Marquie-Beck J, Capparelli E, Best B, Clifford D, Collier AC, et al. Validation of the CNS Penetration-Effectiveness rank for quantifying antiretroviral penetration into the central nervous system. Arch Neurol. 2008;65(1):65–70. pmid:18195140
  70. 70. Damouche A, Lazure T, Avettand-Fenoel V, Huot N, Dejucq-Rainsford N, Satie AP, et al. Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection. PLoS Pathog. 2015;11(9):e1005153. Epub 20150924. pmid:26402858
  71. 71. Cardozo EF, Piovoso MJ, Zurakowski R. Increased inflammation in sanctuary sites may explain viral blips in HIV infection. IET Syst Biol. 2016;10(4):153–66. pmid:27444025
  72. 72. Sorstedt E, Nilsson S, Blaxhult A, Gisslen M, Flamholc L, Sonnerborg A, et al. Viral blips during suppressive antiretroviral treatment are associated with high baseline HIV-1 RNA levels. BMC Infect Dis. 2016;16:305. Epub 20160621. pmid:27329293
  73. 73. Pollack RA, Jones RB, Pertea M, Bruner KM, Martin AR, Thomas AS, et al. Defective HIV-1 Proviruses Are Expressed and Can Be Recognized by Cytotoxic T Lymphocytes, which Shape the Proviral Landscape. Cell Host Microbe. 2017;21(4):494–506 e4. pmid:28407485
  74. 74. Ho YC, Shan L, Hosmane NN, Wang J, Laskey SB, Rosenbloom DI, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell. 2013;155(3):540–51. Epub 20131024. pmid:24243014
  75. 75. Imamichi H, Smith M, Adelsberger JW, Izumi T, Scrimieri F, Sherman BT, et al. Defective HIV-1 proviruses produce viral proteins. Proc Natl Acad Sci U S A. 2020;117(7):3704–10. Epub 20200206. pmid:32029589
  76. 76. Nixon CC, Mavigner M, Sampey GC, Brooks AD, Spagnuolo RA, Irlbeck DM, et al. Systemic HIV and SIV latency reversal via non-canonical NF-kappaB signalling in vivo. Nature. 2020;578(7793):160–5. Epub
  77. 77. McBrien JB, Mavigner M, Franchitti L, Smith SA, White E, Tharp GK, et al. Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8(+) cells. Nature. 2020;578(7793):154–9. Epub 20200122. pmid:31969705
  78. 78. Kessing CF, Nixon CC, Li C, Tsai P, Takata H, Mousseau G, et al. In Vivo Suppression of HIV Rebound by Didehydro-Cortistatin A, a "Block-and-Lock" Strategy for HIV-1 Treatment. Cell Rep. 2017;21(3):600–11. pmid:29045830
  79. 79. Ong EB, Watanabe N, Saito A, Futamura Y, Abd El Galil KH, Koito A, et al. Vipirinin, a coumarin-based HIV-1 Vpr inhibitor, interacts with a hydrophobic region of VPR. J Biol Chem. 2011;286(16):14049–56. Epub 20110228. pmid:21357691
  80. 80. Watanabe N, Nishihara Y, Yamaguchi T, Koito A, Miyoshi H, Kakeya H, et al. Fumagillin suppresses HIV-1 infection of macrophages through the inhibition of Vpr activity. FEBS Lett. 2006;580(11):2598–602. Epub 20060419. pmid:16631749
  81. 81. Shimura M, Zhou Y, Asada Y, Yoshikawa T, Hatake K, Takaku F, et al. Inhibition of Vpr-induced cell cycle abnormality by quercetin: a novel strategy for searching compounds targeting Vpr. Biochem Biophys Res Commun. 1999;261(2):308–16. pmid:10425183
  82. 82. Sato H, Murakami T, Matsuura R, Abe M, Matsuoka S, Yashiroda Y, et al. A Novel Class of HIV-1 Inhibitors Targeting the Vpr-Induced G2-Arrest in Macrophages by New Yeast- and Cell-Based High-Throughput Screening. Viruses. 2022;14(6). Epub 20220616. pmid:35746791
  83. 83. Kamata M, Wu RP, An DS, Saxe JP, Damoiseaux R, Phelps ME, et al. Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death. Biochem Biophys Res Commun. 2006;348(3):1101–6. Epub 20060802. pmid:16904642
  84. 84. Win NN, Ito T, Win YY, Ngwe H, Kodama T, Abe I, et al. Quassinoids: Viral protein R inhibitors from Picrasma javanica bark collected in Myanmar for HIV infection. Bioorg Med Chem Lett. 2016;26(19):4620–4. Epub 20160821. pmid:27575477
  85. 85. Win NN, Ito T, Matsui T, Aimaiti S, Kodama T, Ngwe H, et al. Isopimarane diterpenoids from Kaempferia pulchra rhizomes collected in Myanmar and their Vpr inhibitory activity. Bioorg Med Chem Lett. 2016;26(7):1789–93. Epub 20160215. pmid:26916438
  86. 86. Win NN, Kodama T, Lae KZW, Win YY, Ngwe H, Abe I, et al. Bis-iridoid and iridoid glycosides: Viral protein R inhibitors from Picrorhiza kurroa collected in Myanmar. Fitoterapia. 2019;134:101–7. Epub 20190219. pmid:30794917
  87. 87. Refaeli Y, Levy DN, Weiner DB. The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product. Proc Natl Acad Sci U S A. 1995;92(8):3621–5. pmid:7724608
  88. 88. Schafer EA, Venkatachari NJ, Ayyavoo V. Antiviral effects of mifepristone on human immunodeficiency virus type-1 (HIV-1): targeting Vpr and its cellular partner, the glucocorticoid receptor (GR). Antiviral Res. 2006;72(3):224–32. Epub 20060707. pmid:16889838
  89. 89. Chang WT, Hong MY, Chen CL, Hwang CY, Tsai CC, Chuang CC. Mutant glucocorticoid receptor binding elements on the interleukin-6 promoter regulate dexamethasone effects. BMC Immunol. 2021;22(1):24. Epub 20210326. pmid:33771121
  90. 90. Muthumani K, Choo AY, Zong WX, Madesh M, Hwang DS, Premkumar A, et al. The HIV-1 Vpr and glucocorticoid receptor complex is a gain-of-function interaction that prevents the nuclear localization of PARP-1. Nat Cell Biol. 2006;8(2):170–9. Epub 20060122. pmid:16429131
  91. 91. Alves de Souza Rios L, Mapekula L, Mdletshe N, Chetty D, Mowla S. HIV-1 Transactivator of Transcription (Tat) Co-operates With AP-1 Factors to Enhance c-MYC Transcription. Front Cell Dev Biol. 2021;9:693706. Epub 20210630. pmid:34277639
  92. 92. Akbay B, Germini D, Bissenbaev AK, Musinova YR, Sheval EV, Vassetzky Y, et al. HIV-1 Tat Activates Akt/mTORC1 Pathway and AICDA Expression by Downregulating Its Transcriptional Inhibitors in B Cells. Int J Mol Sci. 2021;22(4). Epub 20210204. pmid:33557396
  93. 93. Caccuri F, Giagulli C, Bugatti A, Benetti A, Alessandri G, Ribatti D, et al. HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2. Proc Natl Acad Sci U S A. 2012;109(36):14580–5. Epub 20120817. pmid:22904195
  94. 94. Caccuri F, Rueckert C, Giagulli C, Schulze K, Basta D, Zicari S, et al. HIV-1 matrix protein p17 promotes lymphangiogenesis and activates the endothelin-1/endothelin B receptor axis. Arterioscler Thromb Vasc Biol. 2014;34(4):846–56. Epub 20140130. pmid:24482377
  95. 95. Giagulli C D’Ursi P, He W, Zorzan S, Caccuri F, Varney , et al. A single amino acid substitution confers B-cell clonogenic activity to the HIV-1 matrix protein p17. Sci Rep. 2017;7(1):6555. Epub 20170726. pmid:28747658
  96. 96. Sawaya BE, Khalili K, Gordon J, Taube R, Amini S. Cooperative interaction between HIV-1 regulatory proteins Tat and Vpr modulates transcription of the viral genome. J Biol Chem. 2000;275(45):35209–14. pmid:10931842
  97. 97. Sato A, Yoshimoto J, Isaka Y, Miki S, Suyama A, Adachi A, et al. Evidence for direct association of Vpr and matrix protein p17 within the HIV-1 virion. Virology. 1996;220(1):208–12. pmid:8659115