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. 2019 May 6;15(5):e1007743.
doi: 10.1371/journal.ppat.1007743. eCollection 2019 May.

Kaposi's Sarcoma-Associated Herpesvirus vIRF2 Protein Utilizes an IFN-dependent Pathway to Regulate Viral Early Gene Expression

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Free PMC article

Kaposi's Sarcoma-Associated Herpesvirus vIRF2 Protein Utilizes an IFN-dependent Pathway to Regulate Viral Early Gene Expression

Sandra Koch et al. PLoS Pathog. .
Free PMC article

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi's sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1-4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. KSHV infection increases the number of PML nuclear bodies and associated proteins in endothelial cells.
(A) HUVECs were either infected with rKSHV.219 at an MOI of 20, or HCMV at an MOI 3, or left uninfected (n.i.). 48 h after infection cells were fixed and processed for IFA staining. Infected cells express GFP, PML was detected with a mouse anti-PML antibody and a goat anti-mouse IgG Lissamine Rhodamine (LRSC)-conjugated secondary antibody and DNA was stained with DAPI. Images were acquired using a Carl Zeiss microscope at 100x magnification. For better visualization PML NBs are shown as black dots on a white background and the nuclei were encircled. Bars, 10 μm. (B) For the quantification of PML NBs upon KSHV and HCMV infection, PML NBs were counted in at least 50 cells per sample and a Mann Whitney U test was performed. Boxes indicate 25th to 75th percentile; the central line inside each box indicates the median and the whisker illustrate the 5th and 95th percentile. The dots indicate the outliers. (C) Protein expression of infection markers and PML NBs-associated proteins was analyzed by WB 48 h post infection of HUVECs infected with either KSHV or HCMV. (D) HEK-293 cells were treated with IFN-α for 72 h and PML protein expression was analyzed by WB.
Fig 2
Fig 2. Effects of KSHV vIRF proteins on PML NBs.
(A) HeLa Cells were transfected with 1 μg of either the control vector or one of the vIRF expressing constructs and were fixed 36 h after transfection for IFA. Transfected cells express GFP, PML was detected with a mouse anti-PML antibody and a goat anti-mouse IgG Lissamine Rhodamine (LRSC)-conjugated secondary antibody and DNA was stained with DAPI. Images were acquired using a Carl Zeiss microscope at 100x magnification. For better visualization PML NBs are shown as black dots on a white background and the nuclei were encircled. Bars, 10 μm. (B) Quantification of immunofluorescence data from A, PML NBs were counted in at least 100 cells per each construct and a Man Whitney U test was performed to determine significance. Boxes indicate 25th to 75th percentile; central line inside each box indicates the median and the whisker illustrates the 5th and 95th percentile. The dots indicate the outliers. (C) HUVECs were transduced with either the control or the vIRF2 expressing lentiviral vector and 36 h after transduction cells were lysed and protein expression was analyzed by WB. (D) HUVECs were transduced with either the control or vIRF2 expressing lentivirus and 36 h later cells were lysed for RNA extraction. PML mRNA was quantified by reverse transcription following qPCR using dually labeled probes (Taqman).
Fig 3
Fig 3. Expression of vIRF2 in KSHV-infected cells.
(A) The vIRF genes are located between ORF57 and ORF58 in the KSHV genome. The vIRF2 protein is encoded by the two exons K11.1 and K11. (B) Schematic diagram of the vIRF2 open reading frame, shown in the opposite orientation to its position in the viral genome. ATG start codons are indicated by red amino acid numbers at the positions aa 1, 47, 103, 108 110, 131, 134 in K11.1 and aa 213, 311, 350, 382, 408, 440, 484, 584, 593 in K11. Dotted lines indicate binding sites for siRNAs used in this study: aa7-13 (1), aa193-199 (2) and aa609-615 (3), DBD: DNA binding domain (aa7-114), NLS: nuclear localization signal (aa146-159), R1/R2: repeat region 1/2, E1/2: antibody epitopes. (C) Expression of vIRF2 in different KSHV positive cell lines. The lytic cycle in the PEL-derived cell lines BC1, BC3 and BCBL1 was induced by 100 ng/ml TPA, HuARLT.rKSHV.219 cells were induced using 1.67 mM SB and 10% tissue culture supernatant containing RTA-expressing baculovirus, the BJAB.rKSHV.219 using 2.5 μg/ml goat anti human-IgM. At the indicated time points after induction the cells were lysed and protein expression was analyzed by WB. (D) Subcellular localization of vIRF2 was analyzed by nuclear and cytoplasmic extraction of BJAB or BJAB.rKSHV.219 cells with or without induction of the lytic cycle using 2.5 μg/ml goat anti human-IgM. WB analysis was performed for vIRF2, Calnexin (cytoplasmic marker; C) and Lamin A/C (nuclear marker; N).
Fig 4
Fig 4. KSHV vIRF2 suppresses KSHV lytic protein expression upon reactivation.
(A) HuARLT.rKSHV.219 cells were transduced with either the control GFP or the GFP-T2A-vIRF2 encoding lentivirus. 24 h later, the lytic cycle was induced by 12.5% tissue culture supernatant containing RTA-expressing baculovirus and 1.25 mM SB. After 48 h cells were lysed and protein expression was analyzed by WB. (B) HuARLT.rKSHV.219 cells were microporated with either a non-targeting siRNA (ctr) or three different vIRF2 siRNAs and 24 h later the lytic cycle was induced with 10% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB. The cells were lysed 72 h after induction and protein expression was analyzed by WB. (C) Schematic diagram of the KSHV genome in the BAC16 vector, comparison of the BAC16.KSHV.WT and the BAC16.KSHV.ΔvIRF2 genome in which the whole vIRF2 gene was replaced with a kanamycin resistance gene (KanR). (D) Stable HuARLT.BAC16.KSHV.WT and ΔvIRF2 cells were induced using 12.5% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB. Protein expression was analyzed by WB after lysis of the cells at the indicated time points after induction. (E) Supernatants of the reactivated HuARLT.BAC16.KSHV.WT and ΔvIRF2 cells after 72 h were used to determine infectious viral titers on HEK.293 cells. In three independent experiments titers for KSHV.WT-infected cells ranged from 2.3–4.6×103/ml and for KSHV.ΔvIRF2-infected cells from 2.6–4×104/ml. (F) HuARLT.BAC16.KSHV.WT and ΔvIRF2 cells were transduced with either the control GFP or the GFP-T2A-vIRF2 encoding lentivirus and the lytic cycle was induced as described in panel A. After 48 h cells were lysed and protein expression was analyzed by WB, using antibodies for the indicated proteins.
Fig 5
Fig 5. KSHV vIRF2 inhibits KSHV lytic protein expression after de novo infection.
(A) HuARLT cells were microporated with either a non-targeting siRNA (ctr) or with two different vIRF2 siRNAs (see Figs 3 and 4) and 24 h later cells were infected with rKSHV at a MOI of 5 (titer determined on HEK-293 cells). The cells were lysed 72 h after induction and protein expression was analyzed by WB. ORF45 and K-bZIP protein levels were quantified and normalized to β-actin levels by using the Image studio software. Relative expression levels (in comparison to those seen with the control siRNA) are indicated below the respective blots. (B) HuARLT cells were infected with either the KSHV.WT virus or the KSHV.ΔvIRF2 virus at a MOI of 5. The cells were lysed at the indicated time points and protein expression was analyzed by WB.
Fig 6
Fig 6. Domains of vIRF2 required for the inhibition of early lytic KSHV protein expression.
(A) Schematic diagram of the possible vIRF2 protein forms expressed from KSHV ORFs K11.1 and K11 in different vIRF2 double stop mutants compared to the WT-infected cells. Methionine residues are indicated by red amino acid numbers at the positions aa1, 47, 103, 110, 131, 134 in K11.1 and aa213, 311, 350, 382, 408, 440, 484, 584, 593 in K11. Lines indicate double stop mutations at the positions aa7-8, aa323-324, aa386-387 and aa460-461, DBD: DNA binding domain (aa7-114), NLS: nuclear localization signal (aa146-159), R1/R2: repeat region 1/2, E1/E2: antibody epitope. (B) The different stable HEK-293.BAC16 cell lines were induced using 10% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB. Protein expression was analyzed 72 h post induction by WB after lysis. (C) The different stable HuARLT.BAC16 cells were induced using 12.5% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB for 72 h. Protein expression was analyzed by WB after lysis of the cells. Stop #1, aa7-8; Stop #2, aa323-324; Stop #3, aa386-387; Stop #4, aa460-461. Rev. #1, revertant to Stop #1; Rev. #2, revertant to Stop #2; Rev. #4, revertant to Stop #4.
Fig 7
Fig 7. vIRF2 induces IFN-regulated proteins with tetratricopeptide repeats.
(A) The lytic cycle was induced in the stable HuARLT.BAC16.KSHV.WT and HuARLT.BAC16.KSHV.ΔvIRF2 cells with 12.5% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB. At the indicated time points after induction the cells were lysed and the expression of the IFIT proteins 1, 2 and 3 as well as vIRF2 and K-bZIP was analyzed by WB. (B) Empty HuARLT or HuARLT.rKSHV.219 cells were transduced with a lentivirus expressing either the control GFP or the vIRF2 protein. 24 h after the transduction, reactivation was induced by 12.5% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB and 48 h later cells were lysed and protein expression was analyzed by WB. (C) HuARLT cells were infected with either the KSHV.WT virus or the KSHV.ΔvIRF2 virus at a MOI of 5. The cells were lysed at the indicated time points and protein expression was analyzed by WB. IFIT protein levels were quantified to β-actin levels by using the Image studio software and are indicated below the blots setting the levels at 24 h post infection in WT-infected cells to 1.
Fig 8
Fig 8. IFIT proteins and PML suppress KSHV lytic gene expression.
(A) HuARLT.rKSHV.219 (left) or HuARLT (right) cells were microporated with a pool of four different siRNAs targeting IFIT1. 24 h later either the lytic cycle was induced with 10% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB (left) or HuARLT cells were infected with rKSHV.219 at an MOI of 5 (right). (B) HuARLT cells were microporated with a pool of four different siRNAs targeting IFIT2. 24 h later cells were infected with rKSHV.219 at an MOI of 5. (C) HuARLT.rKSHV.219 cells were microporated with a pool of three different siRNAs targeting IFIT3. 24 h later the lytic cycle was induced with 10% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB. All cells were lysed at the indicated time points and protein expression was analyzed by WB. (D) HuARLT.rKSHV.219 cells were microporated with a pool of three different siRNAs targeting all PML isoforms and 24 h later the lytic cycle was induced with 10% tissue culture supernatant containing RTA-expressing baculovirus and 1.67 mM SB. The cells were lysed at the indicated time points and analyzed by WB.

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This study was supported by the Collaborative Research Centre 900 ‘Chronic Infections’ of the Deutsche Forschungsgemeinschaft (project C1 to TFS) and a personal doctoral fellowship of the German Academic Exchange Service (DAAD) to MD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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