Structure of HOE/BAY 793 Complexed to Human Immunodeficiency Virus (HIV-1) Protease in Two Different Crystal Forms Structure/Function Relationship and Influence of Crystal Packing

ABSTRACT The reverse transcriptase (RT) of all retroviruses is required for synthesis of the viral DNA genome. The human immunodeficiency virus type 1 (HIV-1) RT exists as a heterodimer made up of 51-kDa and 66-kDa subunits. The crystal structure and in vitro biochemical analyses indicate that the p66 subunit of RT is primarily responsible for the enzyme's polymerase and RNase H activities. Since both the p51 and p66 subunits are generated from the same coding region, as part of the Pr160Gag-Pol precursor protein, there are inherent limitations for studying subunit-specific function with intact provirus in a virologically relevant context. Our lab has recently described a novel system for studying the RT heterodimer (p51/p66) wherein a LTR-vpr-p51-IRES-p66 expression cassette provided in trans to an RT-deleted HIV-1 genome allows precise molecular analysis of the RT heterodimer. In this report, we describe in detail the specific approaches, alternative strategies, and pitfalls that may affect the application of this novel assay for analyzing RT subunit structure/function in infectious virions and human target cells. The ability to study HIV-1 RT subunit structure/function in a physiologically relevant context will advance our understanding of both RT and the process of reverse transcription. The study of antiretroviral drugs in a subunit-specific virologic context should provide new insights into drug resistance and viral fitness. Finally, we anticipate that this approach will help elucidate determinants that mediate p51-p66 subunit interactions, which is essential for structure-based drug design targeting RT heterodimerization.

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2P055 Rational design of peptides that inhibit HIV-1 integrase activity by docking simulation(Proteins-structure and structure-function relationship,Poster Presentations)

Seibutsu Butsuri ◽

10.2142/biophys.47.s126_4 ◽

2007 ◽

Vol 47 (supplement) ◽

pp. S126

Author(s):

Hideyoshi Fuji ◽

Emiko Urano ◽

Hua Yan ◽

Toru Nakahara ◽

Hiroshi Tsutsumi ◽

...

Keyword(s):

Structure Function ◽

Rational Design ◽

Docking Simulation ◽

Structure Function Relationship ◽

Function Relationship ◽

Poster Presentations ◽

Hiv 1

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Mechanism of HIV-1 Resistance to Short-Peptide Fusion Inhibitors Targeting the Gp41 Pocket

Journal of Virology ◽

10.1128/jvi.00373-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 5801-5811 ◽

Cited By ~ 23

Author(s):

Yang Su ◽

Huihiui Chong ◽

Zonglin Qiu ◽

Shengwen Xiong ◽

Yuxian He

Keyword(s):

Structure Function ◽

Heptad Repeat ◽

Cross Resistance ◽

Short Peptide ◽

Fusion Inhibitors ◽

Structure Function Relationship ◽

Underlying Mechanisms ◽

Function Relationship ◽

Relationship Of ◽

Hiv 1

ABSTRACTThe deep hydrophobic pocket on the N trimer of HIV-1 gp41 has been considered an ideal drug target. On the basis of the M-T hook structure, we recently developed short-peptide-based HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the pocket site and possess highly potent antiviral activity. In this study, we focused on investigating their resistance pathways and mechanisms by escape HIV-1 mutants to SC22EK, a template peptide for MTSC22 and HP23. Two substitutions, E49K and N126K, located, respectively, at the N- and C-heptad repeat regions of gp41, were identified as conferring high resistance to the inhibitors targeting the pocket and cross-resistance to enfuvirtide (T20) and sifuvirtide (SFT). The underlying mechanisms of SC22EK-induced resistance include the following: (i) significantly reduced binding affinity of the inhibitors, (ii) dramatically enhanced interaction of the viral six-helix bundle, and (iii)severely damaged functionality of the viral Env complex. Our data have provided important information for the structure-function relationship of gp41 and the structure-activity relationship of viral fusion inhibitors.IMPORTANCEEnfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but the problem of resistance significantly limits its use, calling for new strategies or concepts to develop next-generation drugs. On the basis of the M-T hook structure, short-peptide HIV-1 fusion inhibitors specifically targeting the gp41 pocket site exhibit high binding and antiviral activities. Here, we investigated the molecular pathway of HIV-1 resistance to the short inhibitors by selecting and mapping the escape mutants. The key substitutions for resistance and the underlying mechanisms have been finely characterized. The data provide important information for the structure-function relationship of gp41 and its inhibitors and will definitely help our future development of novel drugs that block gp41-dependent fusion.

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A Structurally Disordered Region at the C Terminus of Capsid Plays Essential Roles in Multimerization and Membrane Binding of the Gag Protein of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.77.3.1772-1783.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 1772-1783 ◽

Cited By ~ 57

Author(s):

Chen Liang ◽

Jing Hu ◽

James B. Whitney ◽

Lawrence Kleiman ◽

Mark A. Wainberg

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Particle Production ◽

Gag Protein ◽

Crystal Forms ◽

Extracellular Virus ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Crystal structures of human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) reveal that the last 11 C-terminal amino acids are disordered. This disordered region contains a glycine-rich sequence 353-GVGGP-357 (numbering refers to the initiation methionine of Gag) that is highly conserved within the Gag proteins of HIV-1, HIV-2, and simian immunodeficiency virus, which suggests the importance of this sequence in virus replication. In the present study, we demonstrate that changing any individual residue within this short region in the context of the full-length HIV-1 genome virtually abolishes production of extracellular virus particles, in either the presence or absence of viral protease activity. This severe defect in virus particle production results from impaired Gag multimerization, as well as from decreased Gag association with the cellular membranes, as demonstrated by the results of gradient sedimentation and membrane flotation centrifugation assays. These findings are further supported by the diffuse distribution pattern of the mutant Gag within the cytoplasm, as opposed to the punctate distribution of the wild-type Gag on the plasma membrane. On the basis of these results, we propose that the disordered feature of amino acid stretch 353-GVGGP-357 in the CA crystal forms may have allowed Gag to adopt multiple conformations and that such structural flexibility is needed by Gag in order to construct geometrically complex particles.

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