HIV Viral Infectivity Factor BC-box in complex with ElonginB and ElonginC

David Torres '16 and Joey Duronio '16


I. Introduction

The Human Immunodefiency Virus (HIV) as well as other similar retroviruses contain a viral infectivity factor (Vif) that inhibits certain anti-viral activity in human cells. Vif is used in viral replication by hijacking the Cullin5 E3 ubiquitin ligase, which consists of Cullin5, ElonginBC, andRbx2, and targeting APOBEC3G for degradation. APOBEC3G is a cellular enzyme and part of the APOBEC superfamily of proteins. This family of proteins plays a role in anti-viral immunity. Vif inhibits APOBEC3G from entering the new budding virus and hypermutating the genome to render the virus essentially dead.


Vif is able to hijack the Cullin5 E3 ubiquitin ligase by binding to ElonginBC via the BC-box domain. It then uses the hosts own ubiquitination and degradation system to destroy the APOBEC3G.

General Structure of Elongin-Cullin-SOCS-box (ECS)

          A normal cellular ECS ubiquitin ligase is made up of ElonginB (EloB), ElonginC (EloC), Cullin5 (Cul5), Rbx2, and a SOCS-box protein. SOCS-box is formally known as the suppressor of cytokine signaling box proteins. SOCS box proteins are characterized by a conserved interaction domain, which offers a link between cellular substrates and the E3 ubiquitin ligase. This conserved domain of SOCS box includes the BC box, which binds EloB and EloC (EloBC), and the Cullin box, which is hypothesized to participate in the binding of either Cul5 or Cul2. Crystal structures show that the SOCS box has a conserved region composed of a BC box helix, a small 90 turn, a second short helix ending in a region rich with prolines, a loop, and a third helix. The first helix is considered as the BC box and the rest is referred to as the Cullin box.

Vif Binding APOBEC3G

            Vif uses conserved regions to influence the degradation of APOBEC3G. Vif acts as the substrate of APOBEC3G by mimicking the SOCS box domain of a normal cellular E3 ubiquitin ligase. Research proposes that the Vif N-terminal domain interacts with APOBEC3G, while its C-terimanl domain recruits the E3 ligase through two conserved regions. HIV Vif contains a BC-box region that interacts with EloC and is crucial for recruiting the cellular E3 ubiquitin ligase. A number of Vif residues V142, L145, L148, A149, A152, AND L153 create the hydrophobic face that interacts with EloC. Hydrogen bonding between the backbone carbonyl of Vif G143 and EloC Y76 also contributes to the Vif-EloC interaction. A second E3 ligase-binding domain is a conserved region containing histidine and cysteine residues (this region is called the zinc-binding motif). This zinc-binding motif interacts with Cul5. Mutations of these His or Cys residues destroys the Vif-Cul5 interaction.

            The interaction between a third Vif C-terminal domain, the Vif Cullin box, and the E3 ubiquitin ligase has not been thoroughly studied. However, the Vif-Cul5 interaction has been identified by many experiments. These experiments were not able to identify a direct interaction between the Vif Cullin box and Cul5.

IV. DNA Binding

Once CAP has bound cAMP, it is ready to bind to the DNA. Binding occurs at the conserved sequence of 5'-AAATGTAGATCACATTT-3' Hydrogen bonds between the protein and the DNA phsophates occur at the backbone amide of residue 139, and the side chains of Thr-140, Ser-179, and Thr-182 In addition to these phosphate interactions, the side chains of Glu-181 and Arg-185, both emanating from the recognition helix directly contact the bases within the major groove of the DNA. Because of the way that the protein binds to the DNA, a kink of ~40 degrees occurs between nucleotide base pairs six and seven on each side of the dyad axis, 5'-TG-3' This sequence has been shown to favor DNA flexibility and bending in other systems as well. Because of this kink, an additional five ionic interactions and four hydrogen bonds are able to occur between the protein and the DNA strand. Examples of these new interactions occur between Lys-26, Lys-166, His-199 and the DNA sugar-phosphate backbone The DNA bend is integral to the mechanism of transcription activation. Not only does it place CAP in the proper orientation for interaction with RNA polymerase, but wrapping the DNA around the protein may result in direct contacts between upstream DNA and RNA polymerase. 

V. Activating Regions

Transcription activation by CAP requires more than merely the binding of cAMP and binding and bending of DNA. CAP contains an "activating region" that has been proposed to participate in direct protein-protein interactions with RNA polymerase and/or other basal transcription factors. Specifically, amino acids 156, 158, 159, and 162 have been proposed to be critical for transcription activation by CAP. These amino acids are part of a surface loop composed of residues 152-166 Researchers have concluded that the third and final step in transcription activation is this direct protein-protein contact between amino acids 156-162 of CAP, and RNA polymerase.

VI. References

Gunasekera, Angelo, Yon W. Ebright, and Richard H. Ebright. 1992. DNA Sequence Determinants for Binding of the Escherichia coli Catabolite Gene Activator Protein. The Journal of Biological Chemistry 267:14713-14720.

Schultz, Steve C., George C. Shields, and Thomas A. Steitz. 1991. Crystal Structure of a CAP-DNA complex: The DNA Is Bent by 90 degrees Science 253: 1001-1007.

Vaney, Marie Christine, Gary L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry 28:4568-4574.

Weber, Irene T., Gary L. Gilliland, James G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a Cyclic AMP-independent Mutant of Catabolite Activator Protein. The Journal of Biological Chemistry 262:5630-5636.

Zhou, Yuhong, Ziaoping Zhang, and Richard H. Ebright. 1993. Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation. Proceedings of the National Academy of Sciences of the United States of America 90:6081-6085.

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