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HSV-1 Biology and Life Cycle
Herpes simplex virus type 1 (HSV-1) is a common and important human pathogen that has been studied in a wide variety of contexts for several decades. This book presents chapters on protocols on many strands of HSV-1 research that are currently in use in leading laboratories. This chapter gives a brief overview of HSV-1 biology and life cycle, covering basic aspects of the virus and its replication in cultured cells, the diseases caused by the virus, viral latency, antiviral defenses, and the mechanisms that the virus uses to counteract these defenses. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

In Vivo HSV-1 DNA Transport Studies Using Murine Retinal Ganglion Cells
We present a method for studying the axonal transport of Herpes simplex virus in mature neurons in situ. Using genetically identical mice and carefully controlling the delivery of virus, an investigator can obtain insight into the transport of virus to and from the neuron cell body within the cellular environment of an intact animal. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Cryo-EM Techniques to Resolve the Structure of HSV-1 Capsid-Associated Components
Electron cryo-microscopy has become a routine technique to determine the structure of biochemically purified herpes simplex virus capsid particles. This chapter describes the procedures of specimen preparation by cryopreservation; low dose and low temperature imaging in an electron cryo-microscope; and data processing for reconstruction. This methodology has yielded subnanometer resolution structures of the icosahedral capsid shell where α-helices and β-sheets of individual subunits can be recognized. A relaxation of the symmetry in the reconstruction steps allows us to resolve the DNA packaging protein located ...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Expression, Purification, and Crystallization of HSV-1 Glycoproteins for Structure Determination
HSV glycoproteins play important roles in the viral infectious cycle, particularly viral entry into the cell. Here we describe the protocol for expression, purification, and crystallization of viral glycoproteins based on those developed for the HSV-1 gB and HSV-2 gH/gL ectodomains. These protocols can be used for generating milligram amounts of wild-type (WT) or mutant gB and gH/gL ectodomains or can be adapted to produce purified ectodomains of other HSV glycoproteins for biochemical and structural studies. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Multifluorescence Live Analysis of Herpes Simplex Virus Type-1 Replication
The possibility to label specific viral and cellular structures with live cell markers such as autofluorescent proteins has greatly contributed to our understanding of diverse steps of the virus life cycle, as it allows monitoring virus replication in a spatial and temporal fashion. Here, we describe the multi-fluorescent analysis of the multi-compartment herpes simplex virus type-1 by live-cell confocal laser scanning microscopy. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Preparation of Herpes Simplex Virus-Infected Primary Neurons for Transmission Electron Microscopy
Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus–host cell interaction. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Affinity Purification Combined with Mass Spectrometry to Identify Herpes Simplex Virus Protein–Protein Interactions
The identification and characterization of herpes simplex virus protein interaction complexes are fundamental to understanding the molecular mechanisms governing the replication and pathogenesis of the virus. Recent advances in affinity-based methods, mass spectrometry configurations, and bioinformatics tools have greatly increased the quantity and quality of protein–protein interaction datasets. In this chapter, detailed and reliable methods that can easily be implemented are presented for the identification of protein–protein interactions using cryogenic cell lysis, affinity purification, trypsin digestion, a...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

A Precipitation-Based Assay to Analyze Interactions of Viral Particles with Cytosolic Host Factors
Since viruses are obligate intracellular parasites, viral particles, subviral structures, and viral proteins enlist the support of host proteins to foster intracellular transport, viral gene expression, replication, and evasion from antiviral host responses. We have devised a biochemical in vitro method to analyze specific interactions of cytosolic factors with capsids of herpes simplex virus and to characterize host proteins that specifically coprecipitate with different types of viral particles by immunoblotting, mass spectrometry, and immunoelectron microscopy. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Characterization of Extracellular HSV-1 Virions by Proteomics
The analysis of herpes simplex virus type 1 mature extracellular virions by proteomics requires highly enriched samples to limit false positives and favor the detection of true components. The protocol described below involves the removal of highly contaminating serum proteins and purification of the virions by a series of differential and density centrifugation steps. In addition, L-particles, which are viral particles devoid of genome and capsid but present in the extracellular milieu, are depleted on Ficoll 400 gradients. As previously reported, the resulting viral particles are free of most contaminants and suitable fo...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Using Homogeneous Primary Neuron Cultures to Study Fundamental Aspects of HSV-1 Latency and Reactivation
We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal and explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit o...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Phenotypic and Genotypic Testing of HSV-1 Resistance to Antivirals
Resistance testing of antivirals to herpes simplex virus type 1 can be done by phenotypic and genotypic methods. The determination of a resistant phenotype is based on the calculation of inhibitory concentrations for the antiviral drug, which should be tested. The main advantage is a clear interpretation of laboratory findings, but the method is time consuming and a considerable experience is required for handling infectious virus. Genotypic resistance testing is based on the detection of resistance-related mutations in viral genes encoding the thymidine kinase and DNA polymerase by means of amplification and sequencing. T...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news

Evaluation of p47phox Phosphorylation in Human Neutrophils Using Phospho-Specific Antibodies
In this study, we describe a radioactive-free technique to analyze the phosphorylation of p47phox in cell lysates, based on the use of phospho-specific antibodies, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. This technique could be used to quickly and easily study the phosphorylation of p47phox under different conditions, such as testing the effects of pharmacological agents in this process or assessing the activation status of neutrophils in situ. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news

Affinity Purification and Reconstitution of Human Phagocyte Flavocytochrome b for Detection of Conformational Dynamics in the Membrane
Human flavocytochrome b (Cyt b) is the core electron transferase of the NADPH oxidase in phagocytes and a number of other cell types. The oxidase complex generates superoxide, initiating production of a cascade of reactive oxygen species critical for the killing of infectious agents. Many fundamental questions still remain concerning its structural dynamics and electron transfer mechanisms. In particular, Cyt b structure/function correlates in the membrane have been relatively unstudied. In order to facilitate the direct analysis of Cyt b structural dynamics in the membrane, the following method provides rapid and efficien...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news

Assessment of Priming of the Human Neutrophil Respiratory Burst
Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce reactive oxygen species (ROS) such as superoxide anion (O2 ∙−), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl), a process referred to as the respiratory burst. The enzyme responsible for this process is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40ph...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news

Cell-Free NADPH Oxidase Activation Assays: “In Vitro Veritas”
The superoxide (O2 ∙−)-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 phox ) and four cytosolic regulatory proteins, p47 phox , p67 phox , p40 phox , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 ∙− generation is initiated by engagement of membrane receptors by a va...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news