Hypoxia and HIF-1 Trigger Marek’s Disease Virus Reactivation in Lymphoma-Derived Latently Infected T Lymphocytes

Abstract

Latency is a hallmark of herpesviruses, allowing them to persist into their host without virions production. Acute exposure to hypoxia (below 3% O 2 ) was identified as a trigger of latent-to-lytic switch (reactivation) for human oncogenic gamma-herpesviruses (KSHV and EBV). Therefore, we hypothesized that hypoxia could also induce reactivation of Marek’s disease virus (MDV), sharing biological properties with EBV and KSHV (notably oncogenic properties), into lymphocytes. Acute exposure to hypoxia (1% O 2 ) of two MDV-latently infected cell lines derived from MD tumors (3867K and MSB-1) induced MDV reactivation. A bioinformatic analysis of the RB-1B MDV genome revealed 214 putative hypoxia-response element consensus sequences on 119 open reading frames. RT-qPCR analysis showed five MDV genes strongly upregulated early after hypoxia. In 3867K cells under normoxia, pharmacological agents mimicking hypoxia (MLN4924 and CoCl 2 ) increased MDV reactivation, but to a lower level than real hypoxia. Overexpression of wild-type or stabilized human hypoxia inducible factor-1α (HIF-1α) in MSB-1 cells in normoxia also promoted MDV reactivation. In such conditions, lytic cycle was detected in cells with a sustainable HIF-1α expression, but also in HIF-1α negative cells, indicating that MDV reactivation is mediated by HIF-1, in a direct and/or indirect manner. Lastly, we demonstrated by a reporter assay that HIF-1α overexpression induced the transactivation of two viral promoters, shown upregulated in hypoxia. These results suggest that hypoxia may play a crucial role in the late lytic replication phase observed in vivo in MDV-infected chickens exhibiting tumors, since a hypoxic microenvironment is a hallmark of most solid tumors. IMPORTANCE Latent-to-lytic switch of herpesviruses (aka reactivation) is responsible for pathology recurrences and/or viral shedding. Studying physiological triggers of reactivation is therefore important for health to limit lesions and viral transmission. Marek's disease virus (MDV) is a potent oncogenic alpha-herpesvirus establishing latency in T-lymphocytes and causing lethal T-lymphomas in chickens. In vivo , a second lytic phase is observed during tumoral stage. Hypoxia being a hallmark of tumors, we wondered whether hypoxia induces MDV reactivation in latently-infected T-lymphocytes, like previously shown for EBV and KSHV in B-lymphocytes. In this study, we demonstrated that acute hypoxia (1% O2) triggers MDV reactivation in two MDV transformed T-cell lines. We provide some molecular basis of this reactivation by showing that hypoxia inducible factor (HIF-1) overexpression induces MDV reactivation to a similar extend than hypoxia after 24 hours. Hypoxia is therefore a reactivation stimulus shared by mammalian and avian oncogenic herpesviruses of different genus.

Temperature-induced reactivation of Marek's disease virus-transformed T cells ex vivo

“…Cell viability was measured using DAPI stain exclusion to differentiate live from dead cells ( Figure 2A ). The cell viability of the control (41°C) group for each cell line was consistently lower at 8 h post-treatment (hpt), likely due to hypoxia, serum starvation, and low temperature during Histopaque purification ( 16 ). When comparing each treatment for KJ1063, sodium butyrate (NaB) significantly reduced cell viability from 86.8 ± 1.0% at 8 hpt down to 77.3 ± 1.4, 60.3 ± 0.4, and 61.9 ± 1.5% at 24, 36, and 48 hpt, respectively.…”

“…Still, increased cellular stress through histone modification is suspected to result in induced apoptosis ( 59 ) and G2/M cell cycle arrest ( 60 ), likely resulting in virus escape (reactivation) from the cell. However, chemical treatment is only one of the multiple mechanisms to induce reactivation or fully productive virus replication, including UV light, serum starvation, temperature changes, and hypoxia ( 16 , 23 , 24 , 61 , 62 ). The core body temperature of chickens is approximately 41°C, while the surface body temperature can fluctuate in ambient temperatures and range from 20 to 40°C ( 36 ).…”

“…Reactivation usually happens in response to cell damage or stress, such as apoptosis, hypoxia, or metabolic stress, to initiate lytic replication ( 15 ). A recent study showed that acute exposure to 1% O 2 can cause upregulation of late MDV genes UL47 (VP13/14), UL49 (VP22), and UL27 glycoprotein B (gB), indicating reactivation ( 16 ). Chemicals such as sodium butyrate have been utilized to induce reactivation in herpesviruses ( 17 ), including MDV ( 18 – 20 ).…”

The sulphated polysaccharides extract ulvans from Ulva armoricana limits Marek’s disease virus dissemination in vitro and promotes viral reactivation in lymphoid cells

“…We assessed whether the two ulvans formulations and the VitComp control could have an effect on the maintenance of MDV latency and viral reactivation (corresponding to the late cytolytic phase of infection) in vitro. To this end, we used the 3867 K cells, a lymphoblastoid cell line that expresses the green fluorescent protein (GFP) fused to the tegument protein UL47 upon reactivation, as described previously [ 27 , 28 ]. The 3867 K cells were treated with the vitamin complex, Searup® and ulvans at different concentrations and the rate of viral reactivation (GFP positive cells within the viable cell population) was evaluated by cytometry upon 96 h of treatment (Fig.…”

“…All media supplemented with the different treatments doses were renewed after 48 h of culture. In a second set of experiment, the 3867 K cells were treated with 2 ml/l of VitComp, Searup® or ulvans extract in combination with 0.5 mM of sodium butyrate (NaBut; Sigma-Aldrich), a histone deacetylase inhibitor known to trigger herpesvirus reactivation [ 28 , 51 , 52 ]. Cells were treated for 24 to 96 h depending on the test performed.…”