Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle. IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.

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Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor

Journal of Virology ◽

10.1128/jvi.01411-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 208-219 ◽

Cited By ~ 18

Author(s):

Jiong Shi ◽

Jing Zhou ◽

Upul D. Halambage ◽

Vaibhav B. Shah ◽

Mallori J. Burse ◽

...

Keyword(s):

Amino Acid ◽

Small Molecule ◽

Therapeutic Target ◽

Binding Pocket ◽

Host Protein ◽

Viral Capsid ◽

Inhibitory Protein ◽

Hiv 1 ◽

Ca Protein

ABSTRACTThe HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexesin vitroand permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.IMPORTANCEThe HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

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Interactions between Sindbis virus RNAs and a 68 amino acid derivative of the viral capsid protein further defines the capsid binding site

Nucleic Acids Research ◽

10.1093/nar/22.5.780 ◽

1994 ◽

Vol 22 (5) ◽

pp. 780-786 ◽

Cited By ~ 49

Author(s):

Barbara Weiss ◽

Ute Geigenmüller-Gnirke ◽

Sondra Schlesinger

Keyword(s):

Amino Acid ◽

Binding Site ◽

Capsid Protein ◽

Acid Derivative ◽

Sindbis Virus ◽

Amino Acid Derivative ◽

Viral Capsid ◽

Viral Capsid Protein

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Residues in the HIV-1 Capsid Assembly Inhibitor Binding Site Are Essential for Maintaining the Assembly-competent Quaternary Structure of the Capsid Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m804230200 ◽

2008 ◽

Vol 283 (46) ◽

pp. 32024-32033 ◽

Cited By ~ 53

Author(s):

Vanda Bartonova ◽

Sébastien Igonet ◽

Jana Sticht ◽

Bärbel Glass ◽

Anja Habermann ◽

...

Keyword(s):

Binding Site ◽

Capsid Protein ◽

Quaternary Structure ◽

Capsid Assembly ◽

Inhibitor Binding ◽

Hiv 1

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Inhibition of the Androgen Receptor Amino-Terminal Domain by a Small Molecule as Treatment for Castrate-Resistant Prostate Cancer

10.21236/ada590496 ◽

2013 ◽

Author(s):

Stephen R. Plymate

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Small Molecule ◽

Amino Terminal ◽

Terminal Domain ◽

Castrate Resistant Prostate Cancer ◽

Castrate Resistant ◽

Amino Terminal Domain

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A Single Amino Acid in Human APOBEC3F Alters Susceptibility to HIV-1 Vif

Journal of Biological Chemistry ◽

10.1074/jbc.m110.173161 ◽

2010 ◽

Vol 285 (52) ◽

pp. 40785-40792 ◽

Cited By ~ 42

Author(s):

John S. Albin ◽

Rebecca S. LaRue ◽

Jessalyn A. Weaver ◽

William L. Brown ◽

Keisuke Shindo ◽

...

Keyword(s):

Amino Acid ◽

Single Amino Acid ◽

Hiv 1

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Nuclear Import of Adenovirus DNA Involves Direct Interaction of Hexon with an N-Terminal Domain of the Nucleoporin Nup214

Journal of Virology ◽

10.1128/jvi.02639-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1719-1730 ◽

Cited By ~ 37

Author(s):

Aurélia Cassany ◽

Jessica Ragues ◽

Tinglu Guan ◽

Dominique Bégu ◽

Harald Wodrich ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Nuclear Import ◽

Viral Genome ◽

Molecular Basis ◽

Nuclear Pore ◽

Viral Dna ◽

Terminal Domain ◽

Cytoplasmic Face ◽

The Impact

ABSTRACTIn this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Bothin vitrohexon binding andin vivonuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.IMPORTANCEAdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.

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Characterization of the amino-terminal domain of Mx2/MxB-dependent interaction with the HIV-1 capsid

Protein & Cell ◽

10.1007/s13238-014-0113-5 ◽

2014 ◽

Vol 5 (12) ◽

pp. 954-957 ◽

Cited By ~ 14

Author(s):

Jia Kong ◽

Bo Xu ◽

Wei Wei ◽

Xin Wang ◽

Wei Xie ◽

...

Keyword(s):

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain ◽

Hiv 1 ◽

Dependent Interaction

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High-Throughput Screening Assay for Identification of Small Molecule Inhibitors of Aurora2/STK15 Kinase

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104264071 ◽

2004 ◽

Vol 9 (5) ◽

pp. 391-397 ◽

Cited By ~ 12

Author(s):

Chongbo Sun ◽

Yvette Newbatt ◽

Leon Douglas ◽

Paul Workman ◽

Wynne Aherne ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Small Molecule Inhibitors ◽

Tumor Development ◽

Screening Assay ◽

Amino Terminal ◽

Terminal Domain ◽

High Throughput Screening Assay ◽

Amino Terminal Domain

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, γ-33P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate® to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z′ factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.

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