Two domains of the Borna disease virus p40 protein are required for interaction with the p23 protein.

Berg, Mikael; Ehrenborg, C; Blomberg, Jonas; Pipkorn, Rüdiger; Berg, Anna‐Lena

doi:10.1099/0022-1317-79-12-2957

Cited by 28 publications

(30 citation statements)

References 16 publications

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“…It is likeliest that the cytoplasmic distribution of the P modifies the expression level of this protein in the nucleus, resulting in the suppression of viral transcription and replication. Furthermore, intracellular movement of P may also affect the movement of BDV RNPs in infected cells, because P directly interacts with essential components of RNP, such as N, X, and L (1,14,40,45). The retention of P in the cytoplasm could result in prevention of the reimport of the newly synthesized viral RNPs to the nucleus.…”

Section: Discussionmentioning

confidence: 99%

“…To understand the alteration of subcellular localization of BDV proteins, we examined the intracellular localization of P, because this protein shows clear nuclear localization activity and is known to directly bind to all other components of the viral RNP (1,14,40,45). We demonstrate here that the interaction with X inhibits nuclear localization of the BDV P. Transient-transfection analysis with a cDNA clone corresponding a bicistronic 0.8-kb mRNA that expresses both X and P revealed that P efficiently localizes in the cytoplasm only when X is expressed in the cells.…”

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confidence: 99%

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Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

Kobayashi

Zhang

Lee

et al. 2003

J Virol

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Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that belongs to the Mononegavirales order. Unlike other animal viruses in this order, BDV replicates and transcribes in the nucleus of infected cells. Therefore, regulation of the intracellular movement of virus components must be critical for accomplishing the BDV life cycle in mammalian cells. Previous studies have demonstrated that BDV proteins are prone to accumulate in the nucleus of cells transiently transfected with each expression plasmid of the viral proteins. In BDV infection, however, cytoplasmic distribution of the viral proteins is frequently found in cultured cells and animal brains. In this study, to understand the modulation of subcellular localization of BDV proteins, we investigated the intracellular localization of the viral phosphoprotein (P). Transienttransfection analysis with a cDNA clone corresponding to a bicistronic transcript that expresses both viral X and P revealed that P efficiently localizes in the cytoplasm only when BDV X is expressed in the cells. Furthermore, our analysis revealed that the direct binding between X and P is necessary for the cytoplasmic localization of the P. Interestingly, we showed that X is not detectably expressed in the BDV-infected cells in which P is predominantly found in the nucleus, with little or no signal in the cytoplasm. These observations suggested that BDV P can modulate their subcellular localization through binding to X and that BDV may regulate the expression ratio of each viral product in infected cells to control the intracellular movement of the viral protein complexes. The results presented here provide a new insight into the regulation of the intracellular movement of viral proteins of a unique, nonsegmented, negative-strand RNA virus.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

Kobayashi

Zhang

Lee

et al. 2003

J Virol

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“…Association of N with viral RNA might induce a conformational change in N, which in turn might expose a binding site for the P⅐L complex. In this context, it is of interest to note that two independent binding sites for P have been described (31).…”

Section: Discussionmentioning

confidence: 99%

Overlap of Interaction Domains Indicates a Central Role of the P Protein in Assembly and Regulation of the Borna Disease Virus Polymerase Complex

Schneider

Blechschmidt

Schwemmle

et al. 2004

Journal of Biological Chemistry

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The active polymerase complex of Borna disease virus is composed of the viral proteins N, P, and L. The viral X (negative regulatory factor) protein acts as a regulator of polymerase activity. Interactions of P with N and X were previously studied, but interactions with L were poorly defined. Using a mammalian two-hybrid system, we observed that L specifically interacts with P but not with N, X, or itself. Mapping of the L-binding domain in the P molecule revealed that it overlaps with two adjacent domains required for multimerization and interaction with N. Competition experiments showed that the interaction between L and P was inefficient when N was present, indicating that L may preferentially interact with free P in infected cells. Interestingly, a multimerization-defective P mutant maintained the ability to interact with L, N, and X but failed to support reporter gene expression from an artificial Borna disease virus minigenome. Furthermore, dominant negative effects on minigenome activity were only observed when P mutants with an intact multimerization domain were used, suggesting that P multimers, rather than monomers, exhibit biological activity. P mutants lacking functional interaction domains for L or N still formed complexes with these viral proteins when wild-type P was available as a bridging molecule, indicating that P multimers have the potential to act as scaffolds on which the RNA polymerase complex is assembled.The active transcription and replication complex of non-segmented negative strand RNA viruses, termed ribonucleoprotein complex (1), consists of the viral polymerase (L), 1 the phosphoprotein (P), the nucleoprotein (N), and the viral RNA (2). P is a co-transcriptional factor of L, whereas N encapsidates the viral genome to form N⅐RNA complexes (3). For Sendai virus, a prototype paramyxovirus, it was shown that P acts as a scaffold protein, which brings L into close proximity to the N⅐RNA complex (4), thus allowing RNA synthesis. In the absence of P, the N⅐RNA complexes are not recognized by L (5). The viral P protein exerts this scaffolding function by employing independent binding sites for L and the N⅐RNA complex (6). P of Sendai virus contains an additional but distinct binding domain for free N (not bound to viral RNA) (7) and a domain required for multimer formation (8, 9). Complex formation between P and free N prevents aggregation of N (7). Oligomerization is an essential prerequisite for Sendai virus P function in viral transcription and replication (4). The precise function of oligomeric P is unclear. It is assumed that oligomer formation facilitates movement of P (and most likely of the complete polymerase complex) on the RNA template, thereby maintaining polymerase processivity (4). A requirement of P oligomers for viral RNA synthesis was also described for P proteins of other viruses of the order Mononegavirales, including vesicular stomatitis virus (10, 11) and human parainfluenza virus type 3 (12). For the P protein of Sendai virus and human parainfluenza virus type 3, a de...

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“…The various ORF3-coding regions were inserted into the EcoRI and XhoI sites of the pGEX-5T plasmid (Stratagene) for expression in Escherichia coli BL21 (DE3) as described previously (Timmusk et al, 2006). Briefly, for the GST pulldown studies, the PCV2 full-length ORF3 proteins from SG1, SG2, SG3, the full-length ORF3 protein from PCV1 and the partial ORF3 from PCV1, as well as negative control proteins, were expressed by using the pGEX-5T system (Berg et al, 1998). All proteins were expressed in E. coli BL21 (DE3) and induced with 1 mM IPTG at room temperature overnight.…”

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confidence: 99%

“…The two identical sets of samples were then analysed on two parallel SDS-PAGE gels. Unrelated proteins (ORF2 of PCV2 and bornavirus B33 protein) fused with GST (Timmusk et al, 2006;Berg et al, 1998) were used as negative controls. One of the gels was dried and the binding of ORF3 from various PCV isolates to poRGS16 was measured by phosphoimager analysis.…”

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confidence: 99%

Regulator of G protein signalling 16 is a target for a porcine circovirus type 2 protein

Timmusk

Merlot

Lövgren

et al. 2009

Journal of General Virology

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Interaction studies have suggested that the non-structural protein encoded by open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) binds specifically to a regulator of G protein signalling (RGS) related to human RGS16 (huRGS16). The full-length clone of RGS16 was generated from porcine cells and sequence analysis revealed a close relationship to huRGS16 and murine RGS16. In vitro pull-down experiments verified an interaction between porcine RGS16 (poRGS16) and ORF3 from PCV2. Using GST-linked ORF3 proteins from three different genogroups of PCV2 and from porcine circovirus type 1 (PCV1) in the pull-down experiments indicated that there were differences in their ability to bind poRGS16. Quantitative RT-PCR demonstrated that the expression of poRGS16 mRNA could be induced by a number of cell activators including mitogens (LPS and PHA), interferon inducers (ODN 2216 and poly I : C) and the neurotransmitter norepinephrine. Immunofluorescence labelling confirmed the induced expression of poRGS16 at the protein level and suggested that the PCV2 ORF3 protein colocalized with poRGS16 in LPS-activated porcine PBMC. Furthermore, poRGS16 appeared to participate in the translocation of the ORF3 protein into the cell nucleus, suggesting that the observed interaction may play an important role in the infection biology of porcine circovirus. INTRODUCTIONCircoviruses, belonging to the family Circoviridae and encompassing avian and porcine pathogens (Todd et al., 2001), are non-enveloped, single-stranded, circular DNA viruses, with similarities to the anelloviruses, viruses with unknown pathogenicity that are not yet assigned to any family (Biagini, 2004;Todd et al., 2005). Porcine circovirus type 1 (PCV1) is not known to cause any disease, whereas infection with porcine circovirus type 2 (PCV2) is associated with a number of disease syndromes (Allan et al., 1999;Harding, 2004; Segalés et al., 2004;Opriessnig et al., 2007). Of these, it is generally accepted that postweaning multisystemic wasting syndrome (PMWS) is caused by PCV2, but only if other, still not fully specified, infectious or environmental co-factors are present. The lymphoid organs of pigs with severe PMWS are depleted of lymphoid cells and infiltrated by cells of the myeloid lineage. Consequently, PMWS is associated with a suppression of the host immune response and affected animals are more susceptible to secondary infections (Chae, 2005; Segalés et al., 2005).The genome of PCV2 is among the smallest (1769 nt) of all known viruses and appears to have only four major open reading frames, ORFs 1-4 (Meehan et al., 1998). ORF1 encodes two replicase proteins (Rep and Rep9), corresponding to two different splicing products, and ORF2 encodes a structural protein forming the viral capsid (Cap). The proteins encoded by ORF3 and 4 have to date not been assigned any clear function and it has not been fully proven that these putative polypeptides are expressed in infected cells. However, their overall presence and conserved amino acid composition indicate that th...

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Two domains of the Borna disease virus p40 protein are required for interaction with the p23 protein.

Abstract: Borna disease virus (BDV) has five major open reading frames, which encode the proteins p40, p23, gp18, p57 and p190. By analogy with other negative-strand RNA viruses, p40 is a putative nucleoprotein and p23 is a putative phosphoprotein. These proteins are known to form com-

Cited by 28 publications

References 16 publications

Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

Overlap of Interaction Domains Indicates a Central Role of the P Protein in Assembly and Regulation of the Borna Disease Virus Polymerase Complex

Regulator of G protein signalling 16 is a target for a porcine circovirus type 2 protein

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