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Role of Nuclear Interactions of Paramyxoviral Matrix Proteins: Cellular ANP32B in Host Manipulation and Virus Replication
Project
Project code: FLI-IMVZ-08-Ri-0442, 280662076
Contract period: 01.12.2015
- 30.11.2018
Purpose of research: Experimental development
We recent identified cellular acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) as a nuclear target for Hendra and Nipah virus matrix protein (M). Further preliminary data show that also another member of the paramyxovirus family is able to interact with ANP32B through M. We therefore conclude that targeting of ANP32B is part of a conserved mechanism, at least within the paramxovirinae subfamily. Identification of the detailed role of this interaction in virus replication and host cell manipulation may lead to identification of a so far unknown mechanism involved in replication and pathogenesis. To clarify the role of ANP32B targeting we here investigate (i) which viral M proteins can interact with the nuclear target, (ii) what sequences in the M proteins are required for specific interaction, (iii) how ANP32B influences virus replication and (iv) whether cellular functions of ANP32B in unconventional nuclear export, gene regulation and apoptosis inhibition are modulated by interacting M proteins. Host cell modulatory activities may play a substantial role in virus replication in infected hosts and thus are of high importance to understand virulence and pathogenesis of viruses. Within the project, established protein interaction assays will be combined with systematic mutagenesis of M proteins and generation of recombinant ANP32B-binding defective virus mutants. Influences on ANP32B functions will be addressed by specific assays for nuclear export, gene regulation and apoptosis detection in the presence of plasmid expressed M proteins and in virus infected cells. In vitro identification of specific M / ANP32B dependent molecular mechanisms and development of binding defective virus mutants will offer the outstanding possibility to investigate the role of ANP32B in disease development and immune escape. Whereas the work program of this three years proposal is focused on in vitro characterization of molecular mechanisms, in continuative work the developed virus mutants and knowledge about affected pathways will allow to investigate the role of the cellular interactor for in vivo replication and pathogenesis of henipa- and other paramyxoviruses. As this project focuses on a host-cell interface that may substantially contribute to replication and host manipulation, the proposed work will not only contribute to a better molecular understanding of virus replication but may also offer novel molecular targets for rational interference with virus replication or disease development.
In comparative interaction studies, we were able to show that the interaction of ANP32B with viral matrix proteins is conserved within the Paramxoviridae family, while no evidence of such an interaction with Pneumovirus or Rhabdovirus M proteins could be obtained (Günther et al., 2020. J Gen Virol. 101, 44-58, doi:10.1099/jgv.0.001362). Furthermore, the work showed that the ANP32B dependent nuclear Hendra Virus (HeV) M accumulation depends on the nuclear core import of the ANP32B. Furthermore, the specific coprecipitation of M-ANP32B (no purification of M-ANP32A complexes) showed that the interaction is highly specific and the sole presence of a strongly negatively charged C-terminal region or a leucine-rich N-terminal region, both in all ANP32 proteins are present, are not sufficient for the interaction. Specifically, the detection of an intracellular interaction of ANP32B with M from Newcastle disease virus (NDV, Genus Avulavirus) and Sendai virus (SeV, Genus Respirovirus) is of particular importance, since for the first time paramyxoviruses have a lower security level than henipaviruses can be used in subsequent projects for systematic pathogenesis studies, for example in ANP32B knock out mice. As a natural mouse pathogen, SeV offers enormous potential to investigate the postulated role of ANP32B in viral pathogenesis. Extensive Ala-Scan mutagenesis of the entire HeV M protein and its analysis led to a comprehensive overview of the importance of individual amino acid regions for the ANP32B-dependent core accumulation of the M protein (manuscript in preparation). Despite the identification of 10 intracellularly no longer interacting mutants, the entirety of the data shows that the positive net charge of the protein surface is responsible for highly specific interaction with ANP32B instead of individual amino acid positions. While this presumably makes the generation of recombinant, non-binding virus mutants impossible, the knowledge gained here is an important contribution to the further scientific processing of virus-host interactions, especially with regard to the broad occurrence of this virus-host interaction and its potential importance virus replication and pathogenesis.
Section overview
Subjects
- Animal health
- Biotechnology
Framework programme
Funding programme
Excutive institution
FLI - Institute of Molecular Virology and Cell Biology (FLI - IMVZ)
