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Evolutionary Selection of the Nuclear Localization Signal in the Viral Nucleoprotein Leads to Host Adaptation of the Genus Orthobornavirus.

ABSTRACT: Adaptation of the viral life cycle to host cells is necessary for efficient viral infection and replication. This evolutionary process has contributed to the mechanism for determining the host range of viruses. Orthobornaviruses, members of the family Bornaviridae, are non-segmented, negative-strand RNA viruses, and several genotypes have been isolated from different vertebrate species. Previous studies revealed that some genotypes isolated from avian species can replicate in mammalian cell lines, suggesting the zoonotic potential of avian orthobornaviruses. However, the mechanism by which the host specificity of orthobornaviruses is determined has not yet been identified. In this study, we found that the infectivity of orthobornaviruses is not determined at the viral entry step, mediated by the viral glycoprotein and matrix protein. Furthermore, we demonstrated that the nuclear localization signal (NLS) sequence in the viral nucleoprotein (N) has evolved under natural selection and determines the host-specific viral polymerase activity. A chimeric mammalian orthobornavirus, which has the NLS sequence of avian orthobornavirus N, exhibited a reduced propagation efficiency in mammalian cells. Our findings indicated that nuclear transport of the viral N is a determinant of the host range of orthobornaviruses, providing insights into the evolution and host adaptation of orthobornaviruses.

SUBMITTER: Komorizono R

PROVIDER: S-EPMC7698282 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Evolutionary Selection of the Nuclear Localization Signal in the Viral Nucleoprotein Leads to Host Adaptation of the Genus <i>Orthobornavirus</i>.

Komorizono Ryo R Sassa Yukiko Y Horie Masayuki M Makino Akiko A Tomonaga Keizo K

Viruses 20201111 11

Adaptation of the viral life cycle to host cells is necessary for efficient viral infection and replication. This evolutionary process has contributed to the mechanism for determining the host range of viruses. Orthobornaviruses, members of the family <i>Bornaviridae</i>, are non-segmented, negative-strand RNA viruses, and several genotypes have been isolated from different vertebrate species. Previous studies revealed that some genotypes isolated from avian species can replicate in mammalian ce ...[more]

PMID: 33187187

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Project description:BackgroundTwo nuclear localization sequences (NLS) in influenza A virus nucleoprotein (NP) have been demonstrated to be critical for nuclear import of NP and viral ribonucleoprotein complexes. However, a deletion mutant lacking these two signals was still able to localize to the nucleus suggesting the presence of yet another (a third) potential NLS in the NP protein. In order to identify the nature of this potential NLS signal in the NP of a WS/33L influenza virus A strain, we utilized the tools of bioinformatics coupled with functional experimental analyses in the present study.ResultsComparison of the deduced aa sequence of NP of WS/33L strain with the published WS/33 NP sequences revealed that a single amino acid (aa) change (Met to Arg) at position 105 results in converting the flanking regions (between aa position 90-121, a 32-residue stretch) into two classical overlapping bipartite NLS (obpNLS). GenBank search revealed that 9 out of 500 published NP sequences contain a similar Arg at position 105 (instead of Met) with a 100% homology to the obpNLS region. Various NP-green fluorescent protein (GFP) fusion constructs with and without the signal (obpNLS-Arg105) were utilized to understand the functional nature of this signal. We analyzed the transport competency of the expressed chimeric proteins in terms of their cellular localization by confocal immunofluorescence assay. Our analysis revealed that all NP-GFP constructs containing the wild-type (R105) sequence localized predominantly to the nucleus. Constructs lacking the obpNLS or constructs with reverse mutation (R105 to M105) on the other hand exhibited predominant cytoplasmic localization pattern. Interestingly, when the 32 aa obpNLS was fused with an unrelated viral protein (rotavirus NSP6) that has been known to be cytoplasmic protein, the chimeric protein (obpNLS-NSP6) was efficiently transported into the nucleus, indicating an efficient nuclear transport function of the 32-residue obpNLS in the NP of WS/33L strain of influenza A virus.ConclusionThis report while not only establishing a new NLS in the influenza A virus strain, it also reinforces the idea that proper application of bioinformatics-coupled experimental analysis serves as a powerful tool in identifying new functional signals in proteins of interest.

Project description:Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14-3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14-3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of HDAC5, providing a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in other class IIa HDACs-HDAC4 and HDAC9.

Dataset Information

Evolutionary Selection of the Nuclear Localization Signal in the Viral Nucleoprotein Leads to Host Adaptation of the Genus Orthobornavirus.

Publications

Evolutionary Selection of the Nuclear Localization Signal in the Viral Nucleoprotein Leads to Host Adaptation of the Genus <i>Orthobornavirus</i>.

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