ISGF3: The Key Player in Antiviral Defense Mechanisms

Cells rely on intricate signaling pathways to detect and respond to viral infections. A critical player in this defense system is ISGF3, a transcriptional complex that activates antiviral gene expression.

Components And Assembly

The ISGF3 complex forms through the interaction of three proteins—STAT1, STAT2, and IRF9. Each plays a role in assembling the transcription factor that regulates gene expression. Their coordinated activity enables ISGF3 to translocate to the nucleus and bind DNA sequences.

STAT1

Signal Transducer and Activator of Transcription 1 (STAT1) is essential for ISGF3 assembly. It exists in two isoforms, STAT1α (91 kDa) and STAT1β (84 kDa), with the former being dominant in ISGF3 formation. STAT1 contains functional domains for dimerization, DNA binding, and transactivation. Upon phosphorylation at tyrosine 701 by Janus kinases (JAKs), STAT1 undergoes conformational changes that allow it to pair with STAT2. Mutations affecting its SH2 domain can impair ISGF3 assembly, leading to defective transcriptional responses. A 2021 Nature Immunology study showed that STAT1-deficient cells exhibit reduced ISGF3 activity, underscoring its importance.

STAT2

Unlike STAT1, STAT2 is specific to type I and III interferon signaling and does not independently activate transcription. Instead, it acts as a scaffold protein stabilizing ISGF3. STAT2 is phosphorylated at tyrosine 690 by JAK1 and TYK2, facilitating its interaction with STAT1. It also contains a C-terminal transactivation domain that recruits IRF9, enabling ISGF3 to bind interferon-stimulated response elements (ISREs). Research in The Journal of Biological Chemistry (2022) found that STAT2-deficient cells fail to form functional ISGF3, reducing antiviral gene activation. Viral proteins from pathogens like dengue and hepatitis C target STAT2 for degradation, highlighting its significance in host defense.

IRF9

Interferon Regulatory Factor 9 (IRF9) is the non-phosphorylated component of ISGF3, yet it plays a vital role in DNA binding and transcriptional activation. Unlike STAT1 and STAT2, IRF9 does not require phosphorylation. It contains a DNA-binding domain that recognizes ISRE sequences and a STAT2 interaction domain for complex formation. A 2023 Cell Reports study found that IRF9 knockout cells exhibit impaired ISGF3-mediated transcription, reducing antiviral gene expression. Mutations in IRF9 have been linked to immune disorders with defective interferon responses. Given its role in ISGF3 function, IRF9 is a potential therapeutic target for modulating interferon signaling in viral infections and inflammatory diseases.

Activation Through Interferon Signaling

ISGF3 activation begins when type I (IFN-α, IFN-β) and type III (IFN-λ) interferons bind to their respective receptors—IFNAR1/IFNAR2 and IFNLR1/IL10R2—on the cell surface. This interaction induces conformational changes that recruit and activate Janus kinases (JAK1 and TYK2), which phosphorylate tyrosine residues on the receptors, creating docking sites for STAT1 and STAT2.

Once recruited, STAT1 and STAT2 are phosphorylated at tyrosine 701 and tyrosine 690, respectively. These modifications promote their dissociation from the receptor and dimerization in the cytoplasm. The STAT1-STAT2 dimer then interacts with IRF9 via the STAT2 transactivation domain, forming ISGF3. Nuclear localization signals (NLS) in STAT1 and STAT2 facilitate nuclear translocation through importin-α/β transport proteins.

Inside the nucleus, ISGF3 binds chromatin-associated regulatory elements. The DNA-binding domain of IRF9 recognizes ISREs in target gene promoters. Chromatin accessibility, influenced by histone modifications such as acetylation and methylation, affects ISGF3’s ability to bind ISREs. Once bound, ISGF3 recruits transcriptional coactivators like CBP/p300, assembling the transcriptional machinery for precise gene regulation in response to interferon signaling.

DNA Binding And Gene Regulation

ISGF3 navigates chromatin to locate ISREs, specialized DNA sequences that regulate target gene transcription. IRF9’s DNA-binding domain recognizes these sequences, while chromatin accessibility, influenced by histone modifications, determines which genes are activated. Histone acetyltransferases (HATs) promote an open chromatin state, enhancing ISGF3 access, whereas histone deacetylases (HDACs) maintain a condensed state, limiting binding.

Upon binding, ISGF3 recruits transcriptional coactivators such as CBP/p300, which facilitate transcriptional machinery assembly. These coactivators, with histone acetyltransferase activity, further enhance chromatin accessibility, enabling RNA polymerase II to initiate transcription. Chromatin remodelers like the SWI/SNF complex reposition nucleosomes to expose ISREs. Post-translational modifications of ISGF3 subunits, such as SUMOylation or ubiquitination, influence stability and transcriptional potency.

ISGF3-driven transcription is modulated by feedback mechanisms. Negative regulators like protein inhibitor of activated STAT (PIAS) and suppressor of cytokine signaling (SOCS) proteins can inhibit ISGF3 activity. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) also contribute by targeting ISGF3-responsive transcripts for degradation or translational repression. This multilayered control prevents aberrant gene expression that could disrupt cellular homeostasis.

Roles In Antiviral Defense

ISGF3 orchestrates the transcription of interferon-stimulated genes (ISGs) that inhibit viral replication. These ISGs encode proteins that block viral entry, degrade viral RNA, and amplify interferon responses.

One well-characterized ISG is PKR (protein kinase R), which detects double-stranded RNA from viral replication. PKR phosphorylates eukaryotic initiation factor 2α (eIF2α), halting protein synthesis and depriving viruses of the machinery needed for propagation. This mechanism is particularly effective against RNA viruses like influenza and hepatitis C.

Another crucial ISG is MX1 (myxovirus resistance protein 1), a dynamin-like GTPase that disrupts viral nucleocapsid transport. MX1 binds viral ribonucleoproteins, preventing their trafficking to replication sites and blocking genome amplification. Studies indicate MX1 provides protection against orthomyxoviruses, paramyxoviruses, and bunyaviruses.

ISGF3 also regulates ISG15, a ubiquitin-like modifier that enhances viral protein degradation through the host proteasome system. ISG15 conjugation, or ISGylation, restricts the replication of viruses such as Ebola and chikungunya by targeting viral components for degradation.