Marek’s Disease Virus RLORF4 Inhibits Type I Interferon Production by Antagonizing NF-κB Activation

Abstract

Marek’s disease virus (MDV), which causes T cell lymphomas in chickens, is economically important and has contributed to knowledge of herpesvirus-associated oncogenicity. The DNA-sensing pathway induces innate immune responses against DNA virus infection, and nuclear factor κB (NF-κB) signaling is critical for the establishment of innate immunity. Here, we report that RLORF4, an MDV-specific protein directly involved in viral attenuation, is an inhibitor of the DNA-sensing pathway. The results showed that ectopically expressed RLORF4 blocked beta interferon (IFN-β) promoter activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). RLORF4 selectively inhibited the activation of NF-κB but not IFN-regulatory factor 7. RLORF4 was found to bind the endogenous NF-κB subunits p65 and p50, and it also bound to the Rel homology domains of these subunits. Furthermore, RLORF4 suppressed the nuclear translocation of p65 and p50 mediated by tumor necrosis factor alpha and interferon-stimulatory DNA. Finally, deletion of RLORF4 from the MDV genome promoted IFN-β and interleukin-6 (IL-6) production in vitro and in vivo. In the absence of RLORF4, the host cellular immunity was significantly increased, and reduced viral titers were observed during infection of chickens. Our results suggest that the RLORF4-mediated suppression of the host antiviral innate immunity might play an important role in MDV pathogenesis. IMPORTANCE Marek’s disease virus (MDV) RLORF4 has been shown to be directly involved in the attenuation of MDV upon serial passages in vitro; however, the exact function of this protein during viral infection was not well characterized. This study demonstrated that RLORF4 significantly inhibits cGAS-STING-mediated NF-κB activation by binding to the Rel homology domains of the NF-κB subunits p65 and p50, interrupting their translocation to the nuclei and thereby inhibiting IFN-β production. Furthermore, RLORF4 deficiency promoted the induction of IFN-β and downstream IFN-stimulated genes during MDV infection in chickens. Our results suggest that the contribution of RLORF4 to MDV virulence may stem from its inhibition of viral DNA-triggered IFN-β responses.

IFNα and IFNγ Impede Marek’s Disease Progression

“…Moreover, we could demonstrate that IFNα, but not IFNβ, was released into the supernatant upon infection of primary CEC with MDV ( Figure S1). In line with this, it has recently been shown that the MDV-encoded protein RLORF4 inhibits IFNβ production in chicken fibroblasts [36]. These data confirm the IFNα-mediated antiviral response on the protein level-a response that has so far only been shown on the RNA level [9].…”

Redox Homeostasis in Poultry: Regulatory Roles of NF-κB

“…Furthermore, chicken MD virus RLORF4 (a MDV-specific gene) was shown to inhibit type I interferon production by antagonizing NF-κB activation. In fact, RLORF4 binds to the Rel homology domains of the NF-κB subunits p65 and p50, interrupting their translocation to the nuclei and, thus, inhibiting IFN-β production [ 324 ].…”

Pathogenesis of Hypervirulent Fowl Adenovirus Serotype 4: The Contributions of Viral and Host Factors

“…It is worthy to point out that immunosuppression could actually reduce the effects of a cytokine storm and therefore reduce the mortality associated with these infections. In addition to lysis of important cell types Marek’s disease virus can also interact with inflammatory mediators to change the immune response [66,67]. Thus, the differences in expression patterns of proinflammatory cytokines between the stand-alone infection of FAdV-4 and coinfection of FAdV-4 with other immunosuppressive viruses need to be determined.…”