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. 2021 Nov 9;95(23):e0126921.
doi: 10.1128/JVI.01269-21. Epub 2021 Sep 22.

Human Herpesvirus 6A Tegument Protein U14 Induces NF-κB Signaling by Interacting with p65

Salma Aktar  1 , Jun Arii  1 , Lidya Handayani Tjan  1 , Mitsuhiro Nishimura  1 , Yasuko Mori  1
Affiliations

Human Herpesvirus 6A Tegument Protein U14 Induces NF-κB Signaling by Interacting with p65

Salma Aktar et al. J Virol. .

Abstract

Viral infection induces host cells to mount a variety of immune responses, which may either limit viral propagation or create conditions conducive to virus replication in some instances. In this regard, activation of the NF-κB transcription factor is known to modulate virus replication. Human herpesvirus 6A (HHV-6A), which belongs to the Betaherpesvirinae subfamily, is frequently found in patients with neuroinflammatory diseases, although its role in disease pathogenesis has not been elucidated. In this study, we found that the HHV-6A-encoded U14 protein activates NF-κB signaling following interaction with the NF-κB complex protein, p65. Through induction of nuclear translocation of p65, U14 increases the expression of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein 1 transcripts. We also demonstrated that activation of NF-κB signaling is important for HHV-6A replication, since inhibition of this pathway reduced virus protein accumulation and viral genome copy number. Taken together, our results suggest that HHV-6A infection activates the NF-κB pathway and promotes viral gene expression via late gene products, including U14. IMPORTANCE Human herpesvirus 6A (HHV-6A) is frequently found in patients with neuro-inflammation, although its role in the pathogenesis of this disease has not been elucidated. Most viral infections activate the NF-κB pathway, which causes the transactivation of various genes, including those encoding proinflammatory cytokines. Our results indicate that HHV-6A U14 activates the NF-κB pathway, leading to upregulation of proinflammatory cytokines. We also found that activation of the NF-κB transcription factor is important for efficient viral replication. This study provides new insight into HHV-6A U14 function in host cell signaling and identifies potential cellular targets involved in HHV-6A pathogenesis and replication.

Keywords: HHV-6; NF-κB; gene expression; herpes; tegument.

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Figures

FIG 1
FIG 1
Exogenous expression of HHV-6A U14 induces the activation of NF-κB promoter. (A) HEK293T cells were cotransfected with either a NF-κB-luc, IFN-β-luc, ARE-luc, or CRE-luc reporter plasmid, together with pRL-CMV as an internal control plasmid, along with either an empty vector or plasmid expressing HA-U14. The luciferase activity was measured at 24 h posttransfection. The means and standard deviations for each data set are shown (n = 7 for NF-κB, n = 4 for IFN-β, ARE and CRE; ***, P < 0.001 [unpaired Student t test]). (B) HEK293T cells were transfected with HA-U14 expression plasmid or empty plasmid (EV). At 24 h posttransfection, the cells were analyzed using immunoblot. (C to E) HEK293T cells were cotransfected with NF-κB-luc (C and E) or CRE-luc (D) reporter plasmid with (E) or without (C and D) the plasmid expressing HA-U14. At 24 h after transfection, TNF-α (C) or colforsin (D) was added for 3 h, followed by SC75741, QNZ, or IKK16 treatment for another 2 h prior to determine the firefly luciferase activity. The data are shown as means and standard deviations (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001 [Tukey’s test]).
FIG 2
FIG 2
Exogenous expression of HHV-6A U14 induces the nuclear translocation of NF-κB protein p65. (A) HEK293T cells were transfected with the HA-U14 expression plasmid or empty plasmid (EV). At 24 h posttransfection, the cells were fractionated into cytoplasmic and nuclear fractions. (B) The intensities of p65 in each fraction, normalized to those of Lamin A/C or α-tubulin, respectively, are shown as means ± the standard deviations (n = 3; **, P < 0.01 [unpaired Student t test]). (C) The noncanonical RelB subunit and the other canonical NF-κB proteins, c-Rel are also shown.
FIG 3
FIG 3
Exogenous expression of HHV-6A U14 increases the expression of NF-κB- regulated genes. HEK293T cells were transfected with HA-U14 expression plasmid or empty plasmid (EV). At 24 h posttransfection, cells were either treated with 5 μM SC75741 or 10 μM QNZ, while another set of cells was left untreated. Medium was then changed after 10 min to remove the drug, and RNA was extracted from cells at 1 h after treatment. The expression of IL-6, IL-8, MCP1, and HPRT1 mRNA was quantified using quantitative real-time PCR (qRT-PCR) for those treated with SC75741 (A) or QNZ (B). Relative mRNA amounts were normalized to those of β-actin. The data are shown as means ± the standard deviations (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001 [unpaired Student t test]).
FIG 4
FIG 4
HHV-6A U14 interaction with the NF-κB proteins. HEK293T cells were transfected with Strep-Flag-U14 expression plasmid or empty plasmid (EV). At 24 h posttransfection, the cells were lysed, and the extracts were subjected to precipitation using Strep-Tactin beads, followed by immunoblotting.
FIG 5
FIG 5
HHV-6A U14 colocalizes with p65. HEK293T cells were transfected with Strep-Flag-U14 expression plasmid or empty plasmid (EV). At 24 h posttransfection, the cells were observed under a confocal microscope after costaining with anti-FLAG and anti-p65 antibody. Nuclear DNA was stained with Hoechst 33342. The boxed area (a, b, and c) in each left panel is shown in the corresponding panel to the right. Scale bars, 10 μm. Fluorescence line scans along the dotted lines of the merged images are shown beneath the image.
FIG 6
FIG 6
HHV-6A infection induces the nuclear translocation of p65. (A) JJhan cells were either mock infected or infected with U1102. At 72 h postinfection, cells were fractionated into cytoplasmic and nuclear fractions and analyzed by immunoblotting with the indicated antibodies. (B) The intensities of p65 in each fraction were normalized to those of Lamin A/C or α-tubulin, respectively, and are shown as means ± the standard deviations (n = 3; **, P < 0.01 [unpaired Student t test]).
FIG 7
FIG 7
mRNA expression level of different genes in HHV-6A-infected cells. JJhan cells were mock infected or infected with U1102. At 72 h postinfection, the cells were either treated with 5 μM SC75741 (NF-κB inhibitor) or left untreated for 10 min. The medium was changed 10 min later to remove SC75741, and RNA was extracted from the cell at 1 h after treatment. The expression of mRNA of IL-6, IL-8, MCP1, IE1, U14, and HPRT1 was quantified using qRT-PCR. Relative amounts of these mRNA were normalized to that of β-actin. The data are shown as means ± the standard deviations (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001 [unpaired Student t test]).
FIG 8
FIG 8
HHV-6A U14 interacts with the NF-κB protein p65 in HHV-6A-infected cells. JJhan cells were mock infected or infected with U1102. At 72 h postinfection, these cells were lysed, and the extracts were subjected to immunoprecipitation with anti-U14 mouse monoclonal antibody (MAb), followed by immunoblotting.
FIG 9
FIG 9
HHV-6A U14 colocalizes with p65 in HHV-6A-infected cells. JJhan cells were mock infected or infected with U1102. At 72 h postinfection, the cells were analyzed using immunofluorescence with the indicated antibodies and Hoechst 33342. The boxed area in each left panel is shown in the corresponding panel to the right. Scale bars, 10 μm. Fluorescence line scans along the dotted lines of the merge image are shown beneath the image.
FIG 10
FIG 10
Effect of NF-κB inhibitors on the viability of JJhan cells. JJhan cells were either mock infected or infected with U1102. At 24 h after infection, these cells were left untreated or treated with either 5 μM SC75741 or 10 μM QNZ. After 48 h, cell viability was measured. The data are shown as means ± the standard deviations (n = 4 for mock-infected cells and n = 3 for U1102-infected cells [Tukey’s test]).
FIG 11
FIG 11
NF-κB inhibitors reduce virus replication. JJhan cells were mock infected or infected with U1102. At 24 h postinfection, the infected cells were treated with SC75741 or QNZ or mock treated for 48 h. At 72 h after infection, these cells were analyzed using fluorescence microscopy (A) and immunoblotting (B) with the indicated antibodies and Hoechst 33342, while genome copy numbers were measured using qPCR (C) following DNA extraction from the supernatant of the infected cells. The data are shown as means ± the standard deviations (n = 4; *, P < 0.05 [Tukey’s test]).
FIG 12
FIG 12
Proposed model of events involved in HHV-6A-mediated NF-κB signaling. After infection, virion components may induce NF-κB signaling to activate viral promoters. HHV-6 gene expression then occurs in a cascade manner (IE→E→L). Expression of late genes (including U14) initiates the activation of NF-κB signaling that can either trigger the activation of cellular genes, as well as viral gene expression, both of which may influence further viral replication. On the other hand, the NF-κB signaling also induces the expression of proinflammatory cytokines (including IL-6 and IL-8).
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