c-Jun signaling during initial HSV-1 infection modulates latency to enhance later reactivation in addition to directly promoting the progression to full reactivation

Abstract

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates to permit transmission, which can result in clinical manifestations. Viral transactivators required for lytic infection are largely absent during latent infection, and therefore, HSV-1 relies on the co-option of neuronal host signaling pathways to initiate its gene expression. The activation of the neuronal c-Jun N-terminal kinase (JNK) cell stress pathway is central to initiating biphasic reactivation in response to multiple stimuli. However, how host factors work with JNK to stimulate the initial wave of gene expression (known as Phase I) or the progression to full Phase II reactivation remains unclear. Here, we found that c-Jun, the primary target downstream of neuronal JNK cell stress signaling, functions during reactivation but not during the JNK-mediated initiation of Phase I gene expression. Instead, c-Jun was required to transition from Phase I to full HSV-1 reactivation and was detected in viral replication compartments of reactivating neurons. Interestingly, we also identified a role for both c-Jun and enhanced neuronal stress during initial neuronal infection in promoting a more reactivation-competent form of HSV-1 latency. Therefore, c-Jun functions at multiple stages during the HSV latent infection of neurons to promote reactivation but not during the initial JNK-dependent Phase I. Importantly, by demonstrating that initial infection conditions can contribute to later reactivation abilities, this study highlights the potential for latently infected neurons to maintain a molecular scar of previous exposure to neuronal stressors.IMPORTANCEThe molecular mechanisms that regulate the reactivation of herpes simplex virus-1 (HSV-1) from latent infection are unknown. The host transcription and pioneer factor c-Jun is the main target of the JNK cell stress pathway that is known to be important in exit of HSV from latency. Surprisingly, we found that c-Jun does not act with JNK during exit from latency but instead promotes the transition to full reactivation. Moreover, c-Jun and enhanced neuronal stress during initial neuronal infection promoted a more reactivation-competent form of HSV-1 latency. c-Jun, therefore, functions at multiple stages during HSV-1 latent infection of neurons to promote reactivation. Importantly, this study contributes to a growing body of evidence that de novo HSV-1 infection conditions can modulate latent infection and impact future reactivation events, raising important questions on the clinical impact of stress during initial HSV-1 acquisition on future reactivation events and consequences.

Keywords: c-Jun; herpes simplex virus; reactivation; stress response.

Fig 1

c-Jun depletion prior to latency establishment limits both Phase I gene expression and full reactivation. (A) Neurons were transduced with a non-targeting shRNA control lentivirus or one of three independent lentiviruses expressing shRNAs that target c-Jun (sh-cJun1, sh-cJun2, and sh-cJun3). Five days post-transduction, LY294002 was added to neurons for 18 hours, and western blotting for c-Jun or α-tubulin was performed. The percentage knockdown of c-Jun normalized to α-tubulin is shown. (B–H) Following c-Jun depletion with sh-cJun3 (B–E) or sh-cJun2 (F–H), primary neurons were infected with Stayput-GFP at a multiplicity of infection (MOI) of 7.5 PFU/cell in the presence of ACV (50 µM) for 6 days and then reactivated 2 days after the removal of acyclovir with LY294002 (20 µM). (B) Quantification of relative latent viral DNA load at 8 days post-infection. Biological replicates from three separate dissections. (C and F) Quantification of the number of GFP-positive neurons at 48 hours post-stimulus. Individual biological replicates from at least three individual dissections. (D, E, G, and H) Relative viral gene expression at 18 hours post-stimulus compared to latent samples quantified by reverse transcription–quantitative PCR (RT-qPCR) for ICP27 (D, G) and ICP8 (E, H) normalized to cellular control mGAPDH. Statistical comparisons were made using a normal or non-normal (Wilcoxon, B, C, and F) paired t-test. Biological replicates from at least three individual dissections. Individual biological replicates along with the means and SEMs are represented. ^*P  <  0.05; ^**P  <  0.01.

Fig 2

c-Jun is not necessary for Phase I gene expression. (A–K) Latently infected neurons were transduced with a non-targeting shRNA lentivirus or sh-cJun3, sh-cJun2, or sh-cJun1 at 6 days post-infection and reactivated 5 days later. In (A–G), neurons were reactivated with LY294002 and in (H–K) with forskolin. RT-qPCR was carried out at 18 hours post-reactivation; ICP27 (A, E, and H), ICP8 (B, F, and I), gC (C, G, and J), and cellular Jun (D and K) at 18 hours post-stimulus are represented. N = 6 biological replicates from at least three independent dissections. Statistical comparisons were made using a non-normal (Mann–Whitney) t-test. Individual biological replicates along with the means and SEMs are represented. ^*P  <  0.05; ^**P  <  0.01.

Fig 3

c-Jun is necessary for full HSV-1 reactivation. (A–J) Neurons were infected with HSV-1 and transduced 6 days post-infection with a non-targeting shRNA lentivirus or sh-cJun3 (A–F) or sh-cJun2 (G–J) and reactivated 5 days later. Acyclovir was added for the first 6 days post-infection. (A, G) Quantification of Us11-GFP-positive neurons following reactivation with LY294002. (B–E, H, and I) RT-qPCR for viral mRNA transcripts ICP27 (B), ICP8 (C), VP16 (D, H), and gC (E, I) at 48 hours post-reactivation with LY294002. (F, J) Quantification of Us11-GFP-positive neurons following reactivation with forskolin. Individual replicates from at least four separate dissections are shown. Statistics determined by a normal or non-normal (Wilcoxon test, A, F–J) paired t-test. Individual biological replicates along with the means and SEMs are represented. ^*P  <  0.05; ^**P  <  0.01. ns, not significant.

Fig 4

c-Jun is necessary for maximum de novo lytic infection in neurons. (A–H) Neurons were transduced with a non-targeting shRNA lentivirus or sh-cJun3 and infected with HSV-1 Stayput-GFP in the absence of viral DNA replication inhibitors 5 days post-transduction at an MOI of 5 PFU/cell for 48 hours. (A) Quantification of the numbers of GFP-positive neurons. Replicates from three separate dissections are shown; unpaired t-test. (B) qPCR for viral DNA copy number. (C) RT-qPCR for viral mRNAs ICP27, ICP8, VP16, and gC. (D) RT-qPCR for Jun. Replicates from three separate dissections are shown; paired t-test. Individual biological replicates along with the means and SEMs are represented. ^*P  <  0.05; ^**P  <  0.01.

Fig 5

c-Jun co-localizes with replicating viral DNA during HSV-1 reactivation. (A, B) Latently infected neurons were reactivated with LY294002 and pulsed with 10 µM EdC for 1 hour to label viral DNA replication compartments. Nuclear stain Hoechst is shown in blue, and immunofluorescence was performed to visualize c-Jun in green. EdC-Pulse was visualized using click chemistry (shown in red). (A) Representative images of reactivating neurons. Scale bar = 10 µm. Pearson’s coefficient between c-Jun and EdC-Pulse featured in bottom left corner of merge. (B) Compiled Pearson’s coefficients. Thirty-five individual neurons; two biological replicates.

Fig 6

Stress signaling events during de novo HSV-1 infection. (A–C) Neonatal sympathetic cultures were infected with Stayput-GFP in inoculation media with or without NGF for 3.5 hours and subsequently fixed and stained for neuronal marker B III tubulin (magenta) or phosphorylated c-Jun (orange). (A) The representative image of nuclear phosphorylated c-Jun is demonstrated. Scale bar = 10 µm. (B) Mean signal intensity for phosphorylated c-Jun in the nucleus following infection. N = 100 from one biological replicate. (C) Quantification of proportion of neurons with pc-Jun-positive nuclei pooled from several fields of view. (D–G) Neuronal cultures were latently infected. NGF was either included or omitted during the 3.5-hour inoculation period. Cultures were later reactivated with LY294002 20 µM. (D) Latent viral DNA load. Replicates from three dissections shown. The peak number of GFP-positive neurons 48 hours post-stimulus (E) and relative expression of ICP27 (F) or ICP8 (G) transcripts 18 hours post-stimulus were quantified to analyze the full reactivation and Phase I gene expression, respectively. Replicates from three dissections shown. Statistical comparisons were made using a normal or non-normal (Mann–Whitney) (E) t-test. Individual biological replicates along with the means and SEMs are represented. ^*P  <  0.05; ^**P  <  0.01.