Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus

Pyper, J. M.; Gartner, Anton

doi:10.1128/jvi.71.7.5133-5139.1997

Cited by 46 publications

(20 citation statements)

References 40 publications

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“…Of some significance is the presence of two forms of the N protein that are essentially identical except for the presence of an amino-terminal 13-amino-acid extension containing an NLS. However, the BDV N protein derivative lacking the amino-terminal extension sequence is still able to localize to the nucleus, albeit less efficiently (35). Therefore, it appears that multiple NLSs are present in nucleocapsid proteins of several negative-strand viruses that replicate in the nucleus.…”

Section: Discussionmentioning

confidence: 99%

Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

Martins

Johnson

Lawrence

et al. 1998

J Virol

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We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of β-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

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Section: Discussionmentioning

confidence: 99%

Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

Martins

Johnson

Lawrence

et al. 1998

J Virol

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“…At present, the indirect immunofluorescent antibody assay (IFA) is commonly used for the detection of BDV antigens in infected cells (30,33). The IFA method is employed for determination of the viral titers as focusforming units (FFU) or 50% tissue culture infectious dose (TCID,).…”

mentioning

confidence: 99%

Molecular Ratio between Borna Disease Viral‐p40 and ‐p24 Proteins in Infected Cells Determined by Quantitative Antigen Capture ELISA

Watanabe

Zhong

Kobayashi

et al. 2000

Microbiology and Immunology

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We developed the antigen capture enzyme-linked immunosorbent assay (ELISA) systems for quantification of Borna disease virus (BDV) major antigens, p40 and p24. Using these ELISAs, we quantified the two proteins in various BDV-infected materials, including the cell lysates and culture supernatants as well as the homogenates of experimental animal brains. The ELISAs were also applied to measure the infectious titer of BDV in persistently infected cell lines. Quantitative analysis with these ELISAs allowed us to measure the molecular ratio between the p40 and p24 in infected samples. Interestingly, the ratio of p24 to p40 in persistently infected cells was much higher than that observed in acutely infected cells although the ratios in the supernatants from both cell lines were quite similar. BDV-inoculated gerbil brain cells showed a relatively high ratio of p24 to p40 as compared with acutely infected cells. These observations suggested that the molecular ratio between the proteins strongly depended on the infectious status of BDV in the host cells. The ELISA system developed here could be a convenient method for the quantification of BDV infection and may also be beneficial for understanding viral replication and infectious status in the host cells.Key words: Borna disease virus, Antigen capture ELISA, Quantitative, p40 and p24 proteins Borna disease virus (BDV) is a neurotropic, negative-strand RNA virus that causes nonpurulent encephalomyelitis in horses and sheep, and induces neurological symptoms and movement disorders in cattle and cats (6,22). Furthermore, recent epidemiological studies suggested that BDV infection also occurs in humans and is associated with certain psychiatric diseases (11,21,31).The BDV antigenome contains at least five open reading frames (ORFs) (5, 7). Of these ORFs, I and II encode BDV major proteins, p40 (nucleoprotein) and p24 (phosphoprotein), which are highly expressed in virusinfected cells and are commonly detected in BDV-infected cells in vitro and in vivo (1, 2). Furthermore, recent studies have demonstrated that both proteins play important roles in virus replication and transcription (33). Thus, BDV-p40 and -p24 proteins could be good targets for the diagnosis of BDV infection, and quantification of these proteins might be a key step for the successful analysis of BDV infection.At present, the indirect immunofluorescent antibody assay (IFA) is commonly used for the detection of BDV antigens in infected cells (30,33). The IFA method is employed for determination of the viral titers as focusforming units (FFU) or 50% tissue culture infectious dose (TCID,). However, the assay is complicated and is not suitable for large numbers of materials. Furthermore, it is difficult to quantify the level of viral proteins in samples by this IFA method. On the other hand, enzymelinked immunosorbent assay (ELISA) is a sensitive and quantitative method which is commonly used for detecting the antigen in various virus systems. The assay can detect small amounts of viral antigen and al...

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“…The intracellular localization of BoDV ribonucleoproteins (RNPs) is regulated by viral proteins containing NLSs and NESs as well as their interactions. N, P, X, and L harbor NLSs, and N and P contain NESs (11)(12)(13)(14)(15)(16)(17)(18). Previous studies have demonstrated that the interaction of accessory protein X with P enhances the nuclear export of P (19).…”

mentioning

confidence: 99%

“…In contrast, P directly binds to N, leading to the retention of N in the nucleus (13,20). N transcripts encode two isoforms, namely, full-length isoform N (p40) and N-terminally truncated isoform N= (p38), which is translated from the second initiation codon downstream of the NLS (17). While N mainly localizes to the nucleus, the solo expression of N= is found in the cytoplasm (12,13,17).…”

mentioning

confidence: 99%

“…N transcripts encode two isoforms, namely, full-length isoform N (p40) and N-terminally truncated isoform N= (p38), which is translated from the second initiation codon downstream of the NLS (17). While N mainly localizes to the nucleus, the solo expression of N= is found in the cytoplasm (12,13,17). N= can modify the nuclear distribution of P via their interaction (13).…”

mentioning

confidence: 99%

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Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms

Kojima

Sato

Yanai

et al. 2019

J Virol

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Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm, and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results provided not only the mechanism of how mRNA splicing generates viral proteins that have distinct functions but also a novel strategy for replication control of RNA viruses using isoforms with different subcellular localizations. IMPORTANCE Borna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the orthobornavirus genus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than the wild type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small genomes.

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Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus

Cited by 46 publications

References 40 publications

Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

Molecular Ratio between Borna Disease Viral‐p40 and ‐p24 Proteins in Infected Cells Determined by Quantitative Antigen Capture ELISA

Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms

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