Ebola Virus Enters Host Cells by Macropinocytosis and Clathrin-Mediated Endocytosis

The Ebola virus (EBOV) genome encodes a partly conserved 40-residue nonstructural polypeptide, called the delta peptide, that is produced in abundance during Ebola virus disease (EVD). The function of the delta peptide is unknown, but sequence analysis has suggested that delta peptide could be a viroporin, belonging to a diverse family of membrane-permeabilizing small polypeptides involved in replication and pathogenesis of numerous viruses. Full-length and conserved C-terminal delta peptide fragments permeabilize the plasma membranes of nucleated cells of rodent, dog, monkey, and human origin; increase ion permeability across confluent cell monolayers; and permeabilize synthetic lipid bilayers. Permeabilization activity is completely dependent on the disulfide bond between the two conserved cysteines. The conserved C-terminal portion of the peptide is biochemically stable in human serum, and most serum-stable fragments have full activity. Taken together, the evidence strongly suggests that Ebola virus delta peptide is a viroporin and that it may be a novel, targetable aspect of Ebola virus disease pathology.IMPORTANCE During the unparalleled West African outbreak of Ebola virus disease (EVD) that began in late 2013, the lack of effective countermeasures resulted in chains of serial infection and a high mortality rate among infected patients. A better understanding of disease pathology is desperately needed to develop better countermeasures. We show here that the Ebola virus delta peptide, a conserved nonstructural protein produced in large quantities by infected cells, has the characteristics of a viroporin. This information suggests a critical role for the delta peptide in Ebola virus disease pathology and as a possible target for novel countermeasures.

Section: Abstract Delta Peptide Ebola Virus Enterotoxins Permeabilmentioning

confidence: 99%

Ebola Virus Delta Peptide Is a Viroporin

Melnik

Komin

et al. 2017

“…The most commonly used system involves the pseudotyping of retroviruses; however, the validity of this approach has been questioned (42)(43)(44). trVLPs produced from tetracistronic minigenomes represent a viable alternative to this system, with the clear advantage that, in contrast to pseudovirus particles, infectious trVLPs not only exhibit the unique threadlike morphology of Ebola viruses (14,37), which has been suggested to strongly influence the uptake mechanism used by these viruses (44), but also do not contain any components from foreign viruses. As such, trVLPs containing tetracistronic minigenomes most closely resemble authentic Ebola virus particles and mimic their ability to continuously infect target cells over multiple infectious cycles.…”

Section: Figmentioning

confidence: 99%

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

Watt

Moukambi

Banadyga

et al. 2014

Self Cite

145

167

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 ...

“…However, numerous studies have shown that in antigen-presenting cells and in certain tumor cell lines, macropinocytosis is constitutively active such that efficient accumulation of fluid-phase markers is not dependent on stimulation by exogenous factors (42,43,(80)(81)(82). Fluorophore-labeled high-molecular-mass polysaccharides, such as 10-kDa and 70-kDa dextrans, have been used to quantify extracellular fluid uptake during virus entry (63,65,79,(83)(84)(85)(86). We incubated HCMV with SNB-19 cells on ice for 1 h, added FITC-conjugated 10-kDa or 70-kDa dextran to the cells, and shifted the temperature to 37°C for 1 h to allow virus internalization.…”

Section: Figmentioning

confidence: 99%

Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

Fischer

Cohen

2016

Previously we showed that THY-1 has a critical role in the initial stage of infection of certain cell types with human cytomegalovirus (HCMV) and that THY-1 is important for HCMV-mediated activation of phosphatidylinositol 3-kinase (PI3K)/Akt during virus entry. THY-1 is known to interact with integrins and is a major cargo protein of clathrin-independent endocytic vesicles. Since macropinocytosis involves integrin signaling, is PI3K/Akt dependent, and is a clathrin-independent endocytic process, we determined whether THY-1 has a role in HCMV entry by macropinocytosis. Using electron microscopy in two cell lines that support HCMV infection in a THY-1-dependent manner, we found that HCMV enters these cells by a macropinocytosis-like process. THY-1 associated with HCMV virions on the cell surface and colocalized with virus inside macropinosomes. 5-(N-Ethyl-Nisopropyl)amiloride (EIPA) and soluble THY-1 blocked HCMV infection in the cell lines by >80% and 60%, respectively. HCMV entry into the cells triggered increased influx of extracellular fluid, a marker of macropinocytosis, and this increased fluid uptake was inhibited by EIPA and by soluble THY-1. Blocking actin depolymerization, Na ؉ /H ؉ exchange, PI3K, and Pak1 kinase, which are critical for macropinocytosis, impaired HCMV infection. Neither internalized HCMV virions nor THY-1 in virus-infected cells colocalized with transferrin as determined by confocal microscopy, indicating that clathrin-mediated endocytosis was not involved in THY-1-associated virus entry. These results suggest that HCMV has adapted to utilize THY-1, a cargo protein of clathrin-independent endocytotic vesicles, to facilitate efficient entry into certain cell types by a macropinocytosis-like process. IMPORTANCE Human cytomegalovirus (HCMV) infects over half of the population and is the most common infectious cause of birth defects.The virus is the most important infection occurring in transplant recipients. The mechanism of how HCMV enters cells is controversial. In this study, we show that THY-1, a cell surface protein that is critical for the early stage of entry of HCMV into certain cell types, contributes to virus entry by macropinocytosis. Our findings suggest that HCMV has adapted to utilize THY-1 to facilitate entry of HCMV into macropinosomes in certain cell types. Further knowledge about the mechanism of HCMV entry into cells may facilitate the development of novel inhibitors of virus infection. Human cytomegalovirus (HCMV) infects over half of the general population. While the infection is often asymptomatic when occurring in young children, it poses a serious health threat in transplant recipients and in immunocompromised individuals. HCMV is the most common infectious cause of birth defects. The virus infects a variety of host cells in vivo, including epithelial, endothelial, and muscle cells as well as fibroblasts. HCMV encodes multiple glycoproteins, some of which are not shared with other human herpesviruses, such as UL128, UL130, and UL131A. Other glycoproteins ar...