Discovery of dual inhibitors targeting both HIV-1 capsid and human cyclophilin A to inhibit the assembly and uncoating of the viral capsid

ABSTRACTThe viral capsid of HIV-1 interacts with a number of host factors to orchestrate uncoating and regulate downstream events, such as reverse transcription, nuclear entry, and integration site targeting. PF-3450074 (PF74), an HIV-1 capsid-targeting low-molecular-weight antiviral compound, directly binds to the capsid (CA) protein at a site also utilized by host cell proteins CPSF6 and NUP153. Here, we found that the dose-response curve of PF74 is triphasic, consisting of a plateau and two inhibitory phases of different slope values, consistent with a bimodal mechanism of drug action. High PF74 concentrations yielded a steep curve with the highest slope value among different classes of known antiretrovirals, suggesting a dose-dependent, cooperative mechanism of action. CA interactions with both CPSF6 and cyclophilin A (CypA) were essential for the unique dose-response curve. A shift of the steep curve at lower drug concentrations upon blocking the CA-CypA interaction suggests a protective role for CypA against high concentrations of PF74. These findings, highlighting the unique characteristics of PF74, provide a model in which its multimodal mechanism of action of both noncooperative and cooperative inhibition by PF74 is regulated by interactions of cellular proteins with incoming viral capsids.IMPORTANCEPF74, a novel capsid-targeting antiviral against HIV-1, shares its binding site in the viral capsid protein (CA) with the host factors CPSF6 and NUP153. This work reveals that the dose-response curve of PF74 consists of two distinct inhibitory phases that are differentially regulated by CA-interacting host proteins. PF74's potency depended on these CA-binding factors at low doses. In contrast, the antiviral activity of high PF74 concentrations was attenuated by cyclophilin A. These observations provide novel insights into both the mechanism of action of PF74 and the roles of host factors during the early steps of HIV-1 infection.

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Cyclophilin A potentiates TRIM5α inhibition of HIV-1 nuclear import without promoting TRIM5α binding to the viral capsid

PLoS ONE ◽

10.1371/journal.pone.0182298 ◽

2017 ◽

Vol 12 (8) ◽

pp. e0182298 ◽

Cited By ~ 6

Author(s):

Mallori Burse ◽

Jiong Shi ◽

Christopher Aiken

Keyword(s):

Nuclear Import ◽

Cyclophilin A ◽

Viral Capsid ◽

Hiv 1

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Structure-Activity Relationships (SAR) Research of Thiourea Derivatives as Dual Inhibitors Targeting both HIV-1 Capsid and Human Cyclophilin A

Chemical Biology & Drug Design ◽

10.1111/j.1747-0285.2010.00981.x ◽

2010 ◽

Vol 76 (1) ◽

pp. 25-33 ◽

Cited By ~ 9

Author(s):

Kan Chen ◽

Zhiwu Tan ◽

Meizi He ◽

Jiebo Li ◽

Shixing Tang ◽

...

Keyword(s):

Cyclophilin A ◽

Thiourea Derivatives ◽

Structure Activity Relationships ◽

Structure Activity ◽

Dual Inhibitors ◽

Hiv 1

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The Cysteine Residues of HIV-1 Capsid Regulate Oligomerization and Cyclophilin A-Induced Changes

Biophysical Journal ◽

10.1529/biophysj.104.053298 ◽

2005 ◽

Vol 88 (3) ◽

pp. 2078-2088 ◽

Cited By ~ 13

Author(s):

Marjorie Bon Homme ◽

Carol Carter ◽

Suzanne Scarlata

Keyword(s):

Cyclophilin A ◽

Cysteine Residues ◽

Induced Changes ◽

Hiv 1

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Designed antiviral ankyrin – A computational approach to combat HIV-1 via intracellular pathway by targeting the viral capsid of HIV-1

Journal of Molecular Liquids ◽

10.1016/j.molliq.2018.12.030 ◽

2019 ◽

Vol 277 ◽

pp. 63-69 ◽

Cited By ~ 1

Author(s):

Hana Atiqah Abdul Karim ◽

Thanyada Rungrotmongkol ◽

Sharifuddin M. Zain ◽

Noorsaadah Abd Rahman ◽

Chatchai Tayapiwattana ◽

...

Keyword(s):

Computational Approach ◽

Viral Capsid ◽

Intracellular Pathway ◽

Hiv 1

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Host cyclophilin A mediates HIV-1 attachment to target cells via heparans

The EMBO Journal ◽

10.1093/emboj/18.23.6771 ◽

1999 ◽

Vol 18 (23) ◽

pp. 6771-6785 ◽

Cited By ~ 123

Author(s):

A. C.S. Saphire

Keyword(s):

Cyclophilin A ◽

Target Cells ◽

Hiv 1

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MxB sensitivity of HIV-1 is determined by a highly variable and dynamic capsid surface

eLife ◽

10.7554/elife.56910 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Richard J Miles ◽

Claire Kerridge ◽

Laura Hilditch ◽

Christopher Monit ◽

David A Jacques ◽

...

Keyword(s):

Nuclear Import ◽

Cytotoxic T Lymphocyte ◽

Cyclophilin A ◽

Conformational Flexibility ◽

Restriction Factors ◽

Nuclear Entry ◽

Linear Forms ◽

Cofactor Binding ◽

Cofactor Recruitment ◽

Hiv 1

The type one interferon induced restriction factor Myxovirus resistance B (MxB) restricts HIV-1 nuclear entry evidenced by inhibition of 2-LTR but not linear forms of viral DNA. The HIV-1 capsid is the key determinant of MxB sensitivity and cofactor binding defective HIV-1 capsid mutants P90A (defective for cyclophilin A and Nup358 recruitment) and N74D (defective for CPSF6 recruitment) have reduced dependency on nuclear transport associated cofactors, altered integration targeting preferences and are not restricted by MxB expression. This has suggested that nuclear import mechanism may determine MxB sensitivity. Here we have use genetics to separate HIV-1 nuclear import cofactor dependence from MxB sensitivity. We provide evidence that MxB sensitivity depends on HIV-1 capsid conformation, rather than cofactor recruitment. We show that depleting CPSF6 to change nuclear import pathway does not impact MxB sensitivity, but mutants that recapitulate the effect of Cyclophilin A binding on capsid conformation and dynamics strongly impact MxB sensitivity. We demonstrate that HIV-1 primary isolates have different MxB sensitivities due to cytotoxic T lymphocyte (CTL) selected differences in Gag sequence but similar cofactor dependencies. Overall our work demonstrates a complex relationship between cyclophilin dependence and MxB sensitivity likely driven by CTL escape. We propose that cyclophilin binding provides conformational flexibility to HIV-1 capsid facilitating simultaneous evasion of capsid-targeting restriction factors including TRIM5 as well as MxB.

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HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription

10.1101/2021.01.16.426924 ◽

2021 ◽

Author(s):

Sanela Rankovic ◽

Akshay Deshpande ◽

Shimon Harel ◽

Christopher Aiken ◽

Itay Rousso

Keyword(s):

Reverse Transcription ◽

Viral Genome ◽

Morphological Changes ◽

Viral Capsid ◽

Target Cells ◽

Viral Core ◽

Successful Infection ◽

Capsid Shell ◽

Hiv 1

AbstractThe HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. Our results suggest that reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.

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