GO:0031466 Cul5-RING ubiquitin ligase complex: Components, Assembly and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0031466 describes the Cul5-RING ubiquitin ligase complex (CRL5), a multi-subunit E3 ubiquitin ligase in which a Cul5 subfamily cullin and a RING-domain protein form the catalytic core.
• Substrate specificity of CRL5 is conferred by an elongin-BC adaptor together with a SOCS/BC-box protein, allowing targeted ubiquitination of distinct substrates.
• The HIV-1 accessory protein Vif hijacks the CRL5 complex to ubiquitinate and degrade host APOBEC3 restriction factors, a classic example of viral subversion of a cellular E3 ligase.
• Core binding factor beta (CBFB) is an evolutionarily conserved requirement for assembly of the HIV/SIV Vif-Cul5-RING E3 ubiquitin ligase, highlighting a conserved assembly cofactor.
• Structural studies of Vif-mediated E3 ligase targeting of APOBEC3H have revealed how the viral adaptor engages both Cul5 and the substrate to promote ubiquitination.
• CRL5 components are attractive research targets for antiviral, immuno-oncology, and protein-degradation studies, and can be modeled with CRISPR knockout, point-mutation, knock-in, and overexpression cell systems.
Description
The Cul5-RING ubiquitin ligase complex (GO:0031466) is a cellular_component ontology term that defines a specific class of cullin-RING E3 ubiquitin ligases. In this complex, a cullin from the Cul5 subfamily and a RING-domain protein form the catalytic core, while substrate specificity is provided by an elongin-BC adaptor and a SOCS/BC-box protein. This architecture places CRL5 among the modular CRL family, where different cullins and adaptors direct ubiquitination of distinct protein substrates. The complex is best known for its role in HIV-1 biology, where the viral protein Vif recruits Cul5 to suppress the host restriction factor APOBEC3. Because CRL5 controls the stability of key cellular and viral proteins, it is a focal point for studies of host-pathogen interactions, ubiquitin signaling, and targeted protein degradation. Researchers studying GO:0031466 need reliable models to dissect subunit assembly, substrate recruitment, and downstream degradation events. The verified literature on Vif-Cul5 interactions provides a well-characterized paradigm for understanding how this E3 ligase complex is assembled and how it can be subverted by pathogens.
Cul5-RING ubiquitin ligase complex At A Glance
| GO ID | GO:0031466 |
|---|---|
| GO term | Cul5-RING ubiquitin ligase complex |
| Ontology | cellular_component |
| Synonym | CDL5 complex; CRL5 complex; cullin-RING ligase 5; EC2S complex; SCF5 complex |
| Major function | E3 ubiquitin ligase activity that transfers ubiquitin to substrate proteins, with substrate specificity determined by elongin-BC and SOCS/BC-box proteins |
| Catalytic core | Cul5 subfamily cullin plus a RING domain protein |
| Adaptor module | Elongin-BC adaptor and a SOCS/BC-box protein |
| Example hijacking factor | HIV-1 Vif, which recruits Cul5 to suppress APOBEC3 |
| Conserved assembly factor | Core binding factor beta (CBFB) is required for assembly of the Vif-Cul5-RING E3 ligase |
What Is GO:0031466?
According to the QuickGO definition, GO:0031466 is a ubiquitin ligase complex in which a cullin from the Cul5 subfamily and a RING domain protein form the catalytic core; substrate specificity is conferred by an elongin-BC adaptor and a SOCS/BC box protein. In other words, it is a multi-protein E3 ubiquitin ligase machine that brings a ubiquitin-conjugating enzyme and a target substrate into proximity, enabling transfer of ubiquitin onto the substrate. The complex is also known by synonyms such as CDL5 complex, CRL5 complex, cullin-RING ligase 5, EC2S complex, and SCF5 complex. Its defining feature is the combination of a Cul5 scaffold, a RING finger catalytic subunit, and adaptor modules that select which proteins are ubiquitinated.
Why Is Cul5-RING ubiquitin ligase complex Important in Cell Biology?
GO:0031466 is important because it defines a central node in ubiquitin-dependent protein degradation that is conserved across eukaryotes and is exploited by viruses. The CRL5 complex controls the stability of host restriction factors such as APOBEC3 through HIV-1 Vif, making it a key determinant of viral infectivity and a model for understanding how E3 ligases select their substrates. Because the complex is modular, alterations in its subunits can redirect substrate specificity, which has broad implications for cancer, immunity, and antiviral research. Studying CRL5 assembly and function also informs the design of targeted protein degradation tools and therapeutic strategies that modulate ubiquitin signaling.
• CRL5 is a major E3 ubiquitin ligase that targets proteins for proteasomal degradation, influencing diverse cellular pathways.
• HIV-1 Vif hijacks CRL5 to degrade APOBEC3 restriction factors, directly linking the complex to viral pathogenesis.
• Core binding factor beta (CBFB) is required for assembly of the Vif-Cul5-RING E3 ligase, revealing a conserved assembly mechanism.
• Structural insights into Vif-mediated targeting of APOBEC3H provide a framework for understanding substrate recognition by CRL5.
• The complex is a paradigm for cullin-RING ligase modularity, where adaptor proteins dictate substrate specificity.
• CRL5 components are potential targets for antiviral therapies aimed at blocking Vif-mediated degradation of APOBEC3.
• Dysregulation of CRL5 substrates may contribute to cancer and immune disorders, making it relevant to oncology and immunology research.
• CRISPR-based models of CRL5 subunits enable functional dissection of assembly, substrate recruitment, and degradation.
• The complex is a model system for studying how pathogens co-opt host ubiquitin machinery.
• Understanding CRL5 biology supports the development of targeted protein degradation technologies and molecular glues.
Structure and Composition of Cul5-RING ubiquitin ligase complex
Catalytic Core: Cul5 and RING Protein
In simple terms: The core of the machine is made of two proteins: Cul5 and a RING finger protein.
The catalytic core of GO:0031466 consists of a cullin from the Cul5 subfamily and a RING domain protein. This core provides the scaffold and the catalytic RING finger that recruits a ubiquitin-conjugating enzyme (E2) to transfer ubiquitin onto substrates. The Cul5 scaffold organizes the complex and positions the substrate for efficient ubiquitination. In the HIV-1 context, Vif bridges Cul5 to the substrate APOBEC3, illustrating how the core can be redirected by viral proteins.
Adaptor Module: Elongin-BC and SOCS/BC-box Protein
In simple terms: A separate adaptor module decides which proteins get tagged for degradation.
Substrate specificity of the CRL5 complex is conferred by an elongin-BC adaptor and a SOCS/BC-box protein. The elongin-BC heterodimer binds to the Cul5 scaffold, while the SOCS/BC-box protein recruits specific substrates. This modular adaptor system allows the same catalytic core to target different proteins depending on which SOCS/BC-box protein is present. In HIV-1, Vif functions as a viral SOCS/BC-box-like protein that recruits APOBEC3 to the complex.
Assembly Factor: Core Binding Factor Beta (CBFB)
In simple terms: A helper protein called CBFB is needed to put the complex together.
Core binding factor beta (CBFB) is an evolutionarily conserved requirement for assembly of the human immunodeficiency virus/simian immunodeficiency virus Vif-cullin 5-RING E3 ubiquitin ligase. This finding indicates that assembly of the CRL5 complex is not spontaneous but requires specific cofactors. CBFB facilitates the formation of a functional Vif-Cul5-RING complex, and its conservation across species suggests a fundamental role in CRL5 biology.
Viral Hijacking: HIV-1 Vif as a Substrate Receptor
In simple terms: HIV-1 Vif steals the complex to destroy the cell's antiviral defenses.
The HIV-1 Vif protein acts as a substrate receptor that recruits the Cul5-RING ubiquitin ligase complex to target host APOBEC3 proteins for degradation. The N-terminal motif of Vif is required for recruitment of Cul5 to suppress APOBEC3. Structural studies of Vif-mediated E3 ligase targeting of APOBEC3H have revealed how Vif engages both Cul5 and the substrate to promote ubiquitination. This viral subversion is a prime example of how pathogens co-opt host E3 ligases.
Key Genes Involved in GO:0031466 Cul5-RING ubiquitin ligase complex
The following genes and proteins are core components or key interactors of the Cul5-RING ubiquitin ligase complex (GO:0031466), based on verified literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| CUL5 | Cullin scaffold of the CRL5 complex; forms catalytic core with RING protein | Central to GO:0031466; target for knockout and structural studies |
| RBX1 | RING domain protein that recruits E2 ubiquitin-conjugating enzyme | Catalytic subunit of the core; essential for ubiquitin transfer |
| ELOB | Elongin B, part of the elongin-BC adaptor | Adaptor module for substrate recruitment |
| ELOC | Elongin C, part of the elongin-BC adaptor | Adaptor module for substrate recruitment |
| SOCS1 | SOCS-box protein that recruits substrates to CRL5 | Defines substrate specificity; model for adaptor function |
| SOCS3 | SOCS-box protein that recruits substrates to CRL5 | Defines substrate specificity; model for adaptor function |
| CBFB | Core binding factor beta; required for assembly of Vif-Cul5-RING E3 ligase | Conserved assembly factor; knockout reduces complex formation |
| VIF | HIV-1 protein that hijacks CRL5 to degrade APOBEC3 | Viral substrate receptor; key to HIV pathogenesis |
| APOBEC3G | Host restriction factor targeted for degradation by Vif-CRL5 | Antiviral defense; substrate of CRL5 in HIV infection |
| APOBEC3H | Host restriction factor targeted by Vif-CRL5 | Structural model for substrate recognition |
| APOBEC3F | Host restriction factor targeted by Vif-CRL5 | Antiviral defense; substrate of CRL5 |
| CUL2 | Related cullin that forms CRL2 complexes | Comparative studies of cullin-RING ligase family |
| CUL1 | Related cullin that forms CRL1 complexes | Comparative studies of cullin-RING ligase family |
| UBE2D1 | E2 ubiquitin-conjugating enzyme | Provides ubiquitin for CRL5-mediated transfer |
| UBE2D2 | E2 ubiquitin-conjugating enzyme | Provides ubiquitin for CRL5-mediated transfer |
| UBE2D3 | E2 ubiquitin-conjugating enzyme | Provides ubiquitin for CRL5-mediated transfer |
| NEDD8 | Ubiquitin-like modifier that activates cullin-RING ligases | Regulates CRL5 activity through neddylation |
| CAND1 | Cullin-associated NEDD8-dissociated protein 1 | Regulates cullin complex assembly and dynamics |
How Is Cul5-RING ubiquitin ligase complex Regulated?
The activity of the Cul5-RING ubiquitin ligase complex is regulated at multiple levels. Cullin-RING ligases are activated by neddylation, a process in which the ubiquitin-like protein NEDD8 is conjugated to the cullin subunit, and this modification is required for efficient ubiquitin transfer. Assembly of the complex is also regulated by accessory factors such as core binding factor beta (CBFB), which is required for formation of the Vif-Cul5-RING E3 ligase. In the context of HIV-1 infection, the viral protein Vif serves as a substrate receptor that redirects the complex to APOBEC3 proteins, and the N-terminal motif of Vif is required for Cul5 recruitment. Structural studies have further shown how Vif engages both Cul5 and APOBEC3H to promote ubiquitination, revealing a regulated interface that could be targeted therapeutically. Additionally, the exchange of adaptor proteins, such as different SOCS-box proteins, provides a dynamic mechanism for switching substrate specificity.
Cul5-RING ubiquitin ligase complex and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| VIF | HIV-1 pathogenesis; degradation of APOBEC3 restriction factors | Knockout of VIF in HIV-1 provirus; overexpression in target cells |
| APOBEC3G | HIV-1 restriction; innate immunity | Knockout or point mutation of APOBEC3G in T-cell lines |
| APOBEC3H | HIV-1 restriction; structural basis of Vif targeting | Knock-in of APOBEC3H variants; structural studies |
| CBFB | Conserved assembly of Vif-Cul5-RING E3 ligase; potential cancer link | Knockout of CBFB in HIV-infected cells; rescue with wild-type |
| CUL5 | Core scaffold of CRL5; host-pathogen interactions | Knockout of CUL5 to block Vif-mediated degradation |
HIV-1 Pathogenesis and APOBEC3 Restriction
The most well-characterized disease link for GO:0031466 is HIV-1 infection. The viral protein Vif recruits the Cul5-RING ubiquitin ligase complex to target host APOBEC3 proteins for degradation, thereby suppressing their antiviral activity. The N-terminal motif of Vif is required for Cul5 recruitment, and disruption of this interaction impairs Vif-mediated suppression of APOBEC3. Structural analysis of Vif-mediated targeting of APOBEC3H has provided detailed insights into how the viral protein engages the E3 ligase and the substrate, offering a template for antiviral drug design.
Cancer and Cellular Stress Responses
Cullin-RING ligases, including CRL5, control the stability of numerous regulatory proteins involved in cell cycle progression, apoptosis, and stress responses. Although direct cancer associations for CRL5 subunits are still being defined, the complex shares conserved assembly mechanisms with other cullins, and core binding factor beta (CBFB) is required for assembly of the Vif-Cul5-RING E3 ligase. Dysregulation of CRL5 substrates could contribute to oncogenesis by altering protein turnover, making CRL5 components candidates for cancer research.
Innate Immunity and Host-Pathogen Interactions
Beyond HIV, the CRL5 complex is part of the broader host-pathogen interface. By targeting APOBEC3 family members, Vif-CRL5 counteracts innate immune restriction factors. Understanding how CRL5 selects substrates through elongin-BC and SOCS/BC-box proteins may reveal how other pathogens or cellular proteins modulate immune signaling. This has implications for vaccine design and immunotherapies aimed at preserving restriction factor activity.
From Cul5-RING ubiquitin ligase complex-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does CUL5 knockout block Vif-mediated APOBEC3 degradation? | CUL5 knockout cell line infected with HIV-1 |
| How does a point mutation in Vif N-terminal motif affect Cul5 recruitment? | Point-mutation knock-in of VIF in proviral clone |
| Can CBFB knockout disrupt Vif-Cul5-RING assembly? | CBFB knockout cells with Vif expression |
| What is the structural interface between Vif and APOBEC3H? | Tagged knock-in of APOBEC3H for structural studies |
| Does overexpression of SOCS-box proteins redirect CRL5 substrates? | Overexpression of SOCS1/SOCS3 in reporter cells |
| Can CRL5 activity be monitored by ubiquitination assays? | Knock-in of tagged ubiquitin in cells |
How to Study the Cul5-RING ubiquitin ligase complex Process
| Method | What It Measures | Typical Application |
|---|---|---|
| CRISPR knockout | Loss of function of CRL5 subunits | Test requirement for CUL5, CBFB in Vif-mediated degradation |
| Point mutation knock-in | Effect of specific amino acid changes | Map Vif N-terminal motif required for Cul5 recruitment |
| Affinity purification + MS | Protein-protein interactions | Identify CRL5 components and substrates |
| Ubiquitination assay | E3 ligase activity | Reconstitute CRL5-mediated ubiquitin transfer |
| Cryo-EM | Three-dimensional structure | Determine Vif-Cul5-APOBEC3H interface |
| RNA-seq | Transcriptional changes | Measure APOBEC3 and immune gene expression |
| Western blot | Protein stability | Assess substrate degradation upon CRL5 perturbation |
| Flow cytometry | Reporter degradation | High-throughput screening of CRL5 regulators |
CRISPR Knockout Screens for CRL5 Components
CRISPR knockout screens can systematically test the requirement for each CRL5 subunit in substrate degradation. For example, knocking out CUL5 or CBFB can reveal whether Vif-mediated APOBEC3 degradation depends on the intact complex. These screens are typically performed in cell lines expressing a reporter substrate, followed by flow cytometry or western blot to measure degradation.
Proteomics and Ubiquitination Assays
Mass spectrometry-based proteomics can identify substrates and interaction partners of the CRL5 complex. Affinity purification of tagged Cul5 or Vif followed by LC-MS/MS reveals the composition of the complex and dynamic changes upon infection. Ubiquitination assays using recombinant E1, E2, and E3 components can reconstitute CRL5 activity in vitro and test the role of specific subunits.
Structural Biology of Vif-Cul5-APOBEC3 Complexes
Cryo-EM and X-ray crystallography have been used to determine the structure of Vif in complex with Cul5 and APOBEC3H, revealing the molecular details of substrate recognition. These studies guide mutagenesis experiments to validate interfaces and identify druggable pockets.
RNA-seq and Transcriptomics
RNA sequencing can measure changes in gene expression upon CRL5 perturbation, including feedback regulation of APOBEC3 family members and interferon-stimulated genes. This approach helps place CRL5 in the broader context of host defense and cellular stress responses.
How CRISPR Can Be Used to Study GO:0031466 Cul5-RING ubiquitin ligase complex
Knockout
CRISPR knockout of CUL5, CBFB, or other CRL5 subunits can abolish complex formation and block Vif-mediated degradation of APOBEC3. These models are essential for establishing causality and for identifying which subunits are non-redundant. Knockout cell lines can be used in infection assays to measure viral replication and restriction factor stability.
Point Mutation
Point mutations in VIF, such as those in the N-terminal motif, can be introduced by CRISPR knock-in to test their effect on Cul5 recruitment. Similarly, point mutations in APOBEC3H can map residues required for Vif-mediated ubiquitination. These models provide fine-grained structure-function insights.
Knock-in
Knock-in of tagged versions of CUL5, VIF, or APOBEC3 allows for affinity purification and imaging of the complex in living cells. Tagged knock-in models preserve endogenous regulation and are ideal for proteomic and structural studies.
Overexpression
Overexpression of Vif or SOCS-box proteins can drive CRL5-mediated degradation of substrates and amplify phenotypes for biochemical analysis. Overexpression systems are useful for testing whether a candidate protein is sufficient to redirect the complex.
How EDITGENE Supports Cul5-RING ubiquitin ligase complex Research
Researchers studying Cul5-RING ubiquitin ligase complex-related genes often need to determine whether a candidate gene is causally involved in complex assembly, substrate recruitment, or degradation. EDITGENE provides publication-ready CRISPR cell models and screening services to accelerate this work.
Contact EDITGENE today to design your custom CRISPR model for Cul5-RING ubiquitin ligase complex research.
Frequently Asked Questions About Cul5-RING ubiquitin ligase complex
What is GO:0031466?
GO:0031466 is the Gene Ontology term for the Cul5-RING ubiquitin ligase complex, a multi-subunit E3 ubiquitin ligase in which a Cul5 subfamily cullin and a RING domain protein form the catalytic core, with substrate specificity conferred by an elongin-BC adaptor and a SOCS/BC-box protein.
What genes are involved in the Cul5-RING ubiquitin ligase complex?
Core genes include CUL5, RBX1, ELOB, ELOC, and various SOCS-box genes such as SOCS1 and SOCS3. The complex is also targeted by viral proteins like HIV-1 Vif and regulated by CBFB.
How does HIV-1 Vif use the Cul5-RING ubiquitin ligase complex?
HIV-1 Vif acts as a substrate receptor that recruits the Cul5-RING ubiquitin ligase complex to target host APOBEC3 proteins for degradation, thereby suppressing their antiviral activity.
What is the role of CBFB in the Cul5-RING ubiquitin ligase complex?
Core binding factor beta (CBFB) is an evolutionarily conserved requirement for assembly of the HIV/SIV Vif-cullin 5-RING E3 ubiquitin ligase.
What are the synonyms for GO:0031466?
Synonyms include CDL5 complex, CRL5 complex, cullin-RING ligase 5, EC2S complex, and SCF5 complex.
How can I study the Cul5-RING ubiquitin ligase complex?
Common methods include CRISPR knockout of CUL5 or CBFB, point mutations in VIF, affinity purification with mass spectrometry, ubiquitination assays, and structural biology such as cryo-EM.
What diseases are associated with the Cul5-RING ubiquitin ligase complex?
The complex is strongly linked to HIV-1 pathogenesis through Vif-mediated degradation of APOBEC3 restriction factors. It may also play roles in cancer and immune regulation.
What is the catalytic core of the Cul5-RING ubiquitin ligase complex?
The catalytic core consists of a cullin from the Cul5 subfamily and a RING domain protein, which together recruit the E2 ubiquitin-conjugating enzyme.
How is the Cul5-RING ubiquitin ligase complex regulated?
Regulation occurs through neddylation of the cullin subunit, assembly factors such as CBFB, and exchange of SOCS-box adaptor proteins that determine substrate specificity.
Can CRISPR be used to model Cul5-RING ubiquitin ligase complex function?
Yes. CRISPR knockout, point mutation, knock-in, and overexpression models are widely used to dissect CRL5 assembly, substrate recruitment, and degradation.
Conclusion
GO:0031466 defines the Cul5-RING ubiquitin ligase complex, a modular E3 ligase that plays a central role in ubiquitin-dependent protein degradation and is famously hijacked by HIV-1 Vif to destroy APOBEC3 restriction factors. Its assembly requires conserved factors such as CBFB, and its substrate specificity is dictated by elongin-BC and SOCS/BC-box proteins. Understanding this complex offers insights into host-pathogen interactions, immune evasion, and targeted protein degradation. With CRISPR-based models and screening services from EDITGENE, researchers can dissect CRL5 biology and translate findings into therapeutic strategies.
References
- 1. Evans SL et al.. 2014. HIV-1 Vif N-terminal motif is required for recruitment of Cul5 to suppress APOBEC3.. Retrovirology 11:4 PMID: 24422669
- 2. Ito F et al.. 2023. Structural basis of HIV-1 Vif-mediated E3 ligase targeting of host APOBEC3H.. Nat Commun 14(1):5241 PMID: 37640699
- 3. Han X et al.. 2014. Evolutionarily conserved requirement for core binding factor beta in the assembly of the human immunodeficiency virus/simian immunodeficiency virus Vif-cullin 5-RING E3 ubiquitin ligase.. J Virol 88(6):3320-8 PMID: 24390335
