Fig 2: HSV-1 infection requires JNK/MAPK pathway signaling.A, HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B, chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D, construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting (C) and for infectious virus production (D). E, ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, JNK2, or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.

Fig 3: Effect of bortezomib on JNK activation and on HSV-1 infection in mouse models.A, diagram of a mouse model of acute HSV-1 infection. Female Balb/C mice were infected by corneal scarification with 2 × 106 PFU of HSV-1 virus (F strain). B–D, virus shedding in the tear swabs collected on day 1 (B) and day 3 (C) and detected by qPCR. A fragment of HSV-1 UL30 DNA was used as a standard for calculation of genome copy number (genome equivalent). D, virus titers in tear swabs (n = 6) collected on day 3 determined by plaque forming assay. E, representative images of H&E staining of brain tissues from HSV-1 infected mice. The histopathological structure of the hippocampus regions (CA1 and CA3) and the dentate gyrus (DG) (left panel) and pathological conditions of the hippocampal region CA1 (right panel) were examined after H&E staining. Arrows indicate the inflammatory cell infiltration and necrosis of neuronal cells. F, numeration of damaged neuronal cells by H&E staining. Data represent mean ± SD of counting from 3 randomly selected fields counted by 2 investigators. G, induction of JNK phosphorylation from HSV-1 infected mouse brain tissues. The brain tissues (n = 2) collected from mock-infected or HSV-1 infected mice treated with vehicle or bortezomib at 2 h after the last administration were used for detection of JNK activation by immunoblotting. The antiviral drug acyclovir (50 mg/kg) was included as an antiviral control. GAPDH was used as a loading control. H, schematic diagram of bortezomib-induced HSV-1 reactivation. Balb/C female mice were inoculated by the corneal scarification with 3 × 105 PFU of HSV-1. After the establishment of viral latency, the mice were treated with bortezomib (1.5 mg/kg, on day 0 and day 1) or by UVB-irradiation. I, the trigeminal ganglia were collected on days as indicated post infection (I) and used for detection of LAT transcript or viral gene expression by RT-qPCR. The data are shown as mean ± SD (n = 3). The absence of lytic genes on day 35 shows the establishment of viral latency. J and K, HSV-1 reactivation in the ganglia determined by RT-qPCR. The ganglia (n = 3) were removed on day 3 and used for detection of viral lytic gene VP16, ICP8, ICP27 (J) and LAT (K) transcripts. Data are shown as mean ± SD. ∗, p ≤ 0.05, ∗∗, p ≤ 0.01, ∗∗∗, p ≤ 0.001.

Fig 4: Diagram of HSV-1 infection and stress signaling on BRD4 function transition. HSV-1 infection triggers JNK activation, resulting in BRD4 release from chromosome association to gene transcription regulation. Genetic ablation or chemical inhibition of JNK blocks HSV-1 infection by impeding BRD4 release. Chemotherapeutic agents that can induce JNK activation have the ability to promote HSV-1 infection and reactivation through BRD4 function transition.

Fig 5: HSV-1 infection promotes BRD4 release from chromatin-targeting dependent on JNK signaling.A, schematic diagram for chromatin-targeting and release of BRD4 by fractionation. B, HSV-1 infection releases BRD4 from chromatin-targeting. HeLa cells were infected with HSV-1 (MOI = 1) for times as indicated and used for fractionation. BRD4 in the LSF and LSEN fractions was detected by immunoblotting. GAPDH, Lamin B and HEXIM1 were used as controls for fractionation. C, JNK inhibition on HSV-1 induced BRD4 release. HeLa cells were infected with HSV-1 for 6 h, and were then mock-treated or treated with SP600125 for another hour prior to fractionation. BRD4 in the LSF and LSEN fractions was detected by immunoblotting. GAPDH and Lamin B were used as controls. D and E, suppression of JNK expression by siRNA on HSV-1-induced BRD4 release (D) and HSV-1 infection (E).The experiments were performed 3 times independently, The experiments were performed 3 times independently. The titration data are presented as mean ± SD from one experiment. ns, no significant, ∗∗, p ≤ 0.01, ∗∗∗, p ≤ 0.001.