Feedback loop regulation between viperin and viral hemorrhagic septicemia virus through competing protein degradation pathways

Background

The host innate immune response can be activated by multiple pathways, including directly by viruses or through the action of interferon (IFN)¹. Interferons regulate the immune response and part of such regulation is the induction of IFN-stimulated genes (ISGs). Although there are many ISGs, viperin has been found to be highly upregulated and acts as an antiviral ISG² against DNA and RNA viruses.

Viperin is comprised of three structural domains: an N-terminal amphipathic domain, a central S-adenosyl-L-methionine (SAM) binding domain, and the C-terminal domain.  It is regulated through several pathways, including IFN-dependent and IFN-independent pathways³. Viperin induction is a highly conserved immune response, however viruses have evolved mechanisms that allow them to evade this host immune response, including degradation of transcription factors that upregulate viperin expression. This is a significant reason as to why such viruses can be hard to treat.

Viral hemorrhagic septicemia (VHS) is a highly contagious fish disease causedby the viral hemorrhagic septicemia virus, affecting over 80 different species of fish. Even with the recent insights into bony fish immune responses, the relationship between VHSV proteins and viperin is still unknown. There are currently no effective treatments available for this disease¹ and providing insight into the mechanistic details of the VHSV induced immune response will allow for possible development of treatments.

In this preprint, Kuntong Jia and his research team set out to uncover the molecular interactions between viperin and VHSV. This paper demonstrates that the sea perch viperin (Ljviperin) interacts with and degrades the N and P proteins of the virus through autophagy to inhibit viral replication. It also demonstrates that the N protein of VHSV mediates degradation of IRF1 and IRF9 to block Ljviperin expession¹ (Figure 1).

Key Findings

Induction of Ljviperin Expression Through IFN-Dependent and -Independent Pathways:

This study focused on the induction pathways of Ljviperin, an important antiviral protein, in Lateolabrax japonicus fry (LJF) cells, with the goal of learning more about its regulation. The study found that Ljviperin expression can be induced via both IFN-dependent and IFN-independent mechanisms, implying that it is evolutionary conserved among vertebrates. Experiments revealed that MAVS can upregulate both Ljviperin and IRF1 transcription, with IRF1 subsequently activating the Ljviperin promoter. Notably, full-length IRF1 is required for this activation, as could be shown by using deletion mutants. Furthermore, ChIP experiments demonstrated that IRF1 binds directly to the Ljviperin promoter.

Regarding the IFN-dependent pathway, IFNc, IFNh, and IFN-γ promoted Ljviperin expression via STAT1, STAT2, and IRF9. Luciferase reporter experiments revealed IRF9’s critical role in Ljviperin expression, as domain-truncated IRF9 mutants failed to activate Ljviperin. ChIP experiments demonstrated IRF9’s direct binding to the Ljviperin promoter. However, not all IFN subtypes were successful at upregulating Ljviperin, indicating selectivity in the induction mechanism.

Overall, these findings shed light on the complicated regulatory mechanisms that control Ljviperin expression in fish, with implications for understanding immunological responses across vertebrates.

Antiviral Activity of Ljviperin Against VHSV:

Ljviperin was shown to interact with and deplete viral proteins, specifically the N and P proteins of Viral Hemorrhagic Septicemia Virus (VHSV), offering insights into its antiviral activities. Co-immunoprecipitation experiments showed that Ljviperin binds to the N and P proteins of VHSV through its SAM-binding and C-terminal domains. Furthermore, Ljviperin was shown to inhibit the homodimerization of N proteins and the heterodimerization of N and P proteins, limiting essential phases of viral propagation. Furthermore, Ljviperin degraded N and P proteins in a dose-dependent manner, indicating its role in viral suppression. This degradation process proceeds via the autophagy pathway, as indicated by the inhibition of protein degradation by 3-MA (inhibits autophagosome pathways) but not MG132 (inhibits the ubiquitin-proteasome).

These findings imply that Ljviperin’s antiviral effect may extend beyond fish, as both human and zebrafish viperins demonstrate similar viral protein degradation patterns and reduce VHSV proliferation. The findings also emphasize the importance of Ljviperin as a crucial actor in the innate immune response to viral infections, as well as open the possibility for antiviral treatments that target the autophagy pathway.

Viral Evasion Strategies Targeting Ljviperin

Lu and colleagues next aimed to elucidate the methods by which the Viral Hemorrhagic Septicemia Virus (VHSV) N protein evades host immune responses, with a particular focus on the antiviral protein Ljviperin. They used luciferase reporter assays and expression studies to demonstrate the ability of the N protein to inhibit Ljviperin expression. It can do so by interfering with the functions of essential transcription factors IRF1 and IRF9, which are important in promoting antiviral gene expression – in fact, the N protein promotes the ubiquitination of IRF1 and IRF9 resulting in their proteasomal destruction.. Furthermore, the authors revealed the N protein’s larger impact on the host immune response by extending its inhibitory effects to other Interferon-Stimulated Genes (ISGs).

By targeting critical antiviral immunity regulators such as IRF1 and IRF9, the N protein supports a complex strategy used by VHSV to undermine host defenses, emphasizing the importance of developing techniques to fight viral immune evasion mechanisms to ensure successful antiviral treatments.

What we like

This study clarifies viperin’s function in VHSV infection using genetic and biochemical techniques. The use of well-established virological procedures adds legitimacy to the investigation.. . A better understanding of viperin’s function in VHSV infection can lead to more efficacious antiviral tactics. Specifically, the preprint authors have shown that targeting the N protein of VHSV may provide intriguing therapeutic options. Considering that VHSV may infect both freshwater and marine fish, such therapies could have a big impact on aquaculture management.

Future directions

The authors suggest several possibilities for further investigation, including exploring the therapeutic potential of targeting the VHSV N protein to control VHSV infection and elucidating the specific mechanisms through which the N protein regulates viperin expression. Additionally, they plan to investigate viperin’s antiviral activity against VHSV, focusing on ddhCTP-independent mechanisms and their role in promoting autophagic degradation of VHSV N and P proteins. To further understand the significance of the N protein on diverse marine species under various conditions (e.g. temperature change, bacterial infections or other stimulating factors) studies may involve testing N protein deletion mutants.

It is crucial to investigate other proteins or pathways related to environmental sensing and how they interact with the N protein-mediated pathway. Recognizing the effects of removing the N protein and other virions on basic cellular functions under different environmental circumstances could provide valuable insights into viral pathogenesis and host-virus interactions.

Tags: disease, pathogenesis, treatment, viperin, viruses

doi: Pending

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