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    圓二色光譜在冠狀病毒研究上的一個應用 (鼠Coronavirus泛素樣結構域對木瓜蛋白酶的穩定性和病毒的發病機理是至關重要的))

    日期:2022-05-25 05:21
    瀏覽次數:1014
    摘要:ABSTRACT Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, vira
    Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis
    Anna M. Mielech,a Xufang Deng,a Yafang Chen,b Eveline Kindler,c,d Dorthea L. Wheeler,e Andrew D. Mesecar,b Volker Thiel,c,d
    Stanley Perlman,e,f Susan C. Bakera
    Department of Microbiology & Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USAa; Purdue University, Department of Biological
    Sciences, Lafayette, Indiana, USAb; Federal Institute of Virology and Immunology, Bern and Mittelh?usern, Switzerlandc; Vetsuisse Faculty, University of Bern, Bern,

    Switzerlandd; Interdisciplinary Program in Immunology, University of Iowa, Iowa City, Iowa, USAe; Department of Microbiology, University of Iowa, Iowa City, Iowa, USA

    IMPORTANCE
    Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the
    Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected
    viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the
    viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant
    Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl
    domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating
    coronavirus replication and pathogenesis.
    Coronaviruses are emerging human pathogens. Severe acute
    respiratory syndrome coronavirus (SARS-CoV) caused the
    epidemic of 2002 to 2003, with a 10% case fatality ratio (1).
    Middle East respiratory syndrome coronavirus (MERS-CoV) is a
    pathogenic virus that was first identified in humans in 2012 (2). As
    of 3 February 2015, there have been 965 confirmed cases and 357
    deaths (http://www.who.int/csr/don/03-february-2015-mers/en/).
    For SARS-CoV, the virus emerged from a reservoir in bats, replicated
    in an intermediate host (civet cats), and spread to humans.
    The epidemic strain of SARS-CoV evolved for efficient human-tohuman
    spread (3–5). Public health measures of isolation of infected
    individuals led to the cessation of the epidemic in humans;
    however, SARS-like viruses remain in bat reservoirs (6–8). For
    MERS-CoV, dromedary camels now are suspected as the zoonotic
    source for transmission to humans, since MERS-CoV sequences
    with 99% nucleotide identity to human MERS-CoV isolates have
    been detected in respiratory samples from camels (9). Although
    there are reports of human-to-human transmission of MERSCoV
    (10, 11), current strains seem to cause mostly lower respiratory
    tract disease and are not as highly transmissible as SARS-CoV
    (12). Other human coronaviruses (HCoV-229E, HCoV-OC43,

    HCoV-NL63, and HCoV-HKU1) are common in the human
    population and are the causative agents of upper and lower respiratory
    tract disease and croup (13–16). To date, there are no FDAapproved
    antiviral drugs or vaccines to fight human coronavirusinduced
    disease. In addition, the potential exists for coronaviruses
    to emerge into the human population from reservoirs in bats or
    other animals (17). Identifying viral components critical for efficient
    replication and manifestation of disease will facilitate antiviral
    drug and vaccine development.

    Thermal Tm analysis using CD. Thermal melting analyses of the wildtype
    PLP2 and V787S mutant was carried out with a Chirascan circular
    dichroism (CD) spectrometer (Applied Photophysics) equipped with a
    temperature control system (Quantum Northwest Inc.) by monitoring
    theCDsignal at 220nmwhile increasing the temperature at a step interval
    of 0.4°C and at a rate 0.5°C/min. Two ml of protein samples at 1 M in
    buffer with 0.1 M potassium phosphate (pH 7.5) was contained in a
    10-mm quartz cell (Starna Cells) with magnetic stirring. Thermal scans
    were performed in three independent experiments for both wild-type and
    V787S mutant MHV PLP2. The melting temperatures (Tm) were calculated
    as the first derivative peak using the program SigmaPlot.


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