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      Enterovirus 71 3C Protease Cleaves a Novel Target CstF-64 and Inhibits Cellular Polyadenylation

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          Abstract

          Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C pro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C pro. CstF-64 was cleaved in vitro by 3C pro but neither by mutant 3C pro (in which the catalytic site was inactivated) nor by another EV71 protease 2A pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3C pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

          Author Summary

          Many viruses contain specific proteases that are essential for processing their own viral proteins. For an efficient replication within their hosts, on the other hand, viruses also utilize these proteases to cleave a number of key host proteins and hijack cellular machineries. In this study, host proteins are identified as the substrates for enterovirus 71 viral protease by adopting a proteomic strategy. Enterovirus 71 infection is highly associated with neurological complication and mortality in an era when poliovirus has been controlled by vaccination. Investigating the host substrates for enterovirus 71 protease may shed light on the viral–host interaction, ultimately providing further insight into the pathogenesis of EV71 infection. We found that 3C protease (3C pro) cleaved CstF-64, the latter a cleavage stimulation factor in host polyadenylation machinery. 3C pro is known to enter host nucleus, whereas the replication of the virus occurs in cytoplasm. While its role in nucleus has been thought to inhibit host transcription, we found that 3C pro may inhibit host-cell gene expression at host 3′-end pre-mRNA processing and polyadenylation steps. Consequently, less polyadenylated host mRNA is synthesized and more cellular resources, such as the translation factors, would be available for viral RNA expression.

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          Most cited references55

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          An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group.

          Enteroviruses can cause outbreaks of hand-foot-and-mouth disease (characterized by vesicular lesions on the hands, feet, and oral mucosa) or herpangina, usually without life-threatening manifestations. In 1998 an epidemic of enterovirus 71 infection caused hand-foot-and-mouth disease and herpangina in thousands of people in Taiwan, some of whom died. We assessed the epidemiologic aspects of this outbreak. Cases of hand-foot-and-mouth disease or herpangina in ambulatory patients were reported to the Taiwan Department of Health by a mean of 818 sentinel physicians. Severe cases in hospitalized patients were reported by 40 medical centers and regional hospitals. Viruses were isolated by 10 hospital laboratories and the department of health. The sentinel physicians reported 129,106 cases of hand-foot-and-mouth disease or herpangina in two waves of the epidemic, which probably represents less than 10 percent of the estimated total number of cases. There were 405 patients with severe disease, most of whom were five years old or younger; severe disease was seen in all regions of the island. Complications included encephalitis, aseptic meningitis, pulmonary edema or hemorrhage, acute flaccid paralysis, and myocarditis. Seventy-eight patients died, 71 of whom (91 percent) were five years of age or younger. Of the patients who died, 65 (83 percent) had pulmonary edema or pulmonary hemorrhage. Among patients from whom a virus was isolated, enterovirus 71 was present in 48.7 percent of outpatients with uncomplicated hand-foot-and-mouth disease or herpangina, 75 percent of hospitalized patients who survived, and 92 percent of patients who died. Although several enteroviruses were circulating in Taiwan during the 1998 epidemic, enterovirus 71 infection was associated with most of the serious clinical manifestations and with nearly all the deaths. Most of those who died were young, and the majority died of pulmonary edema and pulmonary hemorrhage.
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            An overview of the evolution of enterovirus 71 and its clinical and public health significance.

            Since its discovery in 1969, enterovirus 71 (EV71) has been recognised as a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) associated with severe neurological sequelae in a small proportion of cases. There has been a significant increase in EV71 epidemic activity throughout the Asia-Pacific region since 1997. Recent HFMD epidemics in this region have been associated with a severe form of brainstem encephalitis associated with pulmonary oedema and high case-fatality rates. The emergence of large-scale epidemic activity in the Asia-Pacific region has been associated with the circulation of three genetic lineages that appear to be undergoing rapid evolutionary change. Two of these lineages (B3 and B4) have not been described previously and appear to have arisen from an endemic focus in equatorial Asia, which has served as a source of virus for HFMD epidemics in Malaysia, Singapore and Australia. The third lineage (C2) has previously been identified [Brown, B.A. et al. (1999) J. Virol. 73, 9969-9975] and was primarily responsible for the large HFMD epidemic in Taiwan during 1998. As EV71 appears not to be susceptible to newly developed antiviral agents and a vaccine is not currently available, control of EV71 epidemics through high-level surveillance and public health intervention needs to be maintained and extended throughout the Asia-Pacific region. Future research should focus on (1) understanding the molecular genetics of EV71 virulence, (2) identification of the receptor(s) for EV71, (3) development of antiviral agents to ameliorate the severity of neurological disease and (4) vaccine development to control epidemics. Following the successful experience of the poliomyelitis control programme, it may be possible to control EV71 epidemics if an effective live-attenuated vaccine is developed.
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              Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs.

              Inhibition of the nuclear export of poly(A)-containing mRNAs caused by the influenza A virus NS1 protein requires its effector domain. Here, we demonstrate that the NS1 effector domain functionally interacts with the cellular 30 kDa subunit of CPSF, an essential component of the 3' end processing machinery of cellular pre-mRNAs. In influenza virus-infected cells, the NS1 protein is physically associated with CPSF 30 kDa. Binding of the NS1 protein to the 30 kDa protein in vitro prevents CPSF binding to the RNA substrate and inhibits 3' end cleavage and polyadenylation of host pre-mRNAs. The NS1 protein also inhibits 3' end processing in vivo, and the uncleaved pre-mRNA remains in the nucleus. Via this novel regulation of pre-mRNA 3' end processing, the NS1 protein selectively inhibits the nuclear export of cellular, and not viral, mRNAs.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                September 2009
                September 2009
                25 September 2009
                : 5
                : 9
                : e1000593
                Affiliations
                [1 ]Research Center for Emerging Viral Infections, Chang Gung University, Kwei-Shan Tao-Yuan, Taiwan, R.O.C.
                [2 ]Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Kwei-Shan Tao-Yuan, Taiwan, R.O.C.
                [3 ]Graduate Institute of Biomedical Sciences, Chang Gung University, Kwei-Shan Tao-Yuan,Taiwan, R.O.C.
                [4 ]Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America
                Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Spain
                Author notes

                Conceived and designed the experiments: KFW SRS. Performed the experiments: KFW MLL CTH. Analyzed the data: KFW SRS. Wrote the paper: KFW SRS.

                Article
                09-PLPA-RA-0717R2
                10.1371/journal.ppat.1000593
                2742901
                19779565
                567a11c8-e18a-4191-ba91-245c7c29fecd
                Weng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 4 May 2009
                : 27 August 2009
                Page count
                Pages: 13
                Categories
                Research Article
                Virology
                Virology/Effects of Virus Infection on Host Gene Expression

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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