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. 2016 Oct;22(5):666-673.
doi: 10.1007/s13365-016-0443-6. Epub 2016 May 2.

Cystatin B and HIV regulate the STAT-1 signaling circuit in HIV-infected and INF-β-treated human macrophages

L E Rivera  1 , E Kraiselburd  2 , L M Meléndez  3
Affiliations

Cystatin B and HIV regulate the STAT-1 signaling circuit in HIV-infected and INF-β-treated human macrophages

L E Rivera et al. J Neurovirol. 2016 Oct.

Abstract

Cystatin B is a cysteine protease inhibitor that induces HIV replication in monocyte-derived macrophages (MDM). This protein interacts with signal transducer and activator of transcription (STAT-1) factor and inhibits the interferon (IFN-β) response in Vero cells by preventing STAT-1 translocation to the nucleus. Cystatin B also decreases the levels of tyrosine-phosphorylated STAT-1 (STAT-1PY). However, the mechanisms of cystatin B regulation on STAT-1 phosphorylation in MDM are unknown. We hypothesized that cystatin B inhibits IFN-β antiviral responses and induces HIV replication in macrophage reservoirs through the inhibition of STAT-1 phosphorylation. Macrophages were transfected with cystatin B siRNA prior to interferon-β treatment or infected with HIV-ADA to determine the effect of cystatin B modulation in STAT-1 localization and activation using immunofluorescence and proximity ligation assays. Cystatin B decreased STAT-1PY and its transportation to the nucleus, while HIV infection retained unphosphorylated STAT (USTAT-1) in the nucleus avoiding its exit to the cytoplasm for eventual phosphorylation. In IFN-β-treated MDM, cystatin B inhibited the nuclear translocation of both, USTAT-1 and STAT-1PY. These results demonstrate that cystatin B interferes with the STAT-1 signaling and IFN-β-antiviral responses perpetuating HIV in macrophage reservoirs.

Keywords: Cystatin B; HIV; IFN-β; Macrophages; STAT-1.

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Conflict of interest statement

LMM has a patent application approved for cystatin B, related to this manuscript. Pat No. 8,143,231.

Figures

Fig. 1
Fig. 1. Cystatin B inhibits the nuclear translocation of USTAT-1 in IFN-β treated MDM
Uninfected MDM induced by IFN-β were assayed by immunofluorescence for cystatin B (green) and USTAT-1 (red). Blue fluorescent DAPI was used as nuclear stain. MDM expressing cystatin B before (1–3) and after IFN-β treatment (7–9), showed a cytoplasmic localization of STAT-1. In absence of cystatin B, USTAT-1 was retained in the cytoplasm of untreated MDM (4–6), but USTAT-1 was completely translocated to the nucleus in IFN-β treated MDM after silencing of cystatin B (10–12). Non-targeting siRNA was used as a control for the cystatin B silencing procedure (1–3 and 7–9). Confocal images were obtained on a Zeiss confocal microscope Axiovert 200M with a LSM 510, 40x. The results are representative of experiments performed using three different donors.
Fig. 2
Fig. 2. Cystatin B inhibits the nuclear translocation of USTAT-1 in HIV-infected MDM
Nuclear translocation of STAT-1 was observed in HIV-1 infected MDM treated with cystatin B siRNA. At 12DPI, HIV-infected MDM were assayed by immunofluorescence for cystatin B (green) and USTAT-1 (red). Blue fluorescent DAPI was used as nuclear stain. Non-targeting siRNA was used as siRNA control. Cystatin B was silenced in MDM cultures using siRNA. Uninfected MDM expressing cystatin B showed a predominantly cytoplasmic localization of USTAT-1 (1–3). After cystatin B gene silencing, STAT-1 was retained in the cytoplasm of uninfected MDM (4–6) as well as in HIV infected MDM treated with non- targeting siRNA (7–9). At 12 DPI, USTAT-1 was totally translocated to the nucleus in HIV-infected MDM after cystatin B gene silencing (10–12). Images were obtained on a Nikon Eclipse E400 fluorescence microscope with a SPOT Insight QE camera and SPOT 5.1, 40x. These results are representative of two independent experiments performed using two different donors.
Fig. 3
Fig. 3. The role of cystatin B in STAT-1 Activation in MDM is mediated by STAT-1PY
The effect of cystatin B modulation in STAT-1PY were examined in induced MDM. (A) Cystatin B inhibits the STAT-1PY nuclear translocation in IFN-B-induced MDM. Uninfected MDM induced by IFN-β were assayed with PLA in situ. Non-targeting siRNA and unstimulated MDM were used as controls. Both, unstimulated and IFN-β treated MDM showed a cytoplasmic localization of STAT1-PY. In IFN-β treated MDM, STAT-1PY is translocated to the nucleus after silencing of cystatin B. (B) Cystatin B inhibits STAT-1PY in HIV infected MDM. (1) In situ PLA was used to determine the STAT1PY in HIV infected MDM treated with cystatin B siRNA. siRNA treatment against cystatin B increased the STAT-1 tyrosine phosphorylation in HIV infected MDM. (2) An unpaired two-tailed Student’s t-test was used to compare the PLA signals of Tyrosine-phosphorylated-STAT1 in uninfected and HIV-infected MDM treated with or without anti-cystatin B siRNA. (C) HIVp24 antigen levels from MDM supernatants of two donors at 3,6 and 12 days post-infection shows a productive infection. (D) Experimental controls included: (1) Biological Positive control consisting of uninfected MDM stained for cystatin B/cathepsin B interacting proteins, (2) Biological Negative control consisting of HIV-infected MDM that lost cystatin B/cathepsin B interaction, (3) A Technical control included the absence of the primary antibody and (4) STAT-1PY in IFN-β-treated MDM. Images were obtained on a Nikon Eclipse E400 fluorescence microscope with a SPOT Insight QE camera and SPOT 5.1, 40x. These results are representative of two independent experiments using two different donors.
Fig. 3
Fig. 3. The role of cystatin B in STAT-1 Activation in MDM is mediated by STAT-1PY
The effect of cystatin B modulation in STAT-1PY were examined in induced MDM. (A) Cystatin B inhibits the STAT-1PY nuclear translocation in IFN-B-induced MDM. Uninfected MDM induced by IFN-β were assayed with PLA in situ. Non-targeting siRNA and unstimulated MDM were used as controls. Both, unstimulated and IFN-β treated MDM showed a cytoplasmic localization of STAT1-PY. In IFN-β treated MDM, STAT-1PY is translocated to the nucleus after silencing of cystatin B. (B) Cystatin B inhibits STAT-1PY in HIV infected MDM. (1) In situ PLA was used to determine the STAT1PY in HIV infected MDM treated with cystatin B siRNA. siRNA treatment against cystatin B increased the STAT-1 tyrosine phosphorylation in HIV infected MDM. (2) An unpaired two-tailed Student’s t-test was used to compare the PLA signals of Tyrosine-phosphorylated-STAT1 in uninfected and HIV-infected MDM treated with or without anti-cystatin B siRNA. (C) HIVp24 antigen levels from MDM supernatants of two donors at 3,6 and 12 days post-infection shows a productive infection. (D) Experimental controls included: (1) Biological Positive control consisting of uninfected MDM stained for cystatin B/cathepsin B interacting proteins, (2) Biological Negative control consisting of HIV-infected MDM that lost cystatin B/cathepsin B interaction, (3) A Technical control included the absence of the primary antibody and (4) STAT-1PY in IFN-β-treated MDM. Images were obtained on a Nikon Eclipse E400 fluorescence microscope with a SPOT Insight QE camera and SPOT 5.1, 40x. These results are representative of two independent experiments using two different donors.
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