Springer protocols feed by Immunology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Measuring the Frequency of Latent HIV-1 in Resting CD4+ T Cells Using a Limiting Dilution Coculture Assay
Combination antiretroviral therapy (cART) can reduce HIV-1 viremia to clinically undetectable levels. However, replication competent virus persists in a long-lived latent reservoir in resting, memory CD4+ T cells. The latent reservoir in resting CD4+ T cells is the major barrier to curing HIV-1 infection. The recent case of the Berlin patient has suggested that it may be possible to cure HIV-1 infection in certain situations. As efforts to cure HIV-1 infection progress, it will become critical to measure the latent reservoir in patients participating in clinical trials of eradication strategies. Our laboratory has develope...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
High-Throughput Humanized Mouse Models for Evaluation of HIV-1 Therapeutics and Pathogenesis
Mice cannot be used as a model to evaluate HIV-1 therapeutics because they do not become infected by HIV-1 due to structural differences between several human and mouse proteins required for HIV-1 replication. This has limited their use for in vivo assessment of anti-HIV-1 therapeutics and the mechanism by which cofactors, such as illicit drug use accelerate HIV-1 replication and disease course in substance abusers. Here, we describe the development and application of two in vivo humanized mouse models that are highly sensitive and useful models for the in vivo evaluation of candidate anti-HIV therapeutics. The first model...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Modeling HIV-1 Mucosal Transmission and Prevention in Humanized Mice
The new generation humanized mice (hu-mice) that permit continuous de novo generation of human hematopoietic cells have led to novel strategies in studying HIV-1 pathogenesis, prevention and therapies. HIV-1 infection of hu-mice results in chronic viremia and CD4+ T cell loss, thus mimicking key aspects of the disease progression. In addition, the new generation hu-mice are permissive for HIV-1 sexual transmission by vaginal and rectal routes thus allowing in vivo efficacy testing of new anti-HIV-1 drugs for prevention. Two leading models are currently being used, namely the hu-HSC mice and the BLT mice. Here we describe t...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Visualizing the Behavior of HIV-Infected T Cells In Vivo Using Multiphoton Intravital Microscopy
The introduction of multiphoton microscopy has dramatically broadened the scope of intravital imaging studies and has allowed researchers to validate and refine basic mechanistic concepts in many areas of biology within the context of physiologically relevant tissue microenvironments. This has also led to new insights into the behavior of immune cells at steady state, and how their behaviors are altered during an immune response. At the same time, advances in the humanized mouse model have allowed for in vivo studies of strictly human pathogens, such as HIV-1. Here, we describe in detail an intravital microscopy approach t...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Methods to Study Determinants for Membrane Targeting of HIV-1 Gag In Vitro
Assembly of HIV-1 viral particles is a critical step of the HIV-1 life cycle; yet many details of this complex process are unknown. The Gag polyprotein drives viral particle assembly at the plasma membrane via three different types of interactions: protein-protein, protein-RNA, and protein-membrane interactions. As an approach to tease apart the importance of these interactions during viral particle assembly, in particular at the step of Gag membrane binding, we have developed an in vitro liposome-binding assay. Below we describe how to prepare liposomes, which serve as model membranes, and how to assess their interaction ...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Quantification of HIV-1 Gag Localization Within Virus Producer Cells
Trafficking of newly synthesized Gag protein to the plasma membrane is one of the important steps during HIV-1 assembly. It requires participation of both viral and cellular determinants. Several techniques have been used to measure the amount of Gag that is associated with plasma membrane. Here we describe a microscopy-based method to estimate the distribution of Gag protein within the producer cell. This method can be used in conjunction with other biochemical techniques to quantify the distribution of Gag within a virus-producing cell and its accumulation at the plasma membrane. Since this method is microscopy based, it...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase
HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in in...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations
All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sephar...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Analysis of HIV-1 Gag-RNA Interactions in Cells and Virions by CLIP-seq
Next-generation sequencing-based methodologies have revolutionized the analysis of protein-nucleic acid complexes; yet these novel approaches have rarely been applied in virology. Because it has an RNA genome, RNA-protein interactions play critical roles in human immunodeficiency virus type 1 (HIV-1) replication. In many cases, the binding sites of proteins on HIV-1 RNA molecules in physiologically relevant settings are not known. Cross-linking-immunoprecipitation sequencing (CLIP-seq) methodologies, which combine immunoprecipitation of covalently crosslinked protein-RNA complexes with high-throughput sequencing, is a powe...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Novel Biochemical Tools for Probing HIV RNA Structure
Functional analysis of viral RNA requires knowledge of secondary structure arrangements and tertiary base interactions. Thus, high-throughput and comprehensive methods for assessing RNA structure are highly desirable. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has proven highly useful for modeling the secondary structures of HIV and other retroviral RNAs in recent years. This technology is not without its limitations however, as SHAPE data can be severely compromised when the RNA under study is structurally heterogeneous. In addition, the method reveals little information regarding the three...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Rapid Determination of HIV-1 Mutant Frequencies and Mutation Spectra Using an mCherry/EGFP Dual-Reporter Viral Vector
The high mutation rate of human immunodeficiency virus type-1 (HIV-1) has been a pivotal factor in its evolutionary success as a human pathogen, driving the emergence of drug resistance, immune system escape, and invasion of distinct anatomical compartments. Extensive research has focused on understanding how various cellular and viral factors alter the rates and types of mutations produced during viral replication. Here, we describe a single-cycle dual-reporter vector assay that relies upon the detection of mutations that eliminate either expression of mCherry or enhanced green fluorescent protein (EGFP). The reporter-bas...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
HIV-1 Reverse Transcriptase-Based Assay to Determine Cellular dNTP Concentrations
Deoxynucleoside triphosphates (dNTPs) are the building blocks of DNA and their biosynthesis is tightly regulated in the cell. HPLC-MS and enzyme-based methods are currently employed to determine dNTP concentrations from cellular extracts. Here, we describe a highly efficient, HIV-1 reverse transcriptase (RT)-based assay to quantitate dNTP concentrations. The assay is based on the ability of HIV-1 RT to function at very low dNTP concentrations, thus providing for the high sensitivity of detection. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Detection and Tracking of Dual-Labeled HIV Particles Using Wide-Field Live Cell Imaging to Follow Viral Core Integrity
Live cell imaging is a valuable technique that allows the characterization of the dynamic processes of the HIV-1 life cycle. Here, we present a method of production and imaging of dual-labeled HIV viral particles that allows the visualization of two events. Varying release of the intravirion fluid phase marker reveals virion fusion and the loss of the integrity of HIV viral cores with the use of live wide-field fluorescent microscopy. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
HIV-1 Capsid Stabilization Assay
The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587–10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cy...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Measuring T Cell-to-T Cell HIV-1 Transfer, Viral Fusion, and Infection Using Flow Cytometry
Direct T cell-to-T cell HIV-1 infection is a distinct mode of HIV-1 infection that requires physical contact between an HIV-1-infected “donor” cell and an uninfected, CD4-expressing “target” cell. In vitro studies indicate that HIV-1 cell-to-cell infection is much more efficient than infection by cell-free viral particles; however, the exact mechanisms of the enhanced efficiency of this infection pathway are still unclear. Several assays have been developed to study the mechanism of direct cell-to-cell HIV-1 transmission and to assess sensitivity to neutralizing antibodies and pharmacologic inhibito...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
