HIV-1 CA inhibitors are antagonized by inositol phosphate stabilization of the viral capsid in cells

Journal of Virology ◽

10.1128/jvi.01445-21 ◽

2021 ◽

Author(s):

Gregory A Sowd ◽

Jiong Shi ◽

Christopher Aiken

Keyword(s):

Antiretroviral Therapy ◽

Antiviral Activity ◽

Binding Site ◽

Target Cells ◽

Nuclear Entry ◽

Inositol Hexakisphosphate ◽

Capsid Stability ◽

Hiv 1 ◽

In Cells

The HIV-1 capsid, composed of the CA protein, is the target of the novel antiretroviral drug lenacapavir (LCV). CA inhibitors block host factor binding and alter capsid stability to prevent nuclear entry and reverse transcription (RTN), respectively. Capsid stability is mediated in vitro by binding to the host cell metabolite inositol hexakisphosphate (IP6). IP6 depletion in target cells has little effect on HIV-1 infection. We hypothesized that capsid-altering concentrations of CA inhibitors might reveal an effect of IP6 depletion on HIV-1 infection in target cells. To test this, we studied the effects of IP6 depletion on inhibition of infection by the CA inhibitors PF74 and LCV. At low doses of either compound that affect HIV-1 nuclear entry, no effect of IP6 depletion on antiviral activity was observed. Increased antiviral activity was observed in IP6-depleted cells at inhibitor concentrations that affect capsid stability, correlating with increased RTN inhibition. Assays of uncoating and endogenous RTN of purified cores in vitro provided additional support. Our results show that inositol phosphates stabilize the HIV-1 capsid in target cells, thereby dampening the antiviral effects of capsid-targeting antiviral compounds. We propose that targeting of the IP6-binding site in conjunction with CA inhibitors will lead to robust antiretroviral therapy. Importance HIV-1 infection and subsequent depletion of CD4 + T cells results in AIDS. Antiretroviral therapy (ART) treatment of infected individuals prevents progression to AIDS. The HIV-1 capsid has recently become an ART target. Capsid inhibitors block HIV-1 infection at multiple steps, offering advantages over current ART. The cellular metabolite inositol hexakisphosphate (IP6) binds the HIV-1 capsid, stabilizing it in vitro . However, the function of this interaction in target cells is unclear. Our results imply that IP6 stabilizes the incoming HIV-1 capsid in cells, thus limiting the antiviral efficiency of capsid-destabilizing antivirals. We present a model of capsid inhibitor function and propose that targeting of the IP6-binding site in conjunction with capsid inhibitors currently in development will lead to more robust ART.

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The Structure-Activity Relationships of 2,4(1H,3H)-pyrimidinedione Derivatives as potent HIV type 1 and type 2 inhibitors

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020701800502 ◽

2007 ◽

Vol 18 (5) ◽

pp. 259-275 ◽

Cited By ~ 30

Author(s):

Robert W Buckheit ◽

Tracy L Hartman ◽

Karen M Watson ◽

Ho Seok Kwon ◽

Sun Hwan Lee ◽

...

Keyword(s):

Antiviral Activity ◽

Binding Pocket ◽

Target Cells ◽

Significant Activity ◽

In Vitro Test ◽

Structure Activity Relationships ◽

Structure Activity ◽

Test Concentration ◽

Hiv 1

Since the discovery of the 2,4 (1 H,3 H)-pyrimidinediones as potent non-nucleoside inhibitors of the HIV-1 reverse transcriptase (RT) this class of compounds has yielded a number of N-1 acyclic substituted pyrimidinediones with substantial antiviral activity, which is highly dependent upon their molecular fit into the binding pocket common to this inhibitory class. We have specifically examined the structure activity relationships of compounds with chemical modification made by substituting homocyclic rather than acyclic moieties at N-1 of the pyrimidinedione. Seventy-four compounds were synthesized and evaluated for antiviral activity against HIV-1 and HIV-2. The homocyclic modifications resulted in compounds with significant activity against both HIV-1 and HIV-2, suggesting these compounds represent a new class of non-nucleoside RT inhibitors. The structure-activity relationship (SAR) evaluations indicated that cyclopropyl, phenyl and 1- or 3-cyclopenten-1-yl substitutions at the N-1 of the pyrimidinedione, the addition of a methyl linker between the cyclic moiety and the N-1 and the addition of a benzoyl group at the C-6 of the pyrimidinedione had the greatest contribution to antiviral activity. Five pyrimidinedione analogues with therapeutic indexes (TIs)>450,000 and a specific analogue (1-cyclopropylmethyl-5-isopropyl-6-(3,5-dimethylbenzoyl)-2,4(1 H,3 H)-pyrimidinedione), which exhibited a TI of >2,000,000, were identified. None of the analogues were cytotoxic to target cells at the highest in vitro test concentration, which is the upper limit of compound solubility of the analogues in aqueous solution. Thus, we have identified a series of pyrimidinediones with substantially improved antiviral efficacy and range of action and with significantly reduced cellular cytotoxicity.

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Bicaudal D2 facilitates the cytoplasmic trafficking and nuclear import of HIV-1 genomes during infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712033114 ◽

2017 ◽

Vol 114 (50) ◽

pp. E10707-E10716 ◽

Cited By ~ 35

Author(s):

Adarsh Dharan ◽

Silvana Opp ◽

Omar Abdel-Rahim ◽

Sevnur Komurlu Keceli ◽

Sabrina Imam ◽

...

Keyword(s):

Viral Genome ◽

Target Cells ◽

Adapter Protein ◽

Nuclear Entry ◽

Microtubule Network ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Microtubule Motor ◽

Hiv 1

Numerous viruses, including HIV-1, exploit the microtubule network to traffic toward the nucleus during infection. Although numerous studies have observed a role for the minus-end microtubule motor dynein in HIV-1 infection, the mechanism by which the viral core containing the viral genome associates with dynein and induces its perinuclear trafficking has remained unclear. Here, we report that the dynein adapter protein bicaudal D2 (BICD2) is able to interact with HIV-1 viral cores in target cells. We also observe that BICD2 can bind in vitro-assembled capsid tubes through its CC3 domain. We observe that BICD2 facilitates infection by promoting the trafficking of viral cores to the nucleus, thereby promoting nuclear entry of the viral genome and infection. Finally, we observe that depletion of BICD2 in the monocytic cell line THP-1 results in an induction of IFN-stimulated genes in these cells. Collectively, these results identify a microtubule adapter protein critical for trafficking of HIV-1 in the cytoplasm of target cells and evasion of innate sensing mechanisms in macrophages.

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Dominant Negative MA-CA Fusion Protein Is Incorporated into HIV-1 Cores and Inhibits Nuclear Entry of Viral Preintegration Complexes

Journal of Virology ◽

10.1128/jvi.01118-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 2

Author(s):

Jordan Anderson-Daniels ◽

Parmit K. Singh ◽

Gregory A. Sowd ◽

Wen Li ◽

Alan N. Engelman ◽

...

Keyword(s):

Antiviral Activity ◽

Mutational Analysis ◽

Membrane Binding ◽

Dominant Negative ◽

Target Cells ◽

Virus Infectivity ◽

Nuclear Entry ◽

Gag Polyprotein ◽

Hiv 1 ◽

Ca Protein

ABSTRACT Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. The accurate and efficient cleavage of Gag is essential for virion infectivity; inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by ∼95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectably affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions. IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein results in a strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that the membrane-binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.

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Alkoxy propane prodrugs of foscarnet: effect of alkyl chain length on in vitro antiviral activity in cells infected with HIV-1, HSV-1 and HCMV

Antiviral Research ◽

10.1016/s0166-3542(97)00039-9 ◽

1997 ◽

Vol 36 (1) ◽

pp. 43-53 ◽

Cited By ~ 19

Author(s):

Ganesh D Kini ◽

James R Beadle ◽

Hong Xie ◽

Kathy A Aldern ◽

Douglas D Richman ◽

...

Keyword(s):

Antiviral Activity ◽

Chain Length ◽

Alkyl Chain ◽

Alkyl Chain Length ◽

Hiv 1 ◽

In Cells ◽

Hsv 1

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1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.5.1082 ◽

1997 ◽

Vol 41 (5) ◽

pp. 1082-1093 ◽

Cited By ~ 276

Author(s):

S M Daluge ◽

S S Good ◽

M B Faletto ◽

W H Miller ◽

M H St Clair ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Oral Bioavailability ◽

Human Peripheral Blood ◽

Cross Resistance ◽

Immunodeficiency Virus ◽

Carbocyclic Nucleoside ◽

Hiv 1 ◽

In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

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HIV-1 subverts the complement system in semen to enhance viral transmission

Mucosal Immunology ◽

10.1038/s41385-021-00376-9 ◽

2021 ◽

Author(s):

Bernadien M. Nijmeijer ◽

Marta Bermejo-Jambrina ◽

Tanja M. Kaptein ◽

Carla M. S. Ribeiro ◽

Doris Wilflingseder ◽

...

Keyword(s):

Ex Vivo ◽

Virus Transmission ◽

Sexual Contact ◽

Target Cells ◽

People Living With Hiv ◽

Lectin Receptor ◽

Pre Treatment ◽

Living With Hiv ◽

Hiv 1

AbstractSemen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

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In Vitro Antiviral Activity and Cross-Resistance Profile of PL-100, a Novel Protease Inhibitor of Human Immunodeficiency Virus Type 1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00149-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 4036-4043 ◽

Cited By ~ 21

Author(s):

Serge Dandache ◽

Guy Sévigny ◽

Jocelyn Yelle ◽

Brent R. Stranix ◽

Neil Parkin ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Activity ◽

Highly Active Antiretroviral Therapy ◽

Cross Resistance ◽

Drug Resistant ◽

Resistance Profile ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Despite the success of highly active antiretroviral therapy, the current emergence and spread of drug-resistant variants of human immunodeficiency virus (HIV) stress the need for new inhibitors with distinct properties. We designed, produced, and screened a library of compounds based on an original l-lysine scaffold for their potentials as HIV type 1 (HIV-1) protease inhibitors (PI). One candidate compound, PL-100, emerged as a specific and noncytotoxic PI that exhibited potent inhibition of HIV-1 protease and viral replication in vitro (Ki , ∼36 pM, and 50% effective concentration [EC50], ∼16 nM, respectively). To confirm that PL-100 possessed a favorable resistance profile, we performed a cross-resistance study using a panel of 63 viral strains from PI-experienced patients selected for the presence of primary PI mutations known to confer resistance to multiple PIs now in clinical use. The results showed that PL-100 retained excellent antiviral activity against almost all of these PI-resistant viruses and that its performance in this regard was superior to those of atazanavir, amprenavir, indinavir, lopinavir, nelfinavir, and saquinavir. In almost every case, the increase in the EC50 for PL-100 observed with viruses containing multiple mutations in protease was far less than that obtained with the other drugs tested. These data underscore the potential for PL-100 to be used in the treatment of drug-resistant HIV disease and argue for its further development.

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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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Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic-Site Integrase Inhibitor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02719-13 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3233-3244 ◽

Cited By ~ 47

Author(s):

Craig Fenwick ◽

Ma'an Amad ◽

Murray D. Bailey ◽

Richard Bethell ◽

Michael Bös ◽

...

Keyword(s):

Antiviral Activity ◽

Phase 1 ◽

Integrase Inhibitor ◽

Parallel Synthesis ◽

Cell Permeability ◽

Strand Transfer ◽

Primary Resistance ◽

Catalytic Core ◽

Hiv 1

ABSTRACTBI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3′-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-likein vitroabsorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%;F, 82%), and dog (CL, 8%;F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

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