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Characterizing important flavivirus-host interactions: Replication, assembly, restriction factors and vaccine development
Örebro University, School of Medical Sciences.ORCID iD: 0000-0002-8366-9310
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The genus Flavivirus (family Flaviviridae) consists of important zoonotic viruses that cause morbidity and mortality worldwide. These viruses are enveloped and have a positive-sense single-stranded RNA genome encoding a polyprotein. Cleavages of the polyprotein by host and viral proteases result in individual viral proteins, including the structural capsid (C), pre-membrane (prM), envelope (E) proteins, and seven nonstructural proteins. Removal of the C-prM-E genes in the flavivirus genome results in replicons that can replicate in transfected cells but do not generate infectious virus particles. The replicon can be co-expressed with the C-prM-E genes in trans, resulting in packaging of the replicon and generation of replicon virus-like particles (RVPs).

During cellular infection, various host proteins are employed, supporting multiple stages of the virus life cycle. In this thesis, we identified and characterized functions of the host lunapark protein and two members of the Endosomal Sorting Complexes Required for Transport Machinery – ALIX and CHMP4A. We also revealed how the host proteins were recruited by virus proteins during infection.

To counteract the virus infection, virus-infected cells can express antiviral proteins. We demonstrated the antiviral mechanism of interferonstimulated gene (ISG) 15 and the E3 ligase for ISG15 conjugation HERC5, which degrades ALIX and CHMP4A, indirectly targets virus infection. Furthermore, using proteomic screening, we identified tripartite motif-containing proteins (TRIM) – TRIM21 and TRIM14 – as restriction factors to Langat virus and Zika virus.

We also established and characterized an RVP production system based on the West Nile virus (WNV) Kunjin strain. The system was used as a vector to express antigens from Ebola virus (EBOV), which can potentially be developed as a vaccine platform against WNV and EBOV.

Place, publisher, year, edition, pages
Örebro: Örebro University , 2022. , p. 59
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 255
Keywords [en]
Flaviviruses, virus-host cell interaction, lunapark, ESCRT, ALIX, CHMP4A, ISG15, TRIMs, replicon virus-like particles, vaccine
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:oru:diva-95779ISBN: 9789175294261 (print)OAI: oai:DiVA.org:oru-95779DiVA, id: diva2:1617671
Public defence
2022-03-11, Örebro universitet, Campus USÖ, hörsal C1, Södra Grev Rosengatan 32, Örebro, 09:00 (English)
Opponent
Supervisors
Available from: 2021-12-07 Created: 2021-12-07 Last updated: 2024-03-06Bibliographically approved
List of papers
1. Roles of the Endogenous Lunapark Protein during Flavivirus Replication
Open this publication in new window or tab >>Roles of the Endogenous Lunapark Protein during Flavivirus Replication
2021 (English)In: Viruses, E-ISSN 1999-4915, Vol. 13, no 7, article id 1198Article in journal (Other academic) Published
Abstract [en]

The endoplasmic reticulum (ER) of eukaryotic cells is a dynamic organelle, which undergoes continuous remodeling. At the three-way tubular junctions of the ER, the lunapark (LNP) protein acts as a membrane remodeling factor to stabilize these highly curved membrane junctions. In addition, during flavivirus infection, the ER membrane is invaginated to form vesicles (Ve) for virus replication. Thus, LNP may have roles in the generation or maintenance of the Ve during flavivirus infection. In this study, our aim was to characterize the functions of LNP during flavivirus infection and investigate the underlying mechanisms of these functions. To specifically study virus replication, we generated cell lines expressing replicons of West Nile virus (Kunjin strain) or Langat virus. By using these replicon platforms and electron microscopy, we showed that depletion of LNP resulted in reduced virus replication, which is due to its role in the generation of the Ve. By using biochemical assays and high-resolution microscopy, we found that LNP is recruited to the Ve and the protein interacts with the nonstructural protein (NS) 4B. Therefore, these data shed new light on the interactions between flavivirus and host factors during viral replication.

Place, publisher, year, edition, pages
Switzerland: MDPI, 2021
Keywords
Flavivirus, Kunjin virus (WNVKUN), Langat virus (LGTV), Zika virus (ZIKV), replication, replicon-expressing cell line, lunapark (LNP), NS4B
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Biology
Identifiers
urn:nbn:se:oru:diva-92668 (URN)10.3390/v13071198 (DOI)000676903600001 ()34206552 (PubMedID)2-s2.0-85109150313 (Scopus ID)
Funder
Knowledge Foundation, 2019009120200063
Note

This article belongs to the Special Issue Host Cell Organelles in Viral Infections: Friends and Foes. Academic Editors: Daniela Ribeiro and Markus Islinger.

Available from: 2021-06-24 Created: 2021-06-24 Last updated: 2024-03-06Bibliographically approved
2. Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection
Open this publication in new window or tab >>Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection
2022 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 96, no 3, article id e01624-21Article in journal (Refereed) Published
Abstract [en]

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we show that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required for flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrate specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical methodology, we show that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT protein, at essential stages of the virus life cycle.

IMPORTANCE: Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection the virus employs ESCRT protein members for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Other ESCRT protein members such as TSG101 are not required for virus production. The ESCRT, ALIX -CHMP4A complex, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

Place, publisher, year, edition, pages
American Society for Microbiology, 2022
Keywords
tick-borne flaviviruses, ESCRT, TSG101, ALIX, CHMP4A, ISG15, HERC5, virus late domain, replicons, replication, assembly, NS3, envelope
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:oru:diva-95744 (URN)10.1128/JVI.01624-21 (DOI)000766835300001 ()34851141 (PubMedID)2-s2.0-85124289154 (Scopus ID)
Available from: 2021-12-03 Created: 2021-12-03 Last updated: 2024-03-06Bibliographically approved
3. Proteomic screening identifies TRIM21 and TRIM14 as antiviral proteins against Langat virus and Zika virus
Open this publication in new window or tab >>Proteomic screening identifies TRIM21 and TRIM14 as antiviral proteins against Langat virus and Zika virus
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-97558 (URN)
Available from: 2022-02-16 Created: 2022-02-16 Last updated: 2024-03-06Bibliographically approved
4. Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice
Open this publication in new window or tab >>Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice
Show others...
2020 (English)In: Microorganisms, E-ISSN 2076-2607, Vol. 8, no 12, article id 1890Article in journal (Refereed) Published
Abstract [en]

Kunjin virus (KUNV) is an attenuated strain of the severe neurotropic West Nile virus (WNV). The virus has a single-strand positive-sense RNA genome that encodes a polyprotein. Following gene expression, the polyprotein is cleaved into structural proteins for viral packaging and nonstructural proteins for viral replication and expression. Removal of the structural genes generate subgenomic replicons that maintain replication capacity. Co-expression of these replicons with the viral structural genes produces reporter virus-like particles (RVPs) which infect cells in a single round. In this study, we aimed to develop a system to generate multivalent RVPs based on KUNV to elicit an immune response against different viruses. We selected the Ebola virus (EBOV) glycoprotein (GP) and the matrix protein (VP40) genes, as candidates to be delivered by KUNV RVPs. Initially, we enhanced the production of KUNV RVPs by generating a stable cell line expressing the KUNV packaging system comprising capsid, precursor membrane, and envelope. Transfection of the DNA-based KUNV replicon into this cell line resulted in an enhanced RVP production. The replicon was expressed in the stable cell line to produce the RVPs that allowed the delivery of EBOV GP and VP40 genes into other cells. Finally, we immunized BALB/cN mice with RVPs, resulting in seroconversion for EBOV GP, EBOV VP40, WNV nonstructural protein 1, and WNV E protein. Thus, our study shows that KUNV RVPs may function as a WNV vaccine candidate and RVPs can be used as a gene delivery system in the development of future EBOV vaccines.

Place, publisher, year, edition, pages
Basel, Switzerland: MDPI, 2020
Keywords
Reporter virus-like particles (RVPs), replicons, Kunjin virus, Ebola virus, glycoprotein (GP), matrix protein (VP40), packaging system, stable cell line, seroconversion, vaccines
National Category
Other Medical Biotechnology
Research subject
Molecular Biology; Biomedicine; Molecular Cellbiology; Biomedical Laboratory Science
Identifiers
urn:nbn:se:oru:diva-87720 (URN)10.3390/microorganisms8121890 (DOI)000602425000001 ()33260425 (PubMedID)2-s2.0-85097158960 (Scopus ID)
Funder
Knowledge Foundation
Note

Funding Agency:

Faculty for Medicine and Health, Örebro University  

Available from: 2020-12-01 Created: 2020-12-01 Last updated: 2024-03-06Bibliographically approved

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Tran, Pham Tue Hung

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