Normalization strategy for the LC-MS bioanalysis of protein kinetics assays via internal proteolytic analyte utilized as control standard: application in studies of HIV-1 protease cleavage of HIV-1 Gag polyprotein in HIV maturation inhibition research

2017 ◽  
Vol 9 (35) ◽  
pp. 5219-5225
Author(s):  
Joseph L. Cantone ◽  
Zeyu Lin ◽  
Ira B. Dicker ◽  
Dieter M. Drexler
Keyword(s):  
Data Normalization ◽  
Protein Kinetics ◽  
Gag Polyprotein ◽  
Protease Cleavage ◽  
Normalization Strategy ◽  
Standard Application ◽  
Hiv 1

The LC-MS bioanalysis of protein kinetics assays is simplified by a data normalization strategy via internal proteolytic analyte utilized as a control standard.

scholarly journals Correction: Normalization strategy for the LC-MS bioanalysis of protein kinetics assays via internal proteolytic analyte utilized as control standard: application in studies of HIV-1 protease cleavage of HIV-1 Gag polyprotein in HIV maturation inhibition research

2017 ◽  
Vol 9 (37) ◽  
pp. 5557-5557
Author(s):  
Joseph L. Cantone ◽  
Zeyu Lin ◽  
Ira B. Dicker ◽  
Dieter M. Drexler
Keyword(s):  
Protein Kinetics ◽  
Gag Polyprotein ◽  
Protease Cleavage ◽  
Normalization Strategy ◽  
Standard Application ◽  
Hiv 1

Correction for ‘Normalization strategy for the LC-MS bioanalysis of protein kinetics assays via internal proteolytic analyte utilized as control standard: application in studies of HIV-1 protease cleavage of HIV-1 Gag polyprotein in HIV maturation inhibition research’ by Joseph L. Cantone et al., Anal. Methods, 2017, DOI: 10.1039/c7ay01666b.

scholarly journals Structure, Function, and Interactions of the HIV-1 Capsid Protein

Life ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 100
Author(s):  
Eric Rossi ◽  
Megan E. Meuser ◽  
Camille J. Cunanan ◽  
Simon Cocklin
Keyword(s):  
Life Cycle ◽  
Capsid Protein ◽  
Adaptor Proteins ◽  
Restriction Factors ◽  
Gag Polyprotein ◽  
Immunodeficiency Virus ◽  
Host Cell Factors ◽  
Hiv 1

The capsid (CA) protein of the human immunodeficiency virus type 1 (HIV-1) is an essential structural component of a virion and facilitates many crucial life cycle steps through interactions with host cell factors. Capsid shields the reverse transcription complex from restriction factors while it enables trafficking to the nucleus by hijacking various adaptor proteins, such as FEZ1 and BICD2. In addition, the capsid facilitates the import and localization of the viral complex in the nucleus through interaction with NUP153, NUP358, TNPO3, and CPSF-6. In the later stages of the HIV-1 life cycle, CA plays an essential role in the maturation step as a constituent of the Gag polyprotein. In the final phase of maturation, Gag is cleaved, and CA is released, allowing for the assembly of CA into a fullerene cone, known as the capsid core. The fullerene cone consists of ~250 CA hexamers and 12 CA pentamers and encloses the viral genome and other essential viral proteins for the next round of infection. As research continues to elucidate the role of CA in the HIV-1 life cycle and the importance of the capsid protein becomes more apparent, CA displays potential as a therapeutic target for the development of HIV-1 inhibitors.

scholarly journals Phenotypic Susceptibility to Bevirimat in Isolates from HIV-1-Infected Patients without Prior Exposure to Bevirimat

2010 ◽  
Vol 54 (6) ◽  
pp. 2345-2353 ◽  
Author(s):  
Nicolas A. Margot ◽  
Craig S. Gibbs ◽  
Michael D. Miller
Keyword(s):  
Recombinant Viruses ◽  
Gag Polyprotein ◽  
New Class ◽  
Major Mutation ◽  
Hiv Drugs ◽  
The Impact ◽  
First Time ◽  
Hiv 1

ABSTRACT Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

scholarly journals Immature HIV-1 Lattice Assembly Dynamics are Regulated by Scaffolding from Nucleic Acid and the Plasma Membrane

2017 ◽  
Author(s):  
Alexander J. Pak ◽  
John M. A. Grime ◽  
Prabuddha Sengupta ◽  
Antony K. Chen ◽  
Aleksander E. P. Durumeric ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Structural Data ◽  
Single Particle Tracking ◽  
Coarse Grained ◽  
Intermediate Step ◽  
Amino Terminal ◽  
Gag Polyprotein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTThe packaging and budding of Gag polyprotein and viral ribonucleic acid (RNA) is a critical step in the human immunodeficiency virus-1 (HIV-1) lifecycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from sub-nanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while over-expression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.SIGNIFICANCE STATEMENTIn order for human immunodeficiency virus to proliferate, viral proteins and genomic dimers are assembled at host cell membranes and released as immature virions. Disrupting this key intermediate step in viral replication is a potential target for treatment. However, a detailed molecular view of this process remains lacking. Here, we elucidate a network of constitutive interactions that regulate viral assembly dynamics through a combined computational and experimental approach. Specifically, our analysis reveals the active roles of nucleic acid and the membrane as scaffolds that promote the multimerization of Gag polyprotein which proceeds through multi-step and self-correcting nucleation. Our findings also illustrate the functional importance of the N-terminal, C-terminal, and spacer peptide 1 protein domains.

scholarly journals Thegagprecursor contains a specific HIV-1 protease cleavage site between the NC (P7) and P1 proteins

FEBS Letters ◽  
1993 ◽  
Vol 333 (1-2) ◽  
pp. 21-24 ◽  
Author(s):  
Ewald M. Wondrak ◽  
John M. Louis ◽  
Hugues de Rocquigny ◽  
Jean-Claude Chermann ◽  
Bernard P. Roques
Keyword(s):  
Cleavage Site ◽  
Protease Cleavage Site ◽  
Protease Cleavage ◽  
Hiv 1

scholarly journals Data Normalization Strategies for MicroRNA Quantification

2015 ◽  
Vol 61 (11) ◽  
pp. 1333-1342 ◽  
Author(s):  
Heidi Schwarzenbach ◽  
Andreia Machado da Silva ◽  
George Calin ◽  
Klaus Pantel
Keyword(s):  
Mirna Expression ◽  
Small Rnas ◽  
Standard Procedure ◽  
Review Article ◽  
Data Normalization ◽  
Expression Data ◽  
Quantitative Real Time Pcr ◽  
Normalization Strategy ◽  
Mirna Expression Data ◽  
Biological Interpretation

Abstract BACKGROUND Different technologies, such as quantitative real-time PCR or microarrays, have been developed to measure microRNA (miRNA) expression levels. Quantification of miRNA transcripts implicates data normalization using endogenous and exogenous reference genes for data correction. However, there is no consensus about an optimal normalization strategy. The choice of a reference gene remains problematic and can have a serious impact on the actual available transcript levels and, consequently, on the biological interpretation of data. CONTENT In this review article we discuss the reliability of the use of small RNAs, commonly reported in the literature as miRNA expression normalizers, and compare different strategies used for data normalization. SUMMARY A workflow strategy is proposed for normalization of miRNA expression data in an attempt to provide a basis for the establishment of a global standard procedure that will allow comparison across studies.

scholarly journals Development of a novel anti-HIV-1 agent from within: Effect of chimeric Vpr-containing protease cleavage site residues on virus replication

1997 ◽  
Vol 94 (7) ◽  
pp. 3346-3351 ◽  
Author(s):  
D. Serio ◽  
T. A. Rizvi ◽  
M. Cartas ◽  
V. S. Kalyanaraman ◽  
I. T. Weber ◽  
...  
Keyword(s):  
Virus Replication ◽  
Cleavage Site ◽  
Protease Cleavage Site ◽  
Protease Cleavage ◽  
Anti Hiv ◽  
Hiv 1

scholarly journals Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

Retrovirology ◽  
2011 ◽  
Vol 8 (1) ◽  
pp. 30 ◽  
Author(s):  
Maja George ◽  
Torsten Schwecke ◽  
Nadine Beimforde ◽  
Oliver Hohn ◽  
Claudia Chudak ◽  
...  
Keyword(s):  
Cleavage Sites ◽  
Gag Polyprotein ◽  
Protease Cleavage ◽  
Protease Cleavage Sites
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