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Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture.

ABSTRACT: Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.

SUBMITTER: Neumann G

PROVIDER: S-EPMC135697 | biostudies-literature | 2002 Jan

REPOSITORIES: biostudies-literature

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Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture.

Neumann Gabriele G Feldmann Heinz H Watanabe Shinji S Lukashevich Igor I Kawaoka Yoshihiro Y

Journal of virology 20020101 1

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the ...[more]

PMID: 11739705

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Project description:Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels.IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

Project description:Ebola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013-2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection in vitro Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD.IMPORTANCE Treatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune system are characteristic features of EVD, statins could be explored as part of EVD therapeutics.

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Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture.

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Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture.

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