Mutational Alteration of Human Immunodeficiency Virus Type 1 Vif Allows for Functional Interaction with Nonhuman Primate APOBEC3G

Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Journal of Virology (Impact Factor: 4.44). 06/2006; 80(12):5984-91. DOI: 10.1128/JVI.00388-06
Source: PubMed


Human APOBEC3F (hA3F) and APOBEC3G (hA3G) are antiretroviral cytidine deaminases that can be encapsidated during virus assembly to catalyze C-->U deamination of the viral reverse transcripts in the next round of infection. Lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have evolved the accessory protein Vif to induce their degradation before packaging. HIV type 1 (HIV-1) Vif counteracts hA3G but not rhesus macaque APOBEC3G (rhA3G) or African green monkey (AGM) APOBEC3G (agmA3G) because of a failure to bind the nonhuman primate proteins. The species specificity of the interaction is controlled by amino acid 128, which is aspartate in hA3G and lysine in rhA3G. With the objective of overcoming this species restriction, mutations were introduced into HIV-1 Vif at amino acid positions that differed in charge between HIV-1 Vif and SIV Vif. The mutant proteins were tested for the ability to counteract hA3G, rhA3G, and agmA3G. Alteration of the conserved sequence at positions 14 to 17 from DRMR to SERQ, which is the sequence in AGM Vif, caused HIV-1 Vif to functionally interact with rhA3G and agmA3G. Mutation of three residues to the sequence SEMQ allowed interaction with rhA3G. SEMQ Vif also counteracted D128K mutant hA3G and wild-type hA3G. Introduction of the sequence into an infectious molecular HIV-1 clone allowed the virus to replicate productively in human cells that expressed rhA3G or hA3G. These findings provide insight into the interaction of Vif with A3G and are a step toward the development of a novel primate model for AIDS.

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    • "However, due to the incomplete suppression of the tetracycline response element promoter,24 a very small amount of the R88-A3Gm protein was still expressed in the absence of Dox (Figure 1c, lane 1), which could also incorporate into vector particles and impair the vector's transduction efficiency. We thus attempted to promote R88-A3Gm degradation by cotransfecting vector particle–producing cells with the A3GD128K-susceptible HIV-1 VifSEMQ-expressing plasmid.25 Indeed, when VifSEMQ was coexpressed with ProLabel-A3Gm (PL-A3Gm) in 293T cells, the cellular level of the A3Gm protein was reduced by eight times, as measured by ProLabel assay, which is based on the chemiluminescent detection of PL-A3G fusion protein expression (Figure 1d). "
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    ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) drug resistance and the latent reservoir are the two major obstacles to effectively controlling and curing HIV-1 infection. Therefore, it is critical to develop therapeutic strategies specifically targeting these two obstacles. Recently, we described a novel anti-HIV approach based on a modified human intrinsic restriction factor, R88-APOBEC3G (R88-A3G). In this study, we further characterized the antiviral potential of R88-A3GD128K (R88-A3Gm) against drug-resistant strains of HIV-1 and viruses produced from latently infected cells. We delivered R88-A3Gm into target cells using a doxycycline (Dox)-inducible lentiviral vector and demonstrated that its expression and antiviral activity were highly regulated by Dox. In the presence of Dox, R88-A3Gm-transduced T cells were resistant to infection caused by wild-type and various drug-resistant strains of HIV-1. Moreover, when the R88-A3Gm-expressing vector was transduced into the HIV-1 latently infected ACH-2 cell line or human CD4(+) T cells, on activation by phorbol-12-myristate-13-acetate or phytohemaglutinin, R88-A3Gm was able to curtail the replication of progeny viruses. Altogether, these data clearly indicate that R88-A3Gm is a highly potent HIV-1 inhibitor, and R88-A3Gm-based anti-HIV gene therapy is capable of targeting both active and latent HIV-1-infected cells to prevent subsequent viral replication and dissemination.Molecular Therapy-Nucleic Acids (2014) 3, e151; doi:10.1038/mtna.2014.2; published online 4 March 2014.
    Molecular Therapy - Nucleic Acids 03/2014; 3(3):e151. DOI:10.1038/mtna.2014.2 · 4.51 Impact Factor
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    • "This region is essential for directing A3G ubiquitination by the E3 ubiquitin ligase complex and corresponds to a nonlinear region located at the N-terminus [32]. Two sequences appear to be important: the first is the 14DRMR17 motif and interacts with APOBEC3F (A3F) sequence (128–130), which is also located at the N-terminus [33], [34]. The other region that plays a functional role is 40YRHHY44 motif; however, this is important only for A3G interaction [35], [36]. "
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    ABSTRACT: The virion infectivity factor (Vif) is an accessory protein, which is essential for HIV replication in host cells. Vif neutralizes the antiviral host protein APOBEC3 through recruitment of the E3 ubiquitin ligase complex. Fifty thousand Vif models were generated using the ab initio relax protocol of the Rosetta algorithm from sets of three- and nine-residue fragments using the fragment Monte Carlo insertion-simulated annealing strategy, which favors protein-like features, followed by an all-atom refinement. In the protocol, a constraints archive was used to define the spatial relationship between the side chains from Cys/His residues and zinc ions that formed the zinc-finger motif that is essential for Vif function. We also performed centroids analysis and structural analysis with respect to the formation of the zinc-finger, and the residue disposal in the protein binding domains. Additionally, molecular docking was used to explore details of Vif-A3G and Vif-EloBC interactions. Furthermore, molecular dynamics simulation was used to evaluate the stability of the complexes Vif-EloBC-A3G and Vif-EloC. The zinc in the HCCH domain significantly alters the folding of Vif and changes the structural dynamics of the HCCH region. Ab initio modeling indicated that the Vif zinc-finger possibly displays tetrahedral geometry as suggested by Mehle et al. (2006). Our model also showed that the residues L146 and L149 of the BC-box motif bind to EloC by hydrophobic interactions, and the residue P162 of the PPLP motif is important to EloB binding. The model presented here is the first complete three-dimensional structure of the Vif. The interaction of Vif with the A3G protein and the EloBC complex is in agreement with empirical data that is currently available in the literature and could therefore provide valuable structural information for advances in rational drug design.
    PLoS ONE 02/2014; 9(2):e89116. DOI:10.1371/journal.pone.0089116 · 3.23 Impact Factor
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    • "Many efforts have been made to evade from the restriction by the macaque A3 family. Schrofelbauer et al. (2006)showed that mutations of HIV-1 Vif at positions 14–17 from DRMR into SEMQ allowed HIV-1 Vif interaction with A3G from RM. However, this HIV-1 Vif harboring SEMQ remained susceptible to A3B, A3F, and A3H from RM (Virgen and Hatziioannou, 2007), suggesting that the introduction of this sequence in HIV-1 Vif was not sufficient for evading from A3s other than A3G. "
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    Frontiers in Microbiology 07/2013; 4:187. DOI:10.3389/fmicb.2013.00187 · 3.99 Impact Factor
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