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Mutational analysis of the phosphate acceptor sites of the Borna disease virus (BDV) phosphoprotein (P) has suggested a role of phosphorylation for viral spread. However, the studied mutant viruses also had two amino acid exchanges in the X protein, because the reading frames of P and X overlap. To determine the relative contribution of P and X to viral attenuation, we studied a P variant with serine-to-leucine substitutions (P^sub S26L,S28L^) in which the wild-type X sequence was conserved. Viral spread of rBDV-P^sub S26L,S28L^ was impaired in human oligodendroglioma cells and in adult rats. Thus, BDV-P phosphorylation contributes to efficient viral dissemination.[PUBLICATION ABSTRACT]
Arch Virol (2010) 155:789793 DOI 10.1007/s00705-010-0645-9
BRIEF REPORT
Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efcient viral spread
Sonja Schmid Philippe Metz Christine M. A. Prat
Daniel Gonzalez-Dunia Martin Schwemmle
Received: 12 November 2009 / Accepted: 15 January 2010 / Published online: 24 March 2010 Springer-Verlag 2010
Abstract Mutational analysis of the phosphate acceptor sites of the Borna disease virus (BDV) phosphoprotein (P) has suggested a role of phosphorylation for viral spread. However, the studied mutant viruses also had two amino acid exchanges in the X protein, because the reading frames of P and X overlap. To determine the relative contribution of P and X to viral attenuation, we studied a P variant with serine-to-leucine substitutions (PS26L,S28L) in
which the wild-type X sequence was conserved. Viral spread of rBDV-PS26L,S28L was impaired in human oligo
dendroglioma cells and in adult rats. Thus, BDV-P phosphorylation contributes to efcient viral dissemination.
Main text
Borna disease virus (BDV) is a non-segmented negative-strand RNA virus that establishes a persistent infection in
the central nervous system and in cell culture without causing a cytopathic effect [1]. It replicates in the nucleus of the infected cell and uses the splicing machinery for maturation of viral transcripts [2, 3]. The BDV genome encodes six proteins, including the nucleoprotein (N), phosphoprotein (P), X protein, and the RNA-dependent RNA polymerase (L), which are all components of the viral ribonucleoprotein (vRNP) [4]. P is a prime candidate for modulating the polymerase activity, since it is the main scaffold protein of the functional polymerase complex, interacting with N, L, X, and itself [5, 6]. Similar to other members of the order Mononegavirales, P is phosphorylated by cellular kinases, preferentially by protein kinase Ce (PKCe) at serine residues 26 and 28 [7]. Phosphorylation of viral P proteins is generally associated with an increase in viral polymerase activity, as exemplied in detail for the vesicular stomatitis virus (VSV) P protein [8 10]. Surprisingly, a BDV-P mutant (PS26A,S28A) lacking the
PKC phosphate acceptor sites due to substitution of the corresponding serines with alanines leads to an increase in polymerase activity [11]. Thus, in contrast to the P proteins of other members of the Mononegavirales, phosphorylation of BDV-P is a negative regulator of polymerase activity [11]. Furthermore, viral dissemination of rBDV-PS26A,S28A
is severely delayed in various cell culture systems [11], suggesting that regulation of P phosphorylation is a prerequisite for efcient viral spread.
However, since P is expressed from a bicistronic mRNA encoding P and X, the mutations that had been introduced in the open reading frame (ORF) of P when generating PS26A,S28A also resulted in two amino acid substitutions in
the X protein (XI42G,V44G, Fig. 1a). Although viral tran
scription and replication is not dependent on X [12], there is evidence that this protein can modulate viral polymerase activity and prevent apoptosis [13]. Attempts to generate
S. Schmid P. Metz M. Schwemmle (&)
Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Hermann-Herder-Strasse 11, 79104 Freiburg, Germany e-mail: [email protected]
C. M. A. Prat D. Gonzalez-Dunia
INSERM U563, Centre de Physiopathologie de Toulouse Purpan, Universit Paul-Sabatier, Toulouse, France
Present Address:S. SchmidDepartment of Microbiology, Mount Sinai School of Medicine, New York 10029, USA
Present Address:P. MetzDepartment of Molecular Virology, University of Heidelberg, INF 345, 69120 Heidelberg, Germany
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BDV mutants lacking a functional X protein failed [14], further supporting the importance of this protein in the viral life cycle. Thus, the attenuation of rBDV-PS26A,S28A in cell
culture could also be attributed to the mutations in the X protein, although XI42G,V44G showed no obvious differ
ences in its intracellular localization or in its ability to modulate viral polymerase activity [11].
To distinguish between the effects of the destroyed phosphate acceptor sites in P and the mutations in X, we generated a recombinant BDV with serine-to-leucine substitutions at amino acid positions 26 and 28 (rBDVPS26L,S28L). Importantly, these amino acid changes do not affect the amino acid sequence of X (Fig. 1a).
We rst tested the ability of PS26L,S28L to support the
viral polymerase using the BDV minireplicon system [12]. For this purpose, BSR-T7/5 cells constitutively expressing the T7 polymerase [15] were transfected with L-, N- and P-expression constructs coding for either Pwt or the mutant P proteins PS26L,S28L and PS26A,S28A, together with a plasmid
expressing a BDV minireplicon CAT reporter construct. The polymerase cofactor activity of both PS26L,S28L and
PS26A,S28A was higher than that of Pwt (Fig. 1b). To eval
uate whether the increased cofactor activity of PS26L,S28L
was associated with different binding properties with respect to the other vRNP proteins or with changes in its ability to form homo-oligomers, we performed binding studies. To this end, HEK 293T cells were transiently transfected with expression plasmids coding for HA-tagged PS26L,S28L and either N, X, Flag-tagged L, or Flag-tagged
PS26L,S28L. After lysis of the cells, the complexes that were
formed were immunoprecipitated and visualized by western blot as described [11]. As shown in Fig. 1ce, in comparison to Pwt, the efciency of binding between
PS26L,S28L and N, PS26L,S28L and L, and PS26L,S28L and X
was not changed. Furthermore, self-oligomerization of PS26L,S28L was also unaffected (Fig. 1f). These data corre
late with our previous ndings [11] and suggest that the increased cofactor activity of PS26L,S28L is not due to
obvious differences in its binding afnities, especially since the PKC phosphate acceptor sites do not overlap with the known binding sites of the viral nucleocapsid proteins. According to the current model of the polymerase complex of members of the families Paramyxoviridae, Rhabdoviridae and Bornaviridae [4, 16, 17], P plays a dynamic role in transcription and replication and undergoes multiple conformational changes during these processes. Since the PKC phosphorylation sites are located within an intrinsically disordered part of P [18], we speculate that P phosphorylation could interfere with certain conformations of P during transcription and replication.
To check the phosphorylation status of PS26L,S28L in
infected cells, we performed in vivo labeling experiments of Vero cells and human oligodendroglioma (Oligo) cells derived from glial brain tumor cells that were persistently infected with rBDV-Pwt, rBDV-PS26L,S28L, or rBDV
PS26A,S28A. Infected cells were incubated for 22 h with
radioactively labeled orthophosphate. Thereafter, P was immunoprecipitated from whole-cell extracts, and the amount of phosphorylation was quantied by autoradiography as described [11]. As expected, PS26L,S28L as well as
Fig. 1 Inuence of the PKC sites of BDV-P on viral polymerase activity in the minireplicon system. a Bicistronic 0.8 kb mRNA containing two open reading frames (ORF) coding for the P and X protein (-1 frame shift). Arrows indicate the position of the two PKC phosphate acceptor sites in the P-ORF. Amino acids and nucleotides highlighted in bold indicate differences with respect to the wild-type sequence. b The viral polymerase activity with the indicated P mutants was determined using a T7-based BDV minireplicon assay as described [11]. Minireplicon activity was analyzed by CAT ELISA 72 h after transfection. Mean values of four independent experiments are shown. Complete reaction mixtures without P (no P) served as negative control reactions. cf Binding efciency of the BDV-P mutant PS26L, S28L to N, L, X, and itself. HEK 293T cells were transfected with expression plasmids encoding HA-tagged P (HA-P) and N (C), Flag-tagged L (Flag-L) and HA-P(D), HA-P and X (E) and Flag-tagged P (Flag-P) and HA-P(F). Twenty-four hours post-transfection, whole-cell extracts were prepared and subjected to immunoprecipitation (IP) using the indicated antibodies and antisera. Bound proteins were separated by SDS-PAGE and identied by western blot analysis
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Phosphorylation of BDV-P 791
PS26A,S28A was less phosphorylated than Pwt, both in Vero and in Oligo cells (Fig. 2a).
In previous studies, we showed that rBDV-PS26A,S28A
spreads less efciently compared to rBDV-Pwt in all tested
cell culture systems, including primary neurons [19], rat astroglia (C6), Vero, and Oligo cells [11]. Similar to rBDV-PS26A,S28A, the spread of rBDV-PS26L,S28L is
impaired in neurons [19]. To test whether this attenuation was also observed in cells other than neurons, we expanded the analysis and investigated the growth kinetics of rBDVPS26L,S28L in C6, Vero and Oligo cells. Figure 2b shows the
growth kinetics of two different viral stocks from one rescue of rBDV-Pwt and PS26L,S28L, respectively. In con
trast to rBDV-PS26A,S28A, no delay in accomplishing
complete infection by rBDV-PS26L,S28L was observed in C6
and Vero cells (Fig. 2b). However, viral spread was severely delayed in Oligo cells (Fig. 2b). Thus, unlike rBDV-PS26A,S28A, which is attenuated in all cell types
investigated [11], rBDV-PS26L,S28L exhibits a cell-type-
dependent growth disadvantage. Therefore, mutations in P
and X both contribute to the attenuation phenotype of rBDV-PS26A,S28A in cell culture, whereas only the phos
phorylation of P residues 26 and 28 by PKC appears to play a central role for efcient viral spread in Oligo cells (Fig. 2b) and neurons [19].
The cell-type-dependent attenuation of rBDV-PS26L,S28L
could reect differences in the interferon (IFN) system between the various cell lines used. Indeed, IFN does not block replication of BDV in C6 or Vero cells, which lack a functional IFN-b gene [20]. In contrast, BDV growth is affected in Oligo cells after administration of exogenous IFN [20]. Thus, induction of IFN as a result of BDV infection might explain the cell-type specic attenuation of rBDV-PS26L,S28L. However, we did not detect any induc
tion of interferon-stimulated genes (ISG) in acutely infected Oligo cells (data not shown). Furthermore, we did not observe any difference in the sensitivity of rBDV-Pwt,
rBDV-PS26A,S28A or rBDV-PS26L,S28L to IFN-treatment
during acute or persistent infection of Oligo cells (data not shown). Based on these results, we hypothesize that the
Fig. 2 Functional characterization of the BDV mutant rBDVPS26L,S28L. a Uninfected cells or cells that were persistently infected with the indicated viruses were incubated with 32P-labeled ortho-phosphate for 22 h. P was subsequently immunoprecipitated, separated by SDS-PAGE and transferred to a nitrocellulose membrane. The upper panel shows an autoradiograph in which the levels of radioactively labeled P (32P-labeled P) can be seen. The middle panel shows the detection of total P using a P-specic antiserum. The IgG
heavy chain detected by the secondary antibody is indicated. The lower panel gives the phosphorylation efciency of P and P mutants of three independent experiments. The ratio of 32P/total P with Pwt was set to 100%. b C6, Vero, and Oligo cells were simultaneously infected with 1,000 focus-forming units (FFU) of two different viral stocks from one rescue for each indicated BDV strain. The number of infected cells was determined by immunouorescence using an N-specic antiserum as described [11]
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attenuation of rBDV-PS26L,S28L in Oligo cells is primarily
due to the mutations in PS26L,S28L rather than to an IFN-
mediated effect.To evaluate the growth properties of rBDV-PS26L,S28L in
vivo, we infected adult rats with 5000 FFU of BDV by the intranasal route. All rats infected with rBDV-PS26L,S28L
became severely diseased after 3240 days and had to be sacriced (Fig. 3a). They showed typical symptoms including rufed fur and paralysis of the hind limbs accompanied by weight loss (data not shown). Nevertheless, the kinetics of disease development were signicantly delayed, since animals infected with rBDV-Pwt were
moribund at earlier time points. As expected, viral transcripts and proteins (Fig. 3b) were detected in brain samples from all diseased rats, regardless of the BDV strain used. The situation was more complex in rats infected with rBDV-PS26A,S28A. Although some animals also became
sick, with a similar delay in disease onset as rats infected with rBDV-PS26L,S28L, 60% of the rBDV-PS26A,S28A-
infected animals showed no signs of disease throughout the experiment (Fig. 3a) and were free of viral antigen, as determined by western blot analysis (Fig. 3b).
Based on these data, we speculate that the delayed onset of disease symptoms in rBDV-PS26L,S28L or rBDV
PS26A,S28A-infected animals reects the impairment of
these viruses to spread efciently in neurons [19] and presumably results from the mutations introduced at the PKC phosphorylation sites of P. The fact that the phosphorylation mutants of P show increased polymerase activity in the minireplicon but lead to delayed spread of recombinant virus in neurons and rats indicates additional functions of phosphorylated P in the viral life cycle. P might be important for intracellular transport of viral RNPs in neurons. For rabies virus P, another member of the Mononegavirales, interaction with a component of the dynein motor complex has been demonstrated [21, 22]. Furthermore, P could play a role in virus assembly. It has been shown that P phosphorylation of human respiratory syncytial virus regulates budding [23]. In addition, the more pronounced attenuation of rBDV-PS26A,S28A suggests
that wild-type X is also required for efcient infection. In summary, we provide evidence that BDV-P phosphorylation by PKC at serine 26 and 28 contributes to efcient viral spread in a cell-type-dependent manner and to viral virulence in vivo. Whether phosphorylation of BDV-P affects intracellular transport or other steps of the viral life cycle remains to be shown.
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft. We thank members of the Schwemmle and Gonzalez-Dunia labs for critical reading of the manuscript.
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