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            產品分類 PRODCT

            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:2022-07-24 17:03
            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

            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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