Structural investigation of the HIV-1 reverse transcriptase initiation complex by HDX, SAXS, CryoEM and X-ray crystallography

ABSTRACT The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC50] = 2.6 μM) and RNH functions (IC50 = 0.65 μM) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.

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Neutralizing antibodies induced by first-generation gp41-stabilized HIV-1 envelope trimers and nanoparticles

10.1101/2020.12.02.408328 ◽

2020 ◽

Author(s):

Sonu Kumar ◽

Xiaohe Lin ◽

Timothy Ngo ◽

Benjamin Shapero ◽

Cindy Sou ◽

...

Keyword(s):

Cell Sorting ◽

Neutralizing Antibodies ◽

First Generation ◽

Heptad Repeat ◽

X Ray ◽

X Ray Crystallography ◽

Clade C ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Hiv 1

ABSTRACTAntigen-specific B-cell sorting and next-generation sequencing (NGS) were combined to isolate HIV-1 neutralizing antibodies (NAbs) from mice and rabbits immunized with BG505 trimers and nanoparticles. Three mouse NAbs potently neutralize BG505.T332N and recognize a glycan epitope centered at the C3/V4 region, as revealed by electron microscopy (EM), x-ray crystallography, and epitope mapping. Three potent NAbs were sorted from rabbit B cells that target glycan holes on the BG505 envelope glycoprotein (Env) and account for a significant portion of autologous NAb response. We then determined a 3.4Å-resolution crystal structure for the clade C transmitted/founder Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend. This clade C Env, as a soluble trimer and attached to a ferritin nanoparticle, along with a clade A Q482-d12 Env trimer, elicited distinct NAb responses in rabbits. Our study demonstrates that nanoparticles presenting gp41-stabilized trimers can induce potent NAb responses in mice and rabbits with Env-dependent breadth.TEASERMouse and rabbit NAbs elicited by gp41-stabilized trimers and nanoparticles neutralize autologous HIV-1 by targeting different epitopes

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The X-ray Crystal Structure of the Sulfonated Azo Dye Congo Red, a Non-Peptidic Inhibitor of HIV-1 Protease which also Binds to Reverse Transcriptase and Amyloid Proteins

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029500600104 ◽

1995 ◽

Vol 6 (1) ◽

pp. 25-33 ◽

Cited By ~ 28

Author(s):

W. H. Ojala ◽

C. R. Ojala ◽

W. B. Gleason

Keyword(s):

Crystal Structure ◽

Reverse Transcriptase ◽

Congo Red ◽

Azo Dye ◽

Amyloid Proteins ◽

X Ray ◽

Sulfonated Azo Dye ◽

Hiv 1

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Detecting Allosteric Sites of HIV-1 Reverse Transcriptase by X-ray Crystallographic Fragment Screening

Journal of Medicinal Chemistry ◽

10.1021/jm301271j ◽

2013 ◽

Vol 56 (7) ◽

pp. 2738-2746 ◽

Cited By ~ 54

Author(s):

Joseph D. Bauman ◽

Disha Patel ◽

Chhaya Dharia ◽

Marc W. Fromer ◽

Sameer Ahmed ◽

...

Keyword(s):

Reverse Transcriptase ◽

X Ray ◽

Fragment Screening ◽

Allosteric Sites ◽

Hiv 1

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Structural and mechanistic bases for a potent HIV-1 capsid inhibitor

Science ◽

10.1126/science.abb4808 ◽

2020 ◽

Vol 370 (6514) ◽

pp. 360-364 ◽

Cited By ~ 10

Author(s):

Stephanie M. Bester ◽

Guochao Wei ◽

Haiyan Zhao ◽

Daniel Adu-Ampratwum ◽

Naseer Iqbal ◽

...

Keyword(s):

Principal Component ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Capsid Shell ◽

Hydrogen Deuterium ◽

Infected Cells ◽

Long Acting ◽

Structural Insights ◽

Hiv 1

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo–electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

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Structural investigation of HIV-1 nonnucleoside reverse transcriptase inhibitors: 2-Aryl-substituted benzimidazoles

Journal of Molecular Structure ◽

10.1016/j.molstruc.2009.08.005 ◽

2009 ◽

Vol 937 (1-3) ◽

pp. 34-38 ◽

Cited By ~ 3

Author(s):

Natasza E. Ziółkowska ◽

Christopher J. Michejda ◽

Grzegorz D. Bujacz

Keyword(s):

Reverse Transcriptase ◽

Structural Investigation ◽

Reverse Transcriptase Inhibitors ◽

Nonnucleoside Reverse Transcriptase Inhibitors ◽

Hiv 1

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HIV-1 Env structure and vaccine design

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314098829 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C117-C117

Author(s):

Peter Kwong

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Bound States ◽

Vaccine Design ◽

Atomic Level ◽

Alpha Helices ◽

X Ray ◽

X Ray Crystallography ◽

Gp120 Subunit ◽

Hiv 1

Roughly one third of the HIV-1 genome is devoted to the HIV-1 envelope (Env) glycoprotein spike, which comprises three gp120 and three gp41 subunits. Structural characterization of the HIV-1 Env by electron microscopy, NMR, and X-ray crystallography reveals considerable conformational alterations, not only between trimeric ground state, CD4 receptor-bound conformation, and postfusion conformation of the spike, but also between monomeric and trimeric configurations of the subunits as well as between free- and antibody–bound states. One important structure, however, that of the prefusion HIV-1 spike, has resisted atomic level determination. This structure has been on the 10 list of most wanted structure for more than 20 years, because it is the target of the majority of broad HIV-1-neutralizing antibodies – and therefore of importance to vaccine design. In late 2013, the structure of a prefusion HIV-1 spike, based on a BG505 SOSIP.R6.664 construct, was reported by both X-ray crystallography (4.7 Å) and electron microscopy (5.8 Å). While these structures described the trimeric configuration of most of the HIV-1 gp120 subunit, the description of the gp41 subunit was limited to two helical regions comprising only about half the gp41 ectodomain, and the sequence register for the alpha helices was not reported. Recently, we were able to obtain x-ray diffraction data to 3.5 Å resolution on a prefusion crystal structure of the entire HIV-1 spike. The structure utilizes the same BG505 SOSIP.R6.664 construct as previously published, but crystallized in space group P6(3) with the antigen-binding fragments (Fab) of two antibodies, PGT122 and 35O22. The new structure provides atomic-level details for the complete prefusion structure of gp120 and the majority of the trimeric ectodomain of gp41 (up to residue 664). Also visualized are details of the gp120-gp41 interface and of antibodies such as 35O22. In addition to the complete HIV-1 Env ectodomain structure, implications for HIV-1 vaccine design will be described.

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Structural Investigation of Pyribenzoxim Using X-ray Crystallography

Bulletin of the Korean Chemical Society ◽

10.5012/bkcs.2002.23.7.1042 ◽

2002 ◽

Vol 23 (7) ◽

pp. 1042-1044

Keyword(s):

Structural Investigation ◽

X Ray ◽

X Ray Crystallography

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Synthesis, biophysical properties, and RNase H activity of 6’-difluoro[4.3.0]bicyclo-DNA

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.15.9 ◽

2019 ◽

Vol 15 ◽

pp. 79-88 ◽

Cited By ~ 1

Author(s):

Sibylle Frei ◽

Adam K Katolik ◽

Christian J Leumann

Keyword(s):

Building Block ◽

Ring Opening ◽

Structural Investigation ◽

Cyclopropane Ring ◽

Cd Spectroscopy ◽

Rnase H ◽

Biophysical Properties ◽

X Ray ◽

X Ray Crystallography ◽

Cleavage Assay

Here we present the synthesis, the biophysical properties, and the RNase H profile of 6’-difluorinated [4.3.0]bicyclo-DNA (6’-diF-bc4,3-DNA). The difluorinated thymidine phosphoramidite building block was synthesized starting from an already known gem-difluorinated tricyclic glycal. This tricyclic siloxydifluorocyclopropane was converted into the [4.3.0]bicyclic nucleoside via cyclopropane ring-opening through the addition of an electrophilic iodine during the nucleosidation step followed by reduction. The gem-difluorinated bicyclic nucleoside was then converted into the corresponding phosphoramidite building block which was incorporated into oligonucleotides. Thermal denaturation experiments of these oligonucleotides hybridized to complementary DNA or RNA disclosed a significant destabilization of both duplex types (ΔT m/mod = −1.6 to −5.5 °C). However, in the DNA/RNA hybrid the amount of destabilization could be reduced by multiple insertions of the modified unit. In addition, CD spectroscopy of the oligonucleotides hybridized to RNA showed a similar structure than the natural DNA/RNA duplex. Furthermore, since the structural investigation on the nucleoside level by X-ray crystallography and ab initio calculations pointed to a furanose conformation in the southern region, a RNase H cleavage assay was conducted. This experiment revealed that the oligonucleotide containing five modified units was able to elicit the RNase H-mediated cleavage of the complementary RNA strand.

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Post-Catalytic Complexes with Emtricitabine or Stavudine and HIV-1 Reverse Transcriptase Reveal New Mechanistic Insights for Nucleotide Incorporation and Drug Resistance

Molecules ◽

10.3390/molecules25204868 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4868

Author(s):

Nicole Bertoletti ◽

Albert H. Chan ◽

Raymond F. Schinazi ◽

Karen S. Anderson

Keyword(s):

Crystal Structure ◽

Reverse Transcriptase ◽

Highly Active Antiretroviral Therapy ◽

Molecular Mechanisms ◽

X Ray Crystallography ◽

Highly Active ◽

Nucleotide Incorporation ◽

Almost All ◽

Immunodeficiency Syndrome ◽

Hiv 1

Human immunodeficiency virus 1 (HIV-1) infection is a global health issue since neither a cure nor a vaccine is available. However, the highly active antiretroviral therapy (HAART) has improved the life expectancy for patients with acquired immunodeficiency syndrome (AIDS). Nucleoside reverse transcriptase inhibitors (NRTIs) are in almost all HAART and target reverse transcriptase (RT), an essential enzyme for the virus. Even though NRTIs are highly effective, they have limitations caused by RT resistance. The main mechanisms of RT resistance to NRTIs are discrimination and excision. Understanding the molecular mechanisms for discrimination and excision are essential to develop more potent and selective NRTIs. Using protein X-ray crystallography, we determined the first crystal structure of RT in its post-catalytic state in complex with emtricitabine, (-)FTC or stavudine (d4T). Our structural studies provide the framework for understanding how RT discriminates between NRTIs and natural nucleotides, and for understanding the requirement of (-)FTC to undergo a conformation change for successful incorporation by RT. The crystal structure of RT in post-catalytic complex with d4T provides a “snapshot” for considering the possible mechanism of how RT develops resistance for d4T via excision. The findings reported herein will contribute to the development of next generation NRTIs.

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