Identification of Essential Filovirion-associated Host Factors by Serial Proteomic Analysis and RNAi Screen

Spurgers, Kevin B.; Alefantis, Tim; Peyser, Brian D.; Ruthel, Gordon; Bergeron, Alison A.; Costantino, Julie; Enterlein, Sven; Kota, Krishna P.; Boltz, Robert C.; Aman, M. Javad; DelVecchio, Vito G.; Bavari, Sina

doi:10.1074/mcp.m110.003418

Cited by 65 publications

(79 citation statements)

References 75 publications

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“…Most likely, the B5 protein-positive structures represent membranous vesicles that were derived from cellular compartments like the Golgi apparatus, the trans-Golgi network, endosomes, or the plasma membrane. These data confirm previous observations showing that the formation of Ebola virus-like particles at the plasma membrane is a specific process that excludes other proteins from incorporation into the VLPs (13,32,33).…”

Section: Figsupporting

confidence: 92%

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

Schweneker

Laimbacher

Zimmer³

et al. 2017

J Virol

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There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transientexpression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP-and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLPgenerating MVA recombinant.IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines.

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Section: Figsupporting

confidence: 92%

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

Schweneker

Laimbacher

Zimmer³

et al. 2017

J Virol

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“…Such a reduction could inhibit the assembly of enveloped viruses that rely on HSPA5 activity for glycoprotein folding. HSPA5 is incorporated into the EBOV virion (Spurgers et al, 2010), and knocking down HSPA5 in mice conferred protection from lethal challenge with EBOV (Reid et al, 2014). Although OSU-03012 was reported to decrease the expression of NPC1 and LAMP1 (Booth et al, 2015), molecules involved in the entry of EBOV and LASV (Carette et al, 2011;Cote et al, 2011;Jae et al, 2014), it did not inhibit EBOV or LASV glycoprotein-dependent entry in our HIV pseudotype system (Fig.…”

Section: Discussionmentioning

confidence: 99%

Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses

Mohr

McMullan

et al. 2015

Antiviral Research

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Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses. OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382 dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved virus-host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeutic intervention.

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“…For example, cyclophilin A has been shown to be recruited into the virion for the infectivity of HIV-1 (71). It would be interesting to use techniques, such as RNA interference (RNAi), to investigate whether the identified host proteins are involved in baculovirus infection, as has been shown for other viruses (72).…”

Section: Discussionmentioning

confidence: 99%

Comparative Proteomics Reveal Fundamental Structural and Functional Differences between the Two Progeny Phenotypes of a Baculovirus

Hou

Zhang

Deng

et al. 2013

J Virol

103

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The replication of lepidopteran baculoviruses is characterized by the production of two progeny phenotypes: the occlusion-derived virus (ODV), which establishes infection in midgut cells, and the budded virus (BV), which disseminates infection to different tissues within a susceptible host. To understand the structural, and hence functional, differences between BV and ODV, we employed multiple proteomic methods to reveal the protein compositions and posttranslational modifications of the two phenotypes of Helicoverpa armigera nucleopolyhedrovirus. In addition, Western blotting and quantitative mass spectrometry were used to identify the localization of proteins in the envelope or nucleocapsid fractions. Comparative protein portfolios of BV and ODV showing the distribution of 54 proteins, encompassing the 21 proteins shared by BV and ODV, the 12 BV-specific proteins, and the 21 ODV-specific proteins, were obtained. Among the 11 ODV-specific envelope proteins, 8 either are essential for or contribute to oral infection. Twenty-three phosphorylated and 6 N-glycosylated viral proteins were also identified. While the proteins that are shared by the two phenotypes appear to be important for nucleocapsid assembly and trafficking, the structural and functional differences between the two phenotypes are evidently characterized by the envelope proteins and posttranslational modifications. This comparative proteomics study provides new insight into how BV and ODV are formed and why they function differently. Baculoviruses are insect-specific pathogens containing large circular double-stranded DNA genomes. Over millions of years of interdependence between viruses and their natural insect hosts, both have undergone a coevolution, such that lepidopteran baculoviruses have developed a unique biphasic replication cycle that generates two progeny phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are embedded in occlusion bodies (OBs) that offer the virions a certain amount of protection against environmental degradation. Once ingested by a susceptible insect, ODVs are released from OBs within the larval midgut and initiate oral infection. After infecting midgut epithelial cells, BVs are synthesized and released to disseminate systemic infection of different tissues within the larval host. The two phenotypes have been used in a wide range of applications. Due to their expandable genome and the presence of very strong promoters, BVs have been established as successful vectors for the expression of thousands of proteins and have also been studied as potential vectors for gene therapy (1). The OBs of certain baculoviruses have been widely used in agriculture and forestry as viable alternatives to chemical insecticides in insect pest control (2).The broad applications of baculoviruses provide a strong rationale for identifying the proteins associated with both phenotypes and for understanding their roles in baculovirus infection. While previous proteomic studies have elucidated the protein compositions of a...

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Identification of Essential Filovirion-associated Host Factors by Serial Proteomic Analysis and RNAi Screen

Cited by 65 publications

References 75 publications

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses

Comparative Proteomics Reveal Fundamental Structural and Functional Differences between the Two Progeny Phenotypes of a Baculovirus

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