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Comparative Study
. 2020 Feb:541:160-173.
doi: 10.1016/j.virol.2019.12.011. Epub 2019 Dec 30.

Comparative analysis of the viral interferon regulatory factors of KSHV for their requisite for virus production and inhibition of the type I interferon pathway

Gavin Golas  1 , Seung Jin Jang  1 , Nenavath Gopal Naik  1 , Juan D Alonso  1 , Bernadett Papp  2 , Zsolt Toth  3
Affiliations
Comparative Study

Comparative analysis of the viral interferon regulatory factors of KSHV for their requisite for virus production and inhibition of the type I interferon pathway

Gavin Golas et al. Virology. 2020 Feb.

Abstract

Unique among human viruses, Kaposi's sarcoma-associated herpesvirus (KSHV) encodes several homologs of cellular interferon regulatory factors (vIRFs). Since KSHV expresses multiple factors that can inhibit interferon (IFN) signaling to promote virus production, it is still unclear to what extent vIRFs contribute to these specific processes during KSHV infection. To study the function of vIRFs during viral infection, we engineered 3xFLAG-tagged-vIRF and vIRF-knockout recombinant KSHV clones, which were utilized to test vIRF expression, as well as their requirement for viral replication, virus production, and inhibition of the type I IFN pathway in different models of lytic KSHV infection. Our data show that all vIRFs can be expressed as lytic viral proteins, yet were dispensable for KSHV production and inhibition of type I IFN. Nevertheless, as vIRFs were able to suppress IFN-stimulated antiviral genes, vIRFs may still promote the KSHV lytic cycle in the presence of an ongoing antiviral response.

Keywords: BAC16; IFN signaling; Interferon-stimulated genes; KSHV; Lymphatic endothelial cells; Viral immune evasion; Viral replication; vIRFs.

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Conflict of interest statement

Declaration of competing interest There is no conflict of interest.

Figures

Figure 1.
Figure 1.. Construction of recombinant KSHV clones expressing 3xFLAG-tagged vIRFs
(A) Schematic showing the vIRF locus within the KSHV genome and the sites targeted for homologous recombination to engineer 3xFLAG-vIRF and vIRF-KO recombinant KSHV clones. (B) Diagram of the homologous recombination showing the two-step procedure, which was used to make FLAG-tagged vIRF or vIRF knockout clones using BAC16 KSHV. Shown is an example targeting vIRF1 where a 3xFLAG epitope tag was fused to the 5’ end of vIRF1. The ATG start codon of vIRF1 was deleted and instead, ATG of the 3xFLAG epitope tag was utilized. (C) PFGE analysis of SbfI-digested WT and 3xFLAG-vIRF BAC16 DNAs. (D) iSLK cells latently infected by 3xFLAG-vIRF recombinant KSHV were treated with 1 μg/ml Dox and 1 mM NaB to induce lytic reactivation. vIRF protein expression was analyzed from cell lysates by immunoblot at 48 hpi. (E) To assess infectious KSHV production, an equal amount of supernatant from lytically reactivated iSLK-BAC16 vIRF cell lines at 4 dpi was used to infect an equivalent number of 293T cells. BAC16 encodes GFP under the control of the EF1α cellular promoter. The resulting GFP-positive 293T cells at 24 hpi were quantified as readout of virus production using flow cytometry. Error bars represent standard deviation (n=3). Molecular Weight (MW) markers: MW1 for λ DNA-Mono Cut Mix, MW2 for 1 kbp DNA ladder.
Figure 2.
Figure 2.. Kinetics of vIRF expression during lytic KSHV reactivation
To test vIRF expression, 3xFLAG-vIRF BAC16 KSHV was lytically reactivated from latently infected iSLK cell lines using 1 μg/ml Dox and 1 mM NaB and harvested cell lysates at various time points post-induction. (A) vIRF mRNA expression was analyzed by RT-qPCR. Additional viral genes RTA (immediate-early), ORF45 (early), and ORF25 (late) were included as controls. Error bars represent standard deviation (n=3). (B) vIRF protein expression was analyzed by immunoblot using FLAG antibody. Additional viral proteins RTA (IE), ORF6 (early), and K8.1 (late) were included as controls. (C) The iSLK-3xFLAG-vIRF BAC16 cell lines were induced by Dox and NaB for 60 hours in the absence (−) or the presence (+) of 100 uM PAA. The expression of vIRFs was detected by FLAG antibody. The late KSHV protein K8.1 was used as positive control for the PAA treatment.
Figure 3.
Figure 3.. Construction of recombinant KSHV clones lacking vIRFs
(A) WT and vIRF-KO BAC16 DNAs were digested with SbfI and analyzed by PFGE. (B) iSLK cells latently infected by 3xFLAG-vIRF or derivative vIRF-KO recombinant BAC16 clones were treated with 1 μg/ml Dox and 1 mM NaB to induce lytic reactivation for 48 hpi. The loss of vIRF protein expression was confirmed using FLAG-specific immunoblots. RTA and LANA were included as KSHV viral protein controls. (C) To confirm vIRF3 as a lytic protein, 293T cell lines carrying either BAC16-vIRF3–3xFLAG or BAC16-ΔvIRF were treated with 3 mM NaB to induce lytic reactivation. vIRF3 protein expression was determined using FLAG- and vIRF3-specific immunoblots. The asterisk marks a non-specific band in the vIRF3 immunoblot. (D) Immunofluorescence analysis of the expression of vIRF3 using FLAG antibody in latent and reactivated (60 hpi) iSLK-BAC16-vIRF3–3xFLAG cells. (E) Immunofluorescence analysis of vIRF3 expression using vIRF3 antibody in latent and reactivated (60 hpi) iSLK-BAC16 cells.
Figure 4.
Figure 4.. Analyzing the role of vIRFs for virus production during lytic reactivation
The iSLK-BAC16 cell lines in panel A-C were made by infection. (A) Viral DNA load was quantified in latently infected cells at 10 dpi by qPCR. (B) Lytic reactivation was induced with 1 μg/ml Dox and 1 mM NaB for 4 days and viral DNA in the cells was quantified by qPCR. (C) KSHV production was assessed by counting the number of GFP+ 293T cells following infection as described in Fig. 1E. (D) Measuring KSHV production from iSLK-BAC16 cell lines created by transfection of viral DNA. Error bars represent standard deviation (n=3). T-tests were performed between WT and the indicated mutants (*p<0.05, **p<0.01, *** p<0.001).
Figure 5.
Figure 5.. Testing the requirement of vIRFs for KSHV production following de novo lytic infection of HDLMECs
(A) Schematic for the analysis of de novo lytic infection of HDLMECs. (B) Immunoblot analysis of viral protein expression in WT BAC16-infected cells. (C) Input viral DNA level was determined by qPCR at 24 hpi. (D) Infectivity was measured by flow cytometry analysis of GFP+ cells at 24 hpi. (E) KSHV production from infected HDLMECs at 72 hpi was determined by GFP flow cytometry. (F) Total cell viability of KSHV-infected HDLMECs was analyzed by flow cytometry at 72 hpi using a fixable, dead cell discrimination dye. Error bars represent standard deviation (n=3). ns: non-significant. T-tests were performed between WT and the indicated mutants (*p<0.05, **p<0.01, *** p<0.001).
Figure 6.
Figure 6.. Testing the requirement of vIRFs for KSHV production following lytic infection of iSLK-preRTA cells
(A) Schematic for the analysis of de novo lytic infection of iSLK-preRTA cells. Note that RTA was pre-expressed in iSLK cells for 6 hours before KSHV infection. (B) Immunoblot analysis of viral protein expression. The asterisk marks non-specific band. (C) Viral DNA replication was measured by qPCR. (D) Representative GFP images of infected 293T cells. (E) Viral DNA level at 2 hpi was measured by qPCR. (F) Viral DNA replication in cells was measured by qPCR, which was calculated relative to 2 hpi. (G) Measuring KSHV production using GFP flow cytometry. Error bars represent standard deviation (n=3). (ns: non-significant, *p<0.05).
Figure 7.
Figure 7.. vIRFs do not disrupt the CBP-IRF3 interaction and are not needed for efficient silencing of type I IFN expression during lytic reactivation
iSLK-BAC16 and iSLK-BAC16-ΔvIRF cell lines were lytically reactivated using 1 μg/ml Dox and 1 mM NaB for 40 hpi and then challenged with SeV infection. (A) IFNβ gene expression in the absence of SeV. (B) IFNβ gene expression after SeV (1 HA/ml) infection for 8 hours. Error bars represent standard deviation (n=3). (C) Analyzing the expression of phosphorylated IRF3 (pIRF3 S396) in cell lysates. (D) CBP and control IgG immunoprecipitations testing for CBP and IRF3.
Figure 8.
Figure 8.. vIRFs are dispensable for efficient silencing of type I IFN production during de novo lytic infection of iSLK-preRTA cells
(A) Setup of the experiment. iSLK-preRTA cells were infected with WT or ΔvIRF KSHV for 48 hours followed by SeV (1 HA/ml) infection for 6 hours. (B) The early interferon response was analyzed by quantifying IFNβ mRNA 6 hours after SeV infection. (C) The late interferon response was analyzed by assessing the amount of IFNα/β secreted into the supernatant by type I IFN reporter bioassay 24 hours after SeV infection. The concentration of type I IFN was determined using an IFNβ standard curve. Error bars represent standard deviation (n=3). T-test was performed between SeV-treated WT and ΔvIRF samples (ND: not detected, ns: non-significant, *p<0.05).
Figure 9.
Figure 9.. vIRFs are not needed for efficient suppression of type I IFN production during primary lytic infection of HDLMECs
(A) Schematic of the experiment. Primary HDLMECs were infected by WT or ΔvIRF KSHV for 48 hours followed by SeV infection for 6 hours. (B) IFNβ gene expression in the absence of SeV infection was analyzed by RT-qPCR at 54 hpi. (C) IFNβ in the supernatant was measured by bead-based immunoassay. (D) IFNβ gene expression in the presence of SeV infection was measured by RT-qPCR at 54 hpi. Dashed line indicates the limit of assay detection. Error bars represent standard deviation (n=3). ns: non-significant.
Figure 10.
Figure 10.. IFNβ-driven reduction in lytic viral gene expression is more pronounced in BAC16-ΔvIRF-infected cells
Primary HDLMECs were pretreated with IFNβ (500 IU/ml) for 24 hours or left untreated and then were infected by WT or BAC16-ΔvIRF KSHV. (A) KSHV vDNA level was analyzed by qPCR at 2 hpi. (B) RT-qPCR analysis of KSHV gene expression in WT and ΔvIRF KSHV-infected cells at 24 hpi in the absence or presence of IFNβ pretreatment. Error bars represent standard deviation (n=3). T-test was performed between WT and ΔvIRF samples (*p<0.05). (C) Fold inhibition of viral lytic gene expression in the presence of IFNβ relative to samples devoid of IFNβ treatment.
Figure 11.
Figure 11.. Increased ISG expression in BAC16-ΔvIRF KSHV-infected endothelial cells in the presence of IFNβ
Samples described in Fig 10 were used for analyzing the expression of ISGs. (A) RT-qPCR measurement of ISG15 gene expression. (B)Type I IFN pathway-related gene expressions were determined by RT-qPCR array. Error bars represent standard deviation (n=3).
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