A Novel Life Cycle Modeling System for Ebola Virus Shows a Genome Length-Dependent Role of VP24 in Virus Infectivity

Abstract

Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription-and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP 1,2 . This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ϳ500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. IMPORTANCEEbola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories. E bola virus is a negative-sense RNA virus (NSV) and the causative agent of a severe hemorrhagic fever in humans and nonhuman primates, with case fatality rates being up to 90% (1). The VP24 protein is unique to filoviruses and has no ...

Post-exposure treatments for Ebola and Marburg virus infections

“…To model additional aspects of the filovirus life cycle, these systems have been further developed into transcription-competent and replication-competent virus-like particle (trVLP) systems [294][295][296] . A trVLP system also expresses the remaining filovirus proteins, such as VP40, glycoprotein (GP) and VP24.…”

“…trVLP systems either carry a monocistronic (reporter gene) or a multicistronic (reporter gene and viral genes) minigenome. A tetracistronic trVLP system has been developed that expresses a reporter protein as well as VP40, GP and VP24 from the minigenome, which increases the important ratio of infectious to noninfectious trVLPs 296 . If these tetracistronic minigenome-carrying trVLPs are used to infect cells already transfected with plasmids expressing the ribonucleoprotein protein components, VP40, GP and VP24 are produced, leading to new trVLPs that now can be transferred to new target cells, thereby modelling multiple infectious cycles [296][297][298] .…”

“…A tetracistronic trVLP system has been developed that expresses a reporter protein as well as VP40, GP and VP24 from the minigenome, which increases the important ratio of infectious to noninfectious trVLPs 296 . If these tetracistronic minigenome-carrying trVLPs are used to infect cells already transfected with plasmids expressing the ribonucleoprotein protein components, VP40, GP and VP24 are produced, leading to new trVLPs that now can be transferred to new target cells, thereby modelling multiple infectious cycles [296][297][298] . The trVLP systems are powerful tools for high-throughput screening of drugs that interfere with aspects of the filovirus life cycle, including attachment, fusion, entry, replication, transcription, maturation, budding and release 299 .…”

¿Qué deber saber un médico sobre el ébola?

“…El genoma viral codifica una nucleoproteína (NP), la glucoproteína (GP), la ARN polimerasa dependiente de ARN (L), y cuatro proteínas estructurales denominados VP24, VP30, VP35 y VP40. Además, el virus Ébola es capaz de expresar una forma soluble de sGP a través del ARN (Figura 1) 7 . Representa un agente infeccioso, no un ser vivo y el recuerdo de un modelo de conservación de la información genética muy anterior a nuestro propio origen y lo que le ha permitido llegar a producir el daño que ahora nos causa, es su capacidad de adaptación.…”

Changes associated with Ebola virus adaptation to novel species

“…VP24 is involved in the formation of the viral nucleocapsid, the regulation of virus replication, and prevention of interferon signaling Feldmann and Geisbert, 2011;Watt et al, 2014;Reid et al, 2006). VP24 binds to STAT1 and the karyopherins α1 (KPNA1), α5, (KPNA5), and α6 (KPNA6) (Xu et al, 2014).…”