Protocols in Current Issue
    Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy
    Authors:  Sherry T. Shu, Wing Fai Li and Thomas E. Smithgall, date: 07/05/2021, view: 1389, Q&A: 0

    Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide

    A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
    Authors:  Benno Zehnder, Stephan Urban and Thomas Tu, date: 04/20/2021, view: 2609, Q&A: 0

    Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain

    Giant Mimiviridae CsCl Purification Protocol
    Authors:  Lionel Bertaux, Audrey Lartigue and Sandra Jeudy, date: 11/20/2020, view: 997, Q&A: 0
    [Abstract] While different giant viruses’ purification protocols are available, they are not fully described and they use sucrose gradient that does not reach an equilibrium. Here, we report a protocol for the purification of members of the Mimiviridae family virions resulting from Acanthamoeaba castellanii infections. Viruses are ...
    Generation and in Planta Functional Analysis of Potato Virus Y mutants
    [Abstract] Potato virus Y (PVY), the type member of the genus Potyvirus (family Potyviridae), is the most widespread virus affecting potato and is included in the top five most economically detrimental plant viruses. Recently, the structure of the PVY virion has been determined by cryo-electron microscopy, which has opened the doors to ...
    Intestinal Enteroid Culture for Human Astroviruses
    Authors:  Irene A. Owusu, Carmen Mirabelli and Abimbola O. Kolawole, date: 07/20/2020, view: 1420, Q&A: 0
    [Abstract] Human astroviruses (HAstV) are non-enveloped, positive-sense single stranded RNA viruses that typically cause gastroenteritis in children, the elderly and among immunocompromised individuals. Some HAstV species have also been implicated in neurological diseases. It is important to study these viruses to understand the pathogenesis and develop ...
    HIV-CRISPR: A CRISPR/Cas9 Screening Method to Identify Genes Affecting HIV Replication
    Authors:  Ferdinand Roesch and Molly OhAinle, date: 05/05/2020, view: 4693, Q&A: 0
    [Abstract] Screening with CRISPR/Cas9 technology has already led to significant discoveries in the fields of cancer biology, cell biology and virology. Because of the relatively low false discovery rates and the ability to perform high-throughput, pooled approaches, it has rapidly become the assay of choice for screening studies, including whole-genome ...
    Purification of Rice Stripe Virus
    Authors:  Gang Lu, Min Yao, Yijun Zhou and Xiaorong Tao, date: 03/20/2020, view: 1687, Q&A: 0
    [Abstract] Although many spherical and rod-shaped plant virus purification protocols are now available, only a few protocols on filamentous plant virus purification have been published. Here, we report a protocol for large-scale purification of Rice stripe virus (RSV) from RSV-infected rice tissues. RSV virions with high infectivity were first precipitated ...
    Expression and Purification of Adeno-associated Virus Virus-like Particles in a Baculovirus System and AAVR Ectodomain Constructs in E. coli
    [Abstract] Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 ...
    Quantifying HIV-1-Mediated Gut CD4+ T Cell Death in the Lamina Propria Aggregate Culture (LPAC) Model
    Authors:  Stephanie M. Dillon, Kejun Guo, Moriah J. Castleman, Mario L. Santiago and Cara C. Wilson, date: 01/20/2020, view: 2015, Q&A: 0
    [Abstract] Gut CD4 T cells are major targets of HIV-1 and are massively depleted early during infection. To better understand the mechanisms governing HIV-1-mediated CD4 T cell death, we developed the physiologically-relevant Lamina Propria Aggregate Culture (LPAC) model. The LPAC model is ideal for studying CD4 T cell death induced by clinically-relevant ...
    Herpes Simplex Virus Type 1 Propagation, Titration and Single-step Growth Curves
    [Abstract] Given the endemic seroprevalence of herpes simplex viruses (HSV), its associated human diseases, and the emergence of acyclovir-resistant strains, there is a continuous need for better antiviral therapies. Towards this aim, identifying mechanistic details of how HSV-1 manipulates infected cells, how it modulates the immune responses, and how it ...

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