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1.
J Virol ; 94(16)2020 07 30.
Article in English | MEDLINE | ID: mdl-32493822

ABSTRACT

Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry.IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.


Subject(s)
Ebolavirus/metabolism , Niemann-Pick C1 Protein/metabolism , Tumor Suppressor Proteins/metabolism , Biological Transport , Carrier Proteins/metabolism , Ebolavirus/genetics , Ebolavirus/pathogenicity , Endosomes/metabolism , Filoviridae/genetics , Filoviridae Infections/genetics , Filoviridae Infections/metabolism , Glycoproteins/metabolism , Hemorrhagic Fever, Ebola/metabolism , Host-Pathogen Interactions , Membrane Glycoproteins/metabolism , Protein Transport/genetics , Protein Transport/physiology , Receptors, Virus/metabolism , Tumor Suppressor Proteins/genetics , Viral Envelope Proteins/genetics , Virus Internalization/drug effects
2.
J Infect Dis ; 218(suppl_5): S277-S286, 2018 11 22.
Article in English | MEDLINE | ID: mdl-29924324

ABSTRACT

Background: Human and filovirus host interactions remain poorly understood in areas where Ebola hemorrhagic fever outbreaks are likely to occur. In the Bwindi region of Uganda, a hot spot of mammalian biodiversity in Africa, human livelihoods are intimately connected with wildlife, creating potential for exposure to filoviruses. Methods: We tested samples from 331 febrile patients presenting to healthcare facilities near Bwindi Impenetrable Forest, Uganda, by polymerase chain reaction (PCR) analysis and Western blot, using recombinant glycoprotein antigens for Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus. Behavioral data on contact with wildlife were collected to examine risk factors for filovirus seropositivity. Results: All patients were negative for active filovirus infection, by PCR analysis. However, patients were seroreactive to SUDV (4.7%), EBOV (5.3%), and BDBV (8.9%), indicating previous exposure. Touching duikers was the most significant risk factor associated with EBOV seropositivity, while hunting primates and touching and/or eating cane rats were significant risk factors for SUDV seropositivity. Conclusions: People in southwestern Uganda have suspected previous exposure to filoviruses, particularly those with a history of wildlife contact. Circulation of filoviruses in wild animals and subsequent spillover into humans could be more common than previously reported.


Subject(s)
Animals, Wild/virology , Filoviridae Infections/genetics , Filoviridae Infections/virology , Filoviridae/pathogenicity , Adolescent , Adult , Aged , Animals , Animals, Wild/immunology , Antigens, Viral/immunology , Child , Child, Preschool , Female , Filoviridae/immunology , Filoviridae Infections/immunology , Glycoproteins/immunology , Humans , Infant , Infant, Newborn , Male , Middle Aged , Uganda , Young Adult
3.
Methods Mol Biol ; 1628: 133-142, 2017.
Article in English | MEDLINE | ID: mdl-28573616

ABSTRACT

Innate immunity is the first line of defense against virus infections and is marked by production of type I interferons (IFN), a family of cytokines that includes IFN-ß and several IFN-αs. For the filoviruses and many other RNA viruses that replicate in the cytoplasm, the RIG-I-like pattern recognition receptors (RLRs) are potential triggers of IFN production. To counteract such innate antiviral responses, many viruses encode proteins that antagonize RLR signaling. Ebola virus (EBOV) and other filoviruses produce VP35 proteins that block IFN induction via RLR signaling. We describe here cell-based reporter gene assays that quantify the IFN-antagonist function of filovirus VP35 proteins by assessing activation of the IFN-ß promoter.


Subject(s)
Filoviridae Infections/genetics , Filoviridae/genetics , Interferon Type I/genetics , Viral Regulatory and Accessory Proteins/genetics , Filoviridae/pathogenicity , Filoviridae Infections/pathology , Filoviridae Infections/virology , Host-Pathogen Interactions/genetics , Humans , Immunity, Innate/genetics , Interferon Type I/antagonists & inhibitors , Molecular Biology/methods , Signal Transduction/drug effects
4.
Sci Rep ; 6: 33987, 2016 10 05.
Article in English | MEDLINE | ID: mdl-27703185

ABSTRACT

Epigraph is an efficient graph-based algorithm for designing vaccine antigens to optimize potential T-cell epitope (PTE) coverage. Epigraph vaccine antigens are functionally similar to Mosaic vaccines, which have demonstrated effectiveness in preliminary HIV non-human primate studies. In contrast to the Mosaic algorithm, Epigraph is substantially faster, and in restricted cases, provides a mathematically optimal solution. Epigraph furthermore has new features that enable enhanced vaccine design flexibility. These features include the ability to exclude rare epitopes from a design, to optimize population coverage based on inexact epitope matches, and to apply the code to both aligned and unaligned input sequences. Epigraph was developed to provide practical design solutions for two outstanding vaccine problems. The first of these is a personalized approach to a therapeutic T-cell HIV vaccine that would provide antigens with an excellent match to an individual's infecting strain, intended to contain or clear a chronic infection. The second is a pan-filovirus vaccine, with the potential to protect against all known viruses in the Filoviradae family, including ebolaviruses. A web-based interface to run the Epigraph tool suite is available (http://www.hiv.lanl.gov/content/sequence/EPIGRAPH/epigraph.html).


Subject(s)
AIDS Vaccines , Epitopes , Filoviridae Infections , HIV Infections , HIV-1 , Sequence Analysis, Protein/methods , Software , AIDS Vaccines/genetics , AIDS Vaccines/immunology , Epitopes/genetics , Epitopes/immunology , Filoviridae/genetics , Filoviridae/immunology , Filoviridae Infections/genetics , Filoviridae Infections/immunology , Filoviridae Infections/therapy , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/therapy , HIV-1/genetics , HIV-1/immunology , Humans
5.
J Infect Dis ; 212 Suppl 2: S404-9, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26022440

ABSTRACT

Stat1(-/-) mice lack a response to interferon α, ß, and γ, allowing for replication of nonadapted wild-type (wt) Ebolavirus and Marburgvirus. We sought to establish a mouse model for efficacy testing of live attenuated recombinant vesicular stomatitis virus (rVSV)-based filovirus vaccine vectors using wt Ebolavirus and Marburgvirus challenge strains. While infection of immunocompetent mice with different rVSV-based filovirus vectors did not cause disease, infection of Stat1(-/-) mice with the same vectors resulted in systemic infection and lethal outcome for the majority of tested rVSVs. Despite differences in viral loads, organ tropism was remarkably similar between rVSV filovirus vaccine vectors and rVSVwt, with the exception of the brain. In conclusion, Stat1(-/-) mice are not an appropriate immunocompromised mouse model for efficacy testing of live attenuated, replication-competent rVSV vaccine vectors.


Subject(s)
Filoviridae/immunology , STAT1 Transcription Factor/deficiency , STAT1 Transcription Factor/genetics , Vaccines, Attenuated/immunology , Vesicular Stomatitis/immunology , Viral Vaccines/immunology , Animals , Chlorocebus aethiops , Disease Models, Animal , Ebolavirus/immunology , Filoviridae Infections/genetics , Filoviridae Infections/immunology , Filoviridae Infections/virology , Genetic Vectors/genetics , Genetic Vectors/immunology , Hemorrhagic Fever, Ebola/genetics , Hemorrhagic Fever, Ebola/immunology , Hemorrhagic Fever, Ebola/virology , Marburg Virus Disease/genetics , Marburg Virus Disease/immunology , Marburg Virus Disease/virology , Marburgvirus/immunology , Mice , STAT1 Transcription Factor/immunology , Vero Cells , Viral Load/immunology , Virus Replication/genetics , Virus Replication/immunology
6.
Biochem Biophys Res Commun ; 455(3-4): 223-8, 2014 Dec 12.
Article in English | MEDLINE | ID: mdl-25449273

ABSTRACT

Filoviruses, including Ebola and Marburg viruses, cause severe hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Human T-cell immunoglobulin and mucin domain 1 (TIM-1) is one of the host proteins that have been shown to promote filovirus entry into cells. In this study, we cloned TIM-1 genes from three different African green monkey kidney cell lines (Vero E6, COS-1, and BSC-1) and found that TIM-1 of Vero E6 had a 23-amino acid deletion and 6 amino acid substitutions compared with those of COS-1 and BSC-1. Interestingly, Vero E6 TIM-1 had a greater ability to promote the infectivity of vesicular stomatitis viruses pseudotyped with filovirus glycoproteins than COS-1-derived TIM-1. We further found that the increased ability of Vero E6 TIM-1 to promote virus infectivity was most likely due to a single amino acid difference between these TIM-1s. These results suggest that a polymorphism of the TIM-1 molecules is one of the factors that influence cell susceptibility to filovirus infection, providing a new insight into the molecular basis for the filovirus host range.


Subject(s)
Filoviridae Infections/genetics , Filoviridae/pathogenicity , Membrane Glycoproteins/genetics , Polymorphism, Genetic , Receptors, Virus/genetics , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Cloning, Molecular , Flow Cytometry , Genetic Predisposition to Disease , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Kidney/cytology , Kidney/virology , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Mutagenesis , Protein Structure, Tertiary , Receptors, Virus/metabolism , Sequence Homology, Amino Acid , Vero Cells
7.
Nat Med ; 16(9): 991-4, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20729866

ABSTRACT

Currently, no vaccines or therapeutics are licensed to counter Ebola or Marburg viruses, highly pathogenic filoviruses that are causative agents of viral hemorrhagic fever. Here we show that administration of positively charged phosphorodiamidate morpholino oligomers (PMOplus), delivered by various dosing strategies initiated 30-60 min after infection, protects>60% of rhesus monkeys against lethal Zaire Ebola virus (ZEBOV) and 100% of cynomolgus monkeys against Lake Victoria Marburg virus (MARV) infection. PMOplus may be useful for treating these and other highly pathogenic viruses in humans.


Subject(s)
Filoviridae Infections/genetics , Filoviridae Infections/prevention & control , Vaccines, Synthetic/therapeutic use , Viral Vaccines/therapeutic use , Animals , Ebolavirus/isolation & purification , Filoviridae Infections/immunology , Filoviridae Infections/mortality , Hemorrhagic Fever, Ebola/genetics , Hemorrhagic Fever, Ebola/immunology , Hemorrhagic Fever, Ebola/prevention & control , Humans , Macaca mulatta/immunology , Marburg Virus Disease/genetics , Marburg Virus Disease/immunology , Marburg Virus Disease/prevention & control , Marburgvirus/isolation & purification , Primates , Safety
8.
J Infect Dis ; 196 Suppl 2: S382-9, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17940974

ABSTRACT

BACKGROUND: Recent reports indicate the possibility of using small interfering RNAs (siRNAs) to treat filovirus infections; however, they also show that the effectiveness of this approach is highly dependent on target site selection. Therefore, we explored the application of minigenomes as screening tools to identify functional siRNA targets under biosafety level 2 conditions. METHODS: siRNA candidates were screened using the minigenome system to identify those with potential antiviral activity, compared with controls with poor predicted function on the basis of design guidelines, or those that were noncomplementary to Zaire ebolavirus (ZEBOV). These findings were then validated in cell culture by use of a previously developed ZEBOV expressing green fluorescent protein (ZEBOV-GFP), which allowed siRNA function to be easily assessed via flow cytometry or focus formation. RESULTS: The most promising siRNA based on minigenome screening, targeting the nucleoprotein (NP) mRNA (ZNP1), also reduced protein expression and decreased viral titers after infection with ZEBOV-GFP to an extent similar to that reported for an siRNA recently shown to be therapeutic in guinea pigs. CONCLUSIONS: Minigenome screening appears to be an effective and convenient method of evaluating the therapeutic potential of siRNA targets, and findings suggest that its use would increase success rates in later stages of siRNA testing.


Subject(s)
Filoviridae Infections/therapy , Filoviridae/genetics , RNA, Antisense/therapeutic use , RNA, Small Interfering/therapeutic use , RNA, Viral/therapeutic use , Animals , Cell Line , Chlorocebus aethiops , Filoviridae/pathogenicity , Filoviridae Infections/genetics , Genetic Therapy/methods , Genome, Viral , Haplorhini , Humans , Kidney , RNA, Small Interfering/genetics , RNA, Viral/genetics , Vero Cells
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