HERC5 Human

What is HERC5 and what is its fundamental role in human innate immunity?

HERC5 is a 114 kDa multi-domain protein that belongs to both the HECT E3 ubiquitin ligase family and the RCC1 superfamily. It is the main cellular E3 ligase responsible for conjugating ISG15 (Interferon-Stimulated Gene 15) to target proteins, a process known as ISGylation . HERC5 plays a central role in mammalian innate immunity by targeting newly synthesized proteins, including viral proteins, for ISG15 conjugation .

This protein is strongly induced following type I interferon (IFN-α/β) stimulation, which typically occurs during viral infection. Through its ISGylation activity, HERC5 can disrupt various stages of viral replication cycles. Additionally, HERC5 functions to amplify the interferon response by positively regulating ISG induction and preventing premature termination of antiviral responses .

How is HERC5 expression regulated in human cells?

HERC5 expression is primarily regulated by the interferon signaling pathway. Upon viral infection, pattern recognition receptors such as RIG-I (DDX58) detect viral nucleic acids and activate a signaling cascade that leads to the production of type I interferons . The binding of interferons to their receptors activates the JAK-STAT pathway, resulting in the induction of numerous interferon-stimulated genes, including HERC5 .

Research has shown that HERC5 expression can be detected in various cell types following interferon treatment, including PBMCs, Jurkat cells, and dendritic cells . Additionally, epigenetic mechanisms like DNA methylation may influence HERC5 expression, as suggested by studies on related antiviral genes such as DDX58 . Understanding the regulatory mechanisms of HERC5 expression is critical for developing strategies to enhance antiviral responses.

What is ISGylation and how does HERC5 catalyze this process?

ISGylation is a post-translational modification process similar to ubiquitination, where ISG15, a 15.2 kDa ubiquitin-like protein, is covalently attached to target proteins. This process involves a cascade of three enzymes: the E1 activating enzyme UBE1L (Uba7), the E2 conjugating enzyme UBE2L6 (UbcH8), and the E3 ligase HERC5 .

The mechanism of ISGylation mediated by HERC5 begins with the activation of ISG15 by UBE1L using ATP, followed by the transfer of activated ISG15 to UBE2L6. HERC5 then facilitates the final step, conjugating ISG15 to target proteins via an isopeptide bond between the C-terminal glycine of ISG15 and a lysine residue on the target protein . HERC5's HECT domain is essential for this activity, as demonstrated by studies showing that a C994A substitution in this domain completely abrogates its E3 ligase function . HERC5 broadly targets newly synthesized proteins for ISGylation, including many host proteins involved in RNA splicing, chromatin remodeling, cytoskeleton organization, and antiviral responses, as well as viral proteins during infection .

What domains comprise the HERC5 protein and what are their specific functions?

HERC5 contains multiple functional domains that contribute to its role in antiviral immunity:

• N-terminal RCC1-like Domain (RLD): HERC5 contains five regulator of chromatin condensation 1 (RCC1) motifs that collectively form the RLD. This domain plays a direct role in substrate recognition and coordination, particularly for viral substrates such as influenza A virus NS1, hepatitis C virus NS5A, and HIV Gag proteins .

• Spacer Region: Located between the RLD and HECT domains, this region contributes to proper protein folding and might facilitate domain movement during the ISGylation process .

• C-terminal HECT Domain: This domain possesses the catalytic cysteine residue (C994) that is critical for E3 ligase activity. The HECT domain forms a thioester intermediate with ISG15 before transferring it to target proteins .

The multi-domain structure of HERC5 allows it to function efficiently in recognizing diverse viral substrates while maintaining its catalytic activity. Structure-function studies continue to provide insights into how these domains cooperate to mediate HERC5's antiviral effects.

How can researchers experimentally determine HERC5 substrate specificity?

Determining HERC5 substrate specificity involves several complementary approaches:

• Proteomics-based identification: Mass spectrometry techniques, particularly quantitative proteomics, can identify proteins modified by ISG15 in the presence of HERC5. For example, researchers have used labile free proteomics to identify new ISGylation targets in infected cells .

• Co-immunoprecipitation assays: These assays can detect physical interactions between HERC5 and potential substrate proteins. Studies have used this approach to demonstrate HERC5's interaction with HIV-1 Gag .

• Domain mapping experiments: Targeted mutations or truncations in HERC5's domains, particularly the RLD, can help determine regions responsible for substrate recognition. This approach has been used to characterize HERC5's interaction with viral proteins like HIV Gag .

• In vitro ISGylation assays: Reconstituting the ISGylation cascade (UBE1L, UBE2L6, HERC5, and ISG15) in vitro with potential substrates can directly demonstrate HERC5's ability to catalyze ISG15 attachment.

• Confocal microscopy: This technique has been used to study the subcellular co-localization of HERC5 with potential substrates, such as its association with polyribosomes .

What mechanisms determine whether HERC5 targets a protein for ISGylation?

The targeting mechanisms of HERC5 appear to involve several factors:

• Proximity to translation: HERC5 associates with polyribosomes and the 60S ribosomal subunit, suggesting it primarily targets newly synthesized proteins . This association allows HERC5 to access nascent viral proteins during infection.

• RLD domain recognition: The RCC1-like domain of HERC5 appears to recognize specific features or motifs in target proteins, particularly viral proteins. Structure-function studies have shown that this domain is crucial for substrate binding .

• Protein localization: The subcellular localization of HERC5 in cytoplasmic punctate bodies affects which proteins it can access and target. Confocal immunofluorescence microscopy has shown that approximately 60% of HERC5 co-localizes with polyribosomes in interferon-treated cells .

• Interferon induction: Both HERC5 and potential substrate availability are regulated by interferon signaling, creating a coordinated response system during viral infection .

Further structure-function studies are needed to fully elucidate the specificity determinants of HERC5 in recognizing its diverse array of target proteins .

What is the mechanism by which HERC5 restricts HIV-1 replication?

HERC5 restricts HIV-1 replication through multiple mechanisms:

• Inhibition of Gag assembly: HERC5 targets HIV-1 Gag and arrests particle assembly at an early stage at the plasma membrane, as demonstrated by electron microscopy studies . This inhibition occurs via ISGylation of Gag proteins, which interferes with their multimerization.

• Reduction of viral particle release: Expression of HERC5 results in a significant reduction (4 to 10.8-fold) in the release of infectious HIV-1 particles after both single and multiple rounds of replication . Conversely, knockdown of HERC5 using shRNA increases HIV-1 particle release by approximately 1.9-fold.

• Targeting newly synthesized viral proteins: By associating with polyribosomes, HERC5 can target newly synthesized HIV-1 proteins for ISGylation, disrupting their function .

• Distinct mechanism from ISG15 alone: The inhibition of HIV-1 particle production by HERC5 occurs at an earlier stage than the restriction imposed by ISG15 expression alone, which acts during particle release .

These findings suggest that HERC5 represents a potential target for HIV/AIDS therapy, as it targets a stage of viral replication that is not directly counteracted by any known HIV-1 protein .

Beyond HIV, what other viruses are restricted by HERC5 and through what mechanisms?

HERC5 exhibits broad antiviral activity against multiple viruses through various mechanisms:

• Influenza A Virus (IAV): HERC5 binds to and ISGylates the IAV non-structural protein 1 (NS1) through its RLD domain, interfering with viral replication .

• Hepatitis C Virus (HCV): HERC5 targets the HCV non-structural protein 5A (NS5A) for ISGylation, disrupting viral replication complexes .

• Human Papillomavirus (HPV): HERC5 has been shown to ISGylate the major capsid protein L1 of HPV16, reducing the rate of viral replication .

• SARS-CoV-2: Recent studies suggest HERC5 may play a role in restricting SARS-CoV-2 infection, though the specific mechanisms are still being investigated .

• Murine Leukemia Virus (MLV): HERC5 can restrict MLV Gag particle production, demonstrating its effectiveness against evolutionarily divergent retroviruses .

The broad spectrum of HERC5's antiviral activity suggests it targets conserved aspects of viral replication cycles, making it an attractive subject for antiviral therapeutic development .

How does HERC5 modulate the host interferon response during viral infection?

HERC5 plays a dual role in modulating the host interferon response:

• Amplification of ISG induction: HERC5 acts as a positive regulator of the ISGylation feedback loop by amplifying the induction of interferon-stimulated genes during the early stages of viral infection .

• Stabilization of IRF3: HERC5 ISGylates Interferon Regulatory Factor 3 (IRF3), which prevents IRF3 from being targeted for K48 polyubiquitylation and subsequent proteasomal degradation. This increases ISG induction rates in infected cells and prolongs the interferon response .

• Prevention of premature response termination: By enhancing IRF-related signal transduction, HERC5 prevents the premature termination of the antiviral interferon response .

• Enhancement of innate immune signaling: HERC5 interacts with proteins in antiviral signaling pathways, including DDX58 (RIG-I), DHX58, MAVS, and ISG15, suggesting it functions within a broader network of innate immune factors .

What experimental models are most effective for studying HERC5 function?

Several experimental models have proven effective for studying HERC5 function:

• Cell line models:

  • Human 293T and HeLa cells have been extensively used for overexpression and knockdown studies of HERC5 .

  • HOS-CD4/CXCR4 cells support robust HIV-1 replication and are useful for studying HERC5's antiviral effects .

  • Jurkat cells (human T lymphocytes) can be used to study endogenous HERC5 expression and localization after interferon treatment .

• Primary cell models:

  • Peripheral Blood Mononuclear Cells (PBMCs) express HERC5 after interferon treatment and can be used to study physiologically relevant expression patterns .

  • Dendritic cells (such as DC2.4) can be used to examine HERC5 in professional antigen-presenting cells .

• Knockdown/Knockout systems:

  • RNA interference using shRNA against HERC5 has been effective in studying loss-of-function phenotypes .

  • CRISPR-Cas9 genome editing can generate HERC5 knockout cell lines for more complete functional analyses.

• Reconstitution systems:

  • Co-expression of HERC5 with UBE1L and UBE2L6 can reconstitute the ISGylation pathway independent of interferon stimulation, allowing for controlled study of the process .

• Microscopy models:

  • Confocal immunofluorescence microscopy has been used successfully to visualize HERC5 localization and co-localization with substrates .

What techniques are most effective for measuring HERC5-mediated ISGylation?

Several techniques have been developed to effectively measure and analyze HERC5-mediated ISGylation:

• Western blotting:

  • Detection of ISGylated proteins using anti-ISG15 antibodies reveals characteristic laddering patterns or shifts in molecular weight of target proteins .

  • Comparative analysis between wild-type HERC5 and C994A catalytic mutant can confirm HERC5-dependent ISGylation .

• Immunoprecipitation followed by mass spectrometry:

  • Enrichment of ISGylated proteins by immunoprecipitation with anti-ISG15 antibodies followed by mass spectrometry analysis can identify novel substrates .

  • Quantitative labile free proteomics has been used to identify ISGylation targets in infected cells .

• In vitro ISGylation assays:

  • Reconstituted systems using purified components (UBE1L, UBE2L6, HERC5, and ISG15) and potential substrates can directly demonstrate ISGylation activity.

• Confocal microscopy:

  • Co-localization studies using fluorescently labeled HERC5, ISG15, and target proteins can visualize the ISGylation process in cells .

• Reporter systems:

  • Fusion of potential target proteins with reporters like luciferase can provide quantitative readouts of ISGylation effects on protein function.

• shRNA knockdown validation:

  • Comparing ISGylation patterns between control and HERC5-knockdown cells can confirm HERC5-dependent modifications .

What approaches can identify novel HERC5 substrates and interaction partners?

Identifying novel HERC5 substrates and interaction partners requires multifaceted approaches:

• Proteomic approaches:

  • Stable isotope labeling with amino acids in cell culture (SILAC) combined with mass spectrometry can identify proteins differentially ISGylated in the presence or absence of HERC5.

  • Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling can identify proteins in close proximity to HERC5 in cells.

• Network analysis tools:

  • Bioinformatic analysis using tools like GeneMANIA can reveal proteins that correlate with HERC5 function. Research has identified networks including DHX58, MAVS, RAI14, WRNIP1, and ISG15 as HERC5-associated proteins .

• Yeast two-hybrid screening:

  • This technique can identify direct protein-protein interactions between HERC5 and potential partners.

• Co-immunoprecipitation followed by mass spectrometry:

  • Pulling down HERC5 and its associated proteins followed by mass spectrometry analysis can identify interaction partners.

• Domain-specific interaction studies:

  • Using isolated domains of HERC5 (such as the RLD or HECT domains) as bait can identify domain-specific interactions.

• Ribosome profiling:

  • Since HERC5 associates with polyribosomes and targets newly synthesized proteins, ribosome profiling can identify potential substrates being actively translated during HERC5 expression .

What approaches show promise for targeting HERC5 in antiviral therapy development?

Several approaches show potential for leveraging HERC5 in antiviral therapy development:

• Enhancement of HERC5 expression:

  • Small molecules that increase HERC5 expression or activity could boost natural antiviral responses.

  • Targeted delivery of HERC5 mRNA or protein to specific cell types could enhance localized antiviral responses.

• Mimicking HERC5 ISGylation:

  • Developing peptides or small molecules that mimic the effect of HERC5-mediated ISGylation on viral proteins.

  • As noted in research, such approaches could potentially give those with HIV "two extra lines of defense against the spread of the virus" .

• Structure-based drug design:

  • Using the structural information of HERC5's domains, particularly the RLD that recognizes viral substrates, to design molecules that enhance substrate recognition.

  • Targeting the HECT domain to increase catalytic efficiency.

• Combination therapies:

  • Combining HERC5-targeting therapeutics with conventional antivirals for synergistic effects.

  • Exploiting the broad-spectrum antiviral properties of HERC5 to develop therapies effective against multiple viral infections .

• Gene therapy approaches:

  • Delivery of enhanced HERC5 variants that are more effective at restricting viral replication.

  • Utilizing CRISPR-Cas9 to modify endogenous HERC5 for improved antiviral activity.

How might HERC5 genetic variants influence individual susceptibility to viral infections?

HERC5 genetic variants could significantly impact individual responses to viral infections through several mechanisms:

Understanding these genetic factors could help identify individuals at higher risk for severe viral infections and guide personalized antiviral treatment strategies.

What are the major challenges in translating HERC5 research into clinical applications?

Several significant challenges must be addressed to translate HERC5 research into clinical applications:

• Specificity concerns:

  • HERC5 targets newly synthesized proteins broadly, raising concerns about off-target effects if its activity is enhanced therapeutically.

  • Determining how to selectively enhance HERC5's antiviral functions without disrupting normal cellular processes.

• Delivery challenges:

  • Developing effective methods to deliver HERC5-enhancing therapeutics to specific cell types or tissues affected by viral infection.

  • Creating stable formulations of any HERC5-based biologics.

• Timing considerations:

  • HERC5's greatest antiviral potential may be early in infection, requiring rapid diagnosis and treatment.

  • Determining optimal treatment windows for different viral infections.

• Viral evasion mechanisms:

  • Some viruses may develop mechanisms to counteract enhanced HERC5 activity, necessitating combination approaches.

  • Understanding how different viruses interact with or evade HERC5-mediated restriction.

• Translational gaps:

  • Moving from cell culture and animal models to human clinical applications requires addressing differences in HERC5 biology across species.

  • Developing appropriate biomarkers to monitor HERC5 activity and therapeutic efficacy in patients.

• Regulatory pathways:

  • Establishing regulatory frameworks for novel immunomodulatory approaches targeting host factors rather than viral proteins directly.

  • Demonstrating safety profiles for therapies that modify innate immune responses.

Despite these challenges, the broad antiviral activity and unique mechanisms of HERC5 make it a promising candidate for continued therapeutic development efforts.