Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

Virus entry into host cells is the first step of infection and a crucial determinant of pathogenicity. Here we show that Ebola virus-like particles (EBOV-VLPs) composed of the glycoprotein GP(1,2) and the matrix protein VP40 use macropinocytosis and clathrin-mediated endocytosis to enter cells. EBOV-VLPs applied to host cells induced actin-driven ruffling and enhanced FITC-dextran uptake, which indicated macropinocytosis as the main entry mechanism. This was further supported by inhibition of entry through inhibitors of actin polymerization (latrunculin A), Na(+)/H(+)-exchanger (EIPA), and PI3-kinase (wortmannin). A fraction of EBOV-VLPs, however, colocalized with clathrin heavy chain (CHC), and VLP uptake was reduced by CHC small interfering RNA transfection and expression of the dominant negative dynamin II-K44A mutant. In contrast, we found no evidence that EBOV-VLPs enter cells via caveolae. This work identifies macropinocytosis as the major, and clathrin-dependent endocytosis as an alternative, entry route for EBOV particles. Therefore, EBOV seems to utilize different entry pathways depending on both cell type and virus particle size.

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans and non-human primates. While the role of the EBOV major matrix protein VP40 in morphogenesis is well understood, nothing is known about its contributions to the regulation of viral genome replication and/or transcription. Similarly, while it was reported that the minor matrix protein VP24 impairs viral genome replication, it remains unclear whether it also regulates transcription, since all common experimental systems measure the combined products of replication and transcription. We have developed systems that allow the independent monitoring of viral transcription and replication, based on qRT-PCR and a replication-deficient minigenome. Using these systems we show that VP24 regulates not only viral genome replication, but also transcription. Further, we show for the first time that VP40 is also involved in regulating these processes. These functions are conserved among EBOV species and, in the case of VP40, independent of its budding or RNA-binding functions.

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 ...

No abstract

Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions. We successfully used a novel, pocket-sized nanopore sequencer at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak.

Background: Ebola virus VP30 is an essential transcription factor dispensable for viral replication whose activity is regulated via phosphorylation. Results: Phosphorylation of VP30 impacts viral transcription and replication by modulating interaction with the nucleocapsid proteins VP35 and NP. Conclusion: VP30 phosphorylation influences the composition of the viral polymerase complex via phosphorylation-dependent interaction with VP35. Significance: VP30 phosphorylation status modulates both viral transcription and replication.

Infectious virus-like particle (iVLP) systems have recently been established for several negative-strand RNA viruses, including the highly pathogenic Zaire ebolavirus (ZEBOV), and allow study of the viral life cycle under biosafety level 2 conditions. However, current systems depend on the expression of viral helper nucleocapsid proteins in target cells, thus making it impossible to determine whether ribonucleoprotein complexes transferred by iVLPs are able to facilitate initial transcription, an indispensable step in natural infection. Here we describe a ZEBOV iVLP system which overcomes this limitation and show that VP24 is essential for the formation of a functional ribonucleoprotein complex.Ebola virus (EBOV) causes severe hemorrhagic fever in humans, for which treatment is currently unavailable (6, 8). To study particle morphogenesis, genome packaging, and virion entry into target cells under biosafety level 2 conditions, an infectious virus-like particle (iVLP) system for Zaire ebolavirus (ZEBOV) was established (20). This system extends the original minigenome systems (4, 7, 14) by including VP24, VP40, and GP to produce iVLPs which resemble wild-type (WT) virions but contain minigenomes instead of full-length viral genomes. These iVLPs can deliver the minigenomes to target cells, where they are transcribed and replicated in the presence of helper ribonucleoprotein (RNP) components (the nucleoprotein [NP], VP35, VP30, and L components). Similar systems are available for other negative-sense RNA viruses, but RNP components must always be provided to target cells in trans, via either helper virus infection or expression plasmid transfection (12,(15)(16)(17)(19)(20)(21). Therefore, it is impossible to determine whether iVLPs contain fully functional RNP complexes or whether they contain minigenomes but are unable to facilitate initial transcription, as is necessary in virus infection. EBOV VP24 has been described as a minor matrix protein, but its contribution to budding is controversial (10, 13). Electron microscopic studies imply a role in the formation of nucleocapsid-like structures (11), while another study suggests that VP24 is unnecessary for the packaging and delivery of minigenomes by iVLPs (20). To better understand the contribution of VP24 to morphogenesis, we established an iVLP system with naïve target cells to allow assessment of iVLP-associated RNP complex function. As a first step, an iVLP system based on that previously described (20) was established in our laboratory (Fig. 1A). 293T cells (p0) were transfected using FuGENE (Roche) with plasmids encoding each ZEBOV structural protein (125 ng pCAGGS-NP, 125 ng pCAGGS-VP35, 75 ng pCAGGS-VP30, 1,000 ng pCAGGS-L, 250 ng pCAGGS-GP, 250 ng pCAGGS-VP40, and 60 ng pCAGGS-VP24) and a T7-driven minigenome encoding a Renilla luciferase reporter (250 ng), which is detectable in minute amounts (23). Cell supernatant containing released iVLPs was harvested 3 days posttransfection, cleared of cellular debris, and used to infect target 293T cells (p1) previ...