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Journal of Virology, April 2003, p. 4283-4290, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4283-4290.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Selective Virus Resistance Conferred by Expression of Borna Disease Virus Nucleocapsid Components

Till Geib, Christian Sauder, Sascha Venturelli, Christel Hässler, Peter Staeheli,* and Martin Schwemmle*

Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, D-79104 Freiburg, Germany

Received 19 July 2002/ Accepted 27 December 2002


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ABSTRACT
 
Persistent viral infections can render host cells resistant to superinfection with closely related viruses by largely uncharacterized mechanisms. We present evidence for superinfection exclusion in brains of Borna disease virus (BDV)-infected rats and in persistently infected Vero cells, and we suggest that acquired resistance to BDV is due to unbalanced intracellular levels of viral nucleocapsid components. We observed that expression of BDV protein P, N, or X rendered human cells resistant to subsequent challenge with BDV but not with other RNA viruses, indicating that incorrect stoichiometry of nucleocapsid components selectively blocked the polymerase activity of incoming viruses. Vero cells containing high levels of an untranslatable BDV-N transcript remained virus susceptible, demonstrating that viral protein rather than RNA mediated resistance. Transient overexpression of BDV-P in persistently infected Vero cells was also remarkably effective against BDV, indicating that the intracellular balance of viral nucleocapsid components could serve as a target for future therapeutic antiviral strategies.


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INTRODUCTION
 
Borna disease virus (BDV) persistently infects neurons and other cells of the central nervous systems of a broad range of warm-blooded animals and possibly humans (7, 21, 32, 34). Natural and experimental infections with BDV may result in immune system-mediated neurological disease and behavioral abnormalities (17, 28). BDV is a newly classified nonsegmented negative-stranded RNA virus that, unlike other Mononegavirales, replicates in the nuclei of infected cells and employs the splicing machinery for the maturation of some of its transcripts. Its genome codes for at least six viral proteins, of which the phosphoprotein P, the nucleoprotein N, and the RNA polymerase complex-associated protein X are abundantly present in infected cells (19, 31).

It was recently suggested that the remarkable stability of the BDV genome might be a consequence of superinfection exclusion (11). It is well known that certain persistent viral infections can confer resistance to subsequent infection of host cells with the same virus or a closely related virus. This phenomenon, designated superinfection exclusion or homologous interference (1, 20), has been described for retroviruses (5, 9), hepatitis B virus (6), alphaviruses (1, 20), Rift valley fever virus (3), and pestiviruses (23). Superinfection exclusion by retroviruses is primarily due to down-regulation of host cell surface entry receptors (5, 9). A similar mechanism may account for superinfection exclusion in hepatitis B virus-infected duck hepatocytes (6). Poorly defined mechanisms seem to contribute to superinfection exclusion of alphaviruses; one of these is posttranscriptional gene silencing (2). RNA silencing also seems to play a role in the case of Rift valley fever virus, because superinfection exclusion in insect cells could be mimicked by expression of nontranslatable viral RNAs (3).

We now present evidence that superinfection exclusion is operative in brain cells of BDV-infected rats and in persistently infected Vero cells. We further demonstrate that the BDV proteins N, P, and X, but not a nontranslatable viral RNA derived from the N gene, mediated resistance to infection with BDV in human UTA6 cells. Remarkably, transient expression of P also inhibited BDV in persistently infected cells, suggesting that established infections can also be influenced by disturbing the balance of viral nucleocapsid components. These observations pave the way for novel therapeutic strategies against BDV, and possibly other persisting RNA viruses, that target the balanced expression of viral nucleocapsid components.


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MATERIALS AND METHODS
 
Inducible expression constructs. The coding sequences of BDV-N and wild-type or N-terminally flagged BDV-P of strain He/80FR (27), and those of N-terminally flagged nucleoprotein of Thogoto virus (38), were cloned into plasmid pTRE (Clontech, Heidelberg, Germany), which contains promoter elements that permit tetracycline-mediated repression of transgene expression in UTA6 cells. cDNA N-mut, comprising nucleotides 95 to 1167 of the BDV strain He/80FR genome, was generated by first mutating the AUG initiation codon at positions 95 to 97 to ACG and then introducing a -1 frameshift mutation through deletion of nucleotide 122. cDNA encoding BDV-X (strain He/80FR) was cloned into a variant of plasmid pBI (Clontech) that carries a bidirectional promoter permitting simultaneous tetracycline-regulated expression of yellow fluorescent protein (YFP) and the gene of interest.

Cells. A human osteosarcoma cell clone (UTA6) expressing a tetracycline-activated repressor (10) was transfected with the various pTRE or pBI expression plasmids together with pTK-Hyg (Clontech) by using Effectene (Qiagen, Hilden, Germany). Permanently transfected cells were selected in a medium containing G418 (250 µg/ml), hygromycin (250 µg/ml), and tetracycline (1 µg/ml). The resulting cell clones were screened by indirect immunofluorescence analysis (IFA) or autofluorescence to identify those with tightly restricted transgene expression. Vero cells and a Vero cell clone (VA-9) permanently expressing MxA cDNA (12) were infected with BDV strain He/80 or H215 as described previously (26).

Transient cDNA transfections. Semiconfluent monolayers of Vero cells persistently infected with BDV strain He/80 or H215 were transfected with the various expression plasmids by using Effectene (Qiagen). Cells were allowed to grow for 5 to 7 days before viral antigen levels in transfected cells were evaluated by IFA using appropriate mixtures of antibodies which permitted the simultaneous detection of transgene- and virus-encoded proteins.

BDV stocks. Strain H215 was isolated by standard procedures (26) from brain material of a horse with neurological disease (kindly provided by S. Herzog, Giessen, Germany). Virus stocks were obtained from cells persistently infected with strain H215 or He/80FR (27). Stocks of rat-adapted H215 were generated by homogenizing brains of Lewis rats that had been infected with cell culture-derived virus as newborns. Strain RW98 (33) was prepared as described previously (30).

Virus infections. UTA cells were grown in the presence of tetracycline (1 µg/ml) or in its absence as required. Forty-eight hours later, samples of the indicated stocks of BDV were added (5 x 104 focus-forming units/106 cells). Cultures were maintained by splitting at a ratio of 1:3 three times a week. Infection status was monitored by IFA using BDV-specific antisera. For infection by cell-to-cell spread, UTA6 cells persistently infected with BDV strain He/80FR were cocultivated for the indicated times either with uninfected VA-9 cells or with VA-9 cells persistently infected with BDV strain H215. VA-9 cells in the mixed cultures were identified by IFA using a rabbit antiserum to MxA (12), and their infection status was monitored by IFA using monoclonal antibody (MAb) Bo18 (15). This antibody recognizes the epitope LYEPPASLP of BDV-N (4), which is mutated in strain H215. Where indicated, transgenic UTA6 cell lines were infected through coculture with Vero cells persistently infected either with BDV strain He/80 or, in the case of BDV-N expressing cells, with BDV strain H215 to differentiate between transgene- and virus-encoded protein. After 48 h the culture medium was supplemented with hygromycin B and G418 (250 µg/ml each) to selectively kill the Vero cells. Infection of UTA6 cells was monitored by IFA using appropriate antibodies. Intracerebral infection of rats was carried out as described previously (29).

Antibodies. The mouse anti-FLAG ({alpha}-FLAG) MAb M2 was purchased from Sigma (Deisenhofen, Germany), the rabbit anti-green fluorescent protein antibody was purchased from Clontech, rabbit {alpha}-BDV-N was a gift from F. Grässer (Homburg, Germany), rabbit {alpha}-BDV-P IgG was a gift from I. Lipkin (Irvine, Calif.), MAb Bo18, specific for BDV-N (4, 15), and rabbit {alpha}-BDV-X were both gifts from J. Richt (Giessen, Germany), and MAb 21E7, specific for BDV-P, was a gift from L. Stitz (Tübingen, Germany).

Protein and RNA expression studies. For Western blotting, proteins in crude lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12 to 15% gels) before transfer to nitrocellulose membranes. Following treatment with 10% nonfat dry milk or blocking buffer (Sigma-Genosys, Cambridge, United Kingdom), the membranes were incubated with the indicated BDV-specific antibodies. Northern blot analysis was performed by using radiolabeled cDNA fragments originating from BDV-N or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as described previously (35). Immunohistochemical analyses of brains from BDV-infected animals were carried out as described previously (30).


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RESULTS
 
Superinfection resistance of BDV-infected rats. Newborn Lewis rats were infected with BDV strain H215 at postnatal day 1 and challenged 4 (n = 2), 8 (n = 4), 14 (n = 1), 21 (n = 1), or 32 (n = 1) days later with strain RW98. Between 25 and 32 days after the second infection, brains were analyzed by using either MAb Bo18, which recognizes N proteins of all known BDV strains except isolate H215 (4), or a rabbit antiserum that recognizes N proteins of all BDV strains. In brains of rats challenged 4 days after infection with strain H215, only a few scattered Bo18-positive (RW98-infected) brain cells were detected by immunohistochemistry (data not shown). The number of Bo18-positive brain cells was even lower in brains of double-infected rats challenged at day 8 (Fig. 1B). Accordingly, when brain lysates of these rats were subjected to Western blot analysis, only faint RW98-specific signals were detected (Fig. 1A), indicating that the second BDV strain had failed to replicate efficiently. In brains of rats challenged at later time points with RW98, Bo18-positive cells were no longer detectable. In contrast, strain H215 had disseminated efficiently in the rat brains, as evidenced by the fact that the rabbit antiserum detected high levels of viral antigen (Fig. 1A and data not shown) in a large number of brain cells (Fig. 1B and data not shown). Control experiments showed that RW98 replicated very efficiently in brains of naive Lewis rats that were infected at postnatal day 4, 8, 14, 21, or 32 and sacrificed 25 to 32 days later (Fig. 1 and data not shown). Simultaneous infection of a newborn rat with approximately equal doses of BDV strains H215 and RW98 resulted in infection of brain cells with the two strains at comparable frequencies, as judged by staining of consecutive brain sections with the monoclonal or polyclonal N-specific antibody, respectively (Fig. 1C). Thus, both virus strains spread well in rat brains, but superinfection of BDV-infected cells was strongly impaired.



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  		FIG. 1. Brains of Lewis rats are resistant to superinfection with BDV. (A) At postnatal day 1, rats either were infected by the intracerebral route with BDV strain H215 or were left uninfected, as indicated, before challenge with strain RW98 at postnatal day 8. Animals were sacrificed at postnatal day 33, and samples of brain homogenates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting using either a rabbit antiserum ({alpha}N) that recognizes N proteins of both BDV strains or MAb Bo18, which recognizes the N protein of strain RW98 but not that of strain H215. (B) Paraffin sections of rat brains infected either with both BDV strains or with strain RW98 only were subjected to immunohistochemical analysis using either the {alpha}N rabbit antiserum or MAb Bo18. (C) A newborn rat was simultaneously infected at postnatal day 1 with strains RW98 and H215. The animal was sacrificed at postnatal day 33, and consecutive paraffin sections of the brain were immunostained either with a BDV-N-specific rabbit antiserum to detect infection with both strains or with MAb Bo18 to specifically detect N of strain RW98.

BDV-infected cells in culture are resistant to superinfection. To directly test whether BDV-infected cells can be infected by viral cell-to-cell spread, Vero cells persistently infected with BDV strain H215 and UTA6 cells persistently infected with strain He/80 were cocultured at an initial ratio of 1:1 for 21 days. After 7, 14, and 21 days, the culture was screened by double IFA for evidence of He/80 replication in H215-infected Vero cells by using MAb Bo18, which recognizes N of He/80 but not N of H215. Since we used Vero cells that were engineered to constitutively express the marker protein MxA, reliable identification of Vero cells in the mixed culture was easily achieved. Fewer than 1% double-positive Vero cells were found at any time (Fig. 2), indicating that productive infection of H215-infected Vero cells by He/80 had not occurred. In contrast, spreading of He/80 from UTA6 to uninfected Vero cells was very efficient. After 7 days of coculture, more than 70% of these cells were Bo18 positive, and this proportion increased to 97% by day 21 (Fig. 2).



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  		FIG. 2. Vero cells persistently infected with BDV strain H215 cannot be superinfected with strain He/80. He/80-infected UTA6 cells were cocultivated for 21 days with MxA-positive Vero cells that were either persistently infected with BDV strain H215 or uninfected when the coculture experiment was started. Productive infection of Vero cells with He/80 was monitored by double IFA using MAb Bo18, which recognizes N of strain He/80 but not N of strain H215, and an MxA-specific rabbit antiserum. Statistical analysis of the coculture infection experiment was performed at days 7, 14, and 21. Vero cells not infected at the start of the coculture experiment (shaded bars) were compared with Vero cells that were persistently infected with BDV strain H215 from the very beginning (black bars).

Expression of either N, P, or X alone in UTA6 cells confers resistance to BDV infection. To investigate the possibility that components of the BDV ribonucleoprotein complex might play a role in superinfection exclusion, we generated UTA6 cell lines expressing either BDV-N (UTA-N) or BDV-P with an N-terminal FLAG-epitope (UTA-Flag-P) under the control of a tetracycline-dependent repressor. Cocultivation of unrepressed UTA-N cells with Vero cells infected with BDV strain H215 did not result in infection of the transgene-expressing UTA cells, as judged by double IFA at 14 days after the onset of coculture (Fig. 3A). However, cells lacking detectable expression of the transgene were readily infected (Fig. 3A). Similarly, unrepressed UTA-P cells were also highly resistant to infection with BDV strain He/80, as virtually no BDV-N staining was detected in cells expressing Flag-P. In contrast, cells lacking transgene expression were frequently infected (Fig. 3A). A similar picture emerged when UTA6 cells that simultaneously expressed BDV-X and YFP (UTA-X/YFP) were cocultivated with BDV strain He/80-infected Vero cells. YFP-positive cells (which should also express BDV-X) were resistant to infection with BDV. By contrast, cells that did not express the transgenes frequently contained virus-encoded antigen (Fig. 3A), indicating that BDV-X also inhibited BDV replication. UTA6 cells that expressed either YFP alone (Fig. 3A) or the nucleoprotein of Thogoto virus (THOV-NP) (Fig. 3A) contained high levels of BDV antigen at 2 weeks after the onset of the coculture, demonstrating that neither an unrelated protein nor a nucleocapsid protein of a distantly related virus was able to block BDV replication in UTA6 cells. Quantification of the infection rate by counting of double immunofluorescence-positive cells revealed that YFP-expressing cells and THOV-NP-expressing cells contained similarly high frequencies of BDV antigen-positive cells at 7 days after the onset of coculture (69 and 72%, respectively), which further increased to 76 and 81%, respectively, by day 14, indicating that virus infection by cell-to-cell contact was very efficient (Fig. 3B). By contrast, at 2 weeks after the onset of coculture, fewer than 1% of UTA cells expressing BDV-N or BDV-Flag-P and only about 2% of UTA cells expressing BDV-X showed signs of BDV replication.



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  		FIG. 3. Expression of N, P, and X but not of control proteins protects cells from subsequent infection with BDV. UTA6 cells were transfected with plasmids allowing tetracycline-regulated expression of the indicated BDV proteins either alone or under the control of a bidirectional promoter together with YFP (BDV-X/YFP). Cell cultures permanently expressing the various BDV proteins or control proteins were grown in the absence of tetracycline to induce expression of the transgene products. Forty-eight hours later, the various cell cultures were mixed with BDV-infected Vero cells. Two days after coculture, the Vero cells were selectively eliminated by including hygromycin (250 µg/ml) in the culture medium. Surviving UTA6 cells were analyzed by staining for transgene expression and for infection with BDV at 7 and 14 days after the onset of the coculture experiment. (A) Infection by coculture of various UTA6 cells expressing either BDV proteins or control proteins. Transgene expression was analyzed either by IFA with either MAb Bo18 to detect N, the anti-FLAG MAb M2 to detect Flag-P, or FLAG-THOV-NP or by autofluorescence to detect YFP. Virus infection was monitored by IFA using either a rabbit anti-BDV-P antiserum or a rabbit anti-BDV-N antiserum. (B) Statistical analysis. Seven and 14 days after the onset of the coculture experiment, cells were double immunostained and analyzed by IFA. Percentages of cells simultaneously positive for transgene expression and virus infection were determined.

We next infected UTA6 cell lines that permanently expressed either BDV-N (UTA-N) or BDV-P (UTA-P) with He/80 or H215 to test if BDV infection and spread are blocked in these cells. Western blot analysis revealed that in unrepressed cells the steady-state level of P was comparable to that in persistently BDV infected UTA6 cells, while the steady-state level of N was slightly lower (Fig. 4A). Exposure of unrepressed UTA-N cells to BDV strain H215 did not result in persistent infection of the culture, as judged by the almost complete absence of virus-encoded P antigen-positive cells at 3 weeks post-H215 challenge (Fig. 4B, UTA-N on). By contrast, when the H215 challenge was performed with repressed UTA-N cells, more than 70% of the cells in the culture contained BDV-P upon analysis 3 weeks later (Fig. 4B, UTA-N off), indicating successful virus infection. Similarly, BDV challenge experiments with unrepressed UTA-P cells revealed that they were also highly resistant to infection with BDV, as virtually no N-positive cells were detected in such cultures at 3 weeks after infection with strain He/80 (Fig. 4B, UTA-P on). In the repressed state approximately 50% of UTA-P cells were infected at 3 weeks after initial virus challenge (Fig. 4B, UTA-P off). In contrast, approximately 50% of UTA6 cells expressing YFP (UTA-YFP) were infected within 3 weeks, and a similar infection rate was achieved in UTA6 cells lacking YFP (data not shown). It should be noted that the polyclonal serum used to visualize P in this experiment recognizes transgene- and virus-encoded P. However, since uninfected UTA-P cells maintained in tetracycline-containing medium were not stained (data not shown), the signals observed in Fig. 4B must be derived from virus-encoded P. We performed control experiments to determine if unrepressed UTA-N and UTA-P cells gained resistance to unrelated viruses. We found that they remained susceptible to infection with vesicular stomatitis virus and influenza A virus (data not shown).



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  		FIG. 4. Persistent BDV infections cannot be established in cell cultures homogeneously expressing BDV-N or -P. (A) Comparison by Western blot analysis of uninfected (lane 1) and BDV-infected (lane 2) UTA6 cells with UTA6 cells harboring tetracycline-regulated expression plasmids coding for either BDV-P (lanes 3 and 4) or BDV-N (lanes 5 and 6). Transfected cells were cultured in the presence (off) or absence (on) of tetracycline for 48 h prior to harvest. A serum of BDV-infected mice was used to detect the viral gene products N and P. (B) UTA6-N and -P cells were grown in the presence or absence of tetracycline for 48 h before infection with either strain He/80 (UTA-P) or strain H215 (UTA-N). Infection status was assessed 3 weeks later by double IFA. In UTA-P cultures, an {alpha}P rabbit serum was used to detect transgene products and MAb Bo18 was used for virus staining. To visualize N transgene expression in UTA6-N cells, MAb Bo18 was used. Virus staining in these cells was performed with an {alpha}P rabbit antiserum.

Resistance is conferred by viral protein rather than viral RNA. To investigate the possibility that RNA silencing mediated BDV resistance, we established a UTA6 cell culture that simultaneously expressed YFP and a nontranslatable BDV-N transcript (N-mut) carrying a defective initiation codon plus a frameshift mutation. Northern blot analysis using similar amounts of transgene-positive cells demonstrated that the level of mutated N transcripts in UTA-YFP/N-mut cells was only about twofold lower than the level of functional N transcripts in unrepressed UTA-N cells (Fig. 5A). Using a polyclonal {alpha}N antiserum which recognizes various N- and C-terminal epitopes of N (4), we failed to detect N-specific signals in YFP/N-mut-expressing cells by IFA (data not shown), providing evidence that no N protein is expressed from N-mut transcripts. Initial infection experiments with cell-free BDV stocks showed that cells which expressed high levels of YFP (and therefore presumably also contained high levels of N-mut transcripts) were frequently infected with BDV (Fig. 5B). To quantify the rate of infection, a noncloned population of UTA-YFP/N-mut cells was infected by coculture with Vero cells infected with BDV strain He/80 and was analyzed by IFA 7 or 14 days postcocultivation (Fig. 5C). The majority of YFP-positive cells supported virus replication (Fig. 5C and D). Furthermore, the infection rate at 7 or 14 days postcocultivation was similar to that of UTA6 control cells expressing YFP only (Fig. 5D) or to that of UTA6 cells expressing no YFP (data not shown), strongly suggesting that the presence of viral RNA alone was not sufficient to confer BDV resistance.



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  		FIG. 5. BDV resistance is not RNA mediated. (A) UTA6 cells were transfected with an expression plasmid that contains a bidirectional promoter which allows the simultaneous tetracycline-regulated expression of YFP and a nontranslatable BDV-N transcript (N-mut). Samples of RNA from an unrepressed UTA6 cell clone synthesizing wild-type N (UTA-N) and from an unrepressed UTA6 cell clone expressing the bidirectional construct (UTA-YFP/N-mut) were analyzed on Northern blots for BDV-N and GAPDH transcripts. (B) Cells of an unrepressed UTA-YFP/N-mut clone were infected with BDV strain He/80, and successful viral replication in these cells was visualized 3 weeks later by autofluorescence (green) and IFA using an {alpha}BDV-X rabbit antiserum (red). (C) A cell culture stably transfected with the N-mut/YFP plasmid was cultivated in the absence of tetracycline. Forty-eight hours later, the cell culture was cocultivated with Vero cells persistently infected with BDV. Another 48 h later, Vero cells were eliminated with a medium containing hygromycin (250 µg/ml). Cell cultures were screened by IFA for BDV infection 7 and 14 days after the addition of BDV-infected Vero cells. A rabbit serum recognizing P was used to visualize virus infection of YFP-positive cells (red). (D) Coculture infections were performed as described in the legend to panel C with cells expressing either BDV-N, N-mut/YFP, or YFP alone. By use of IFA, the percentages of cells which were simultaneously positive for transgene products and virus at 7 and 14 days after the onset of the coculture experiment were determined.

Different consequences of P, N, and X overexpression in persistently infected cells. We performed transient cDNA transfection experiments with persistently infected Vero cells to determine whether transgenic expression of BDV nucleocapsid components has a negative effect on persisting BDV. Analysis by double IFA at 5 to 7 days posttransfection revealed that 50 to 70% of the persistently infected cells expressing flagged BDV-P contained greatly reduced levels of virus-encoded N antigen (Fig. 6A and B), whereas fewer than 7% of the cells which expressed YFP showed low BDV antigen levels (Fig. 6B). Interestingly, if analysis was performed at earlier times, for example, at 2 to 4 days post-plasmid DNA transfection, we observed grossly altered intracellular distribution of the transgene product (Fig. 6A, upper left panel, inset), indicating that plasmid-encoded P was complexed with other viral proteins. By contrast, no virus inhibition was observed when chronically infected Vero cells were transiently transfected with cDNA constructs encoding either BDV-N (Fig. 6A and B) or BDV-X (Fig. 6B), suggesting that these viral nucleocapsid components are less potent inhibitors than P. We noted that cDNA-encoded N was predominantly located in the cytoplasm under these conditions (compare Fig. 6A and 4B), indicating that it was used by the persisting virus but that, in contrast to the situation with BDV-P, this interaction did not result in virus inhibition. It remains to be determined if the apparent small enhancing effect of N transgene expression on virus-encoded P antigen levels (Fig. 6A, lower left panel) is biologically significant.



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  		FIG. 6. BDV replication in persistently infected Vero cells is inhibited by transient expression of BDV-P but not by expression of other BDV nucleocapsid components. (A) H215-infected Vero cells expressing flagged BDV-P ({alpha}FLAG staining) frequently contained reduced levels of virus-encoded N antigen upon analysis at 5 to 7 days posttransfection. By contrast, cells containing plasmids encoding N (Bo18 staining) frequently contained slightly elevated levels of viral P antigen. Note that plasmid-encoded N, which usually accumulates in the nucleus, was mainly present in the cytoplasm in persistently infected cells. Similarly, plasmid-encoded P accumulated in the cytoplasm at early times (day 3) posttransfection (inset). At later times, when virus inhibition was evident, plasmid-encoded P moved to the nucleus. (B) Vero cells persistently infected with BDV strain He/80 or H215 were transiently transfected with cDNA constructs encoding either Flag-P, N, X and YFP (X/YFP), or YFP only. At 5 to 7 days posttransfection, double IFA was performed with appropriate antibody mixtures, and transgene-expressing cells were classified into three groups according to whether they contained low (blue bars), moderate (gray bars), or high (red bars) levels of virus-encoded antigen.


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DISCUSSION
 
It was recently demonstrated that BDV-infected cells are resistant to superinfection with a closely related BDV strain (11). The failure to reinfect already BDV infected rats with another strain of BDV indicates that superinfection exclusion also occurs in vivo. Although we cannot formally exclude the possibility that infection with the first virus resulted in the production of a soluble factor(s) which might interfere with infection by the second virus, independent additional evidence suggests that the antiviral action of cytokines is not the major mechanism underlying our observation in vivo. It has been shown that de novo infection of rat lung cells, rat C6 glioblastoma cells, and cultured rat hippocampus slices is not efficiently blocked by rat alpha/beta interferon (16, 36; C. Sauder, unpublished data). The observed early block of infection of the brain with the second virus is consistent with the view that infection of neurons with the first virus blocked the second virus from using the same connected neurons for spreading. It is known that BDV spreads axonally and intraneuronally in the brain along defined natural anatomic connections (14). This may also explain why the remaining uninfected cells in infected brains (Fig. 1B, upper panels) do not support replication of the challenge virus. In addition, it remains possible that only certain neuronal populations express the still undefined cellular receptor for BDV at a level that is sufficiently high for virus entry. Our observation that virus-infected cells are also resistant to superinfection in coculture experiments further supports the concept that dissemination of BDV in the brain by cell-to-cell spread is blocked by infected cells.

Viral interference resulting from virus-induced receptor down-regulation probably accounts for superinfection exclusion of noncytolytic murine retroviruses (9) and hepatitis B viruses (6) and may contribute to the efficiency by which cytolytic viruses are able to limit multiple infections of host cells (5). It seems unlikely that a similar mechanism is employed by BDV, because receptor-mediated syncytium formation by infected cells can readily be induced by acidification of the culture medium (13), indicating the presence of functional host cell surface receptors in spite of persistent infection with BDV. Nevertheless, taking into account that the receptor density might be critical for virus entry and that cell fusion at a low pH may not necessarily reflect the natural entry process, we cannot formally exclude this possibility. Superinfection exclusion could alternatively result from interference with other early viral multiplication steps, including nuclear transport of incoming viral ribonucleocapsids, synthesis and transport of viral mRNAs, or viral genome amplification. In the case of BDV, all these steps are mediated by components of the viral RNA polymerase complex, of which proteins N, P, and X are abundantly present in persistently infected cells (31). Because the N/P ratio is known to vary dramatically between cells that are acutely or persistently infected with BDV (37), we tested the hypothesis that unbalanced expression of viral nucleocapsid components might confer superinfection exclusion. Our results showed that human UTA6 cells expressing plasmid-encoded N, P, or X, but not unrelated control proteins, exhibited a high degree of resistance to challenge with BDV. Importantly, this resistance was observed not only after direct challenge with the virus but also in the case of infection through cell-to-cell contact, which presumably represents the major route of viral dissemination in the brain and in cell culture. Since UTA6 cells expressing a nontranslatable transcript from the N gene remained susceptible, it is unlikely that RNA interference phenomena (18) are important in our system. Thus, resistance to BDV is most likely conferred by the specific action of viral proteins and not by RNA silencing.

At present, we can only speculate about where the block of resistance mediated by N, P, and X occurs. Although we cannot exclude receptor down-regulation by these proteins, it remains difficult to envision that all three proteins should have similar capacities in regulating surface expression of a viral receptor(s), especially since P and N show a predominantly nuclear localization when expressed alone, supporting our hypothesis that the critical viral factors block a nuclear step of the BDV replication cycle. Virus inhibition via cell reprogramming by BDV protein also cannot be excluded. However, cells expressing individual virus proteins remained susceptible to infection with other negative-strand RNA viruses, arguing against this possibility. Since N, P, and X are components of the polymerase complex, we hypothesize that expression of the individual proteins interferes directly with the viral polymerase activity. This would be compatible with the view that persisting BDV prevents superinfection of its host cell by a previously undiscovered mechanism, namely, inhibition of the polymerase activity of incoming virus particles.

While cell culture systems for genetic manipulation of negative-strand RNA viruses were being established, it was noted that balanced expression of the various viral nucleocapsid components is important for successful rescue of recombinant viruses (25). However, the inhibitory effects resulting from nonbalanced expression of these viral factors were mostly moderate, except when massive overexpression was evident, as, for example, in the case of the L polymerase of vesicular stomatitis virus (22). It should be noted that although reverse genetic techniques have meanwhile been established for most negative-strand RNA viruses (8), a few exceptional viruses still cannot be rescued successfully. This group of viruses includes BDV and hantaviruses, which persistently infect mammalian cells. It is tempting to speculate that the technical difficulties experienced in efforts to manipulate the genomes of these viruses are at least in part due to the particularly delicate nature of the RNA polymerase complexes of these viruses. We hypothesize that viruses which are capable of successfully establishing a noncytolytic persistent infection might have evolved efficient strategies for controlling the activities of their RNA polymerases. We therefore suspect that BDV, and possibly other viruses which successfully persist in their host cells, can regulate its polymerase activity with a well-balanced ratio of the polymerase complex components, which are strongly inhibitory when present at unfavorable concentrations. Thus, it seems likely that the resulting conditions in persistently infected cells are highly unfavorable for incoming viruses, which probably require a different composition of the polymerase complex for initial genome amplification. It is of interest that a similar, yet mechanistically distinct, concept has been adopted by flaviviruses for mediating superinfection exclusion of their insect host cells. In this case, genomic RNA of incoming flaviviruses is probably not replicated well because a protease encoded by the resident virus efficiently processes newly synthesized viral polymerase precursors, thereby selectively down-regulating the replicase but not the transcriptase activity of the polymerase complex (20).

It is unknown at present whether other persisting RNA viruses might similarly control the replication of their genomes by altering the balance of RNA polymerase accessory factors. It should be noted, however, that viruses which rely on this strategy are probably highly susceptible to therapeutic interventions which target the balanced expression of viral cofactors. Intriguingly, we observed that resident BDV of persistently infected Vero cells was also susceptible to interference by P but resistant to interference by N or X, indicating that P is the most decisive viral factor which, when present at unfavorable concentrations, can efficiently block the activity of the viral polymerase. The observed grossly altered intracellular distribution of the transgene product (Fig. 6A, upper left panel, inset), suggests that plasmid-encoded P was indeed initially employed by the viral polymerase and that this engagement was eventually deleterious for the persisting virus. Since recently improved techniques for efficient transport of large polypeptides across intact biological membranes are quickly becoming available (24), novel strategies for the design of a new generation of drugs against persistent virus infections may soon become feasible.


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ACKNOWLEDGMENTS
 
We thank Darius Moradpour for providing UTA6 cells and for technical advice, Sybille Herzog for providing a sample of horse brain containing BDV strain H215, and Ian Lipkin, Lothar Stitz, Friedrich Grässer, and Jürgen Richt for providing antibodies.

This work was supported by a grant from the Deutsche Forschungsgesellschaft.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany. Phone: 49-761-203-6579. Fax: 49-761-203-5053. E-mail: staeheli{at}ukl.uni-freiburg.de. Back

* Corresponding author. Present address: Institute of Medical Virology, University of Zürich, Gloriastrasse 30, CH-8028 Zürich, Switzerland. Phone: 41-1-634-4906. Fax: 41-1-634-2789. E-mail: mschwemm{at}immv.unizh.ch. Back


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REFERENCES
 
    1
  1. Adams, R. H., and D. T. Brown. 1985. BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus. J. Virol. 54:351-357.[Abstract/Free Full Text]
    2. 2
  2. Adelman, Z. N., C. D. Blair, J. O. Carlson, B. J. Beaty, and K. E. Olson. 2001. Sindbis virus-induced silencing of dengue viruses in mosquitoes. Insect Mol. Biol. 10:265-273.[CrossRef][Medline]
    3. 3
  3. Billecocq, A., M. Vazeille-Falcoz, F. Rodhain, and M. Bouloy. 2000. Pathogen-specific resistance to Rift Valley fever virus infection is induced in mosquito cells by expression of the recombinant nucleoprotein but not NSs non-structural protein sequences. J. Gen. Virol. 81:2161-2166.[Abstract/Free Full Text]
    4. 4
  4. Billich, C., C. Sauder, R. Frank, S. Herzog, K. Bechter, K. Takahashi, H. Peters, P. Staeheli, and M. Schwemmle. 2002. High-avidity human serum antibodies recognizing linear epitopes of Borna disease virus proteins. J. Biol. Psychiatry 15:979-987.
    5. 5
  5. Bour, S., R. Geleziunas, and M. A. Wainberg. 1994. The role of CD4 and its downmodulation in establishment and maintenance of HIV-1 infection. Immunol. Rev. 140:147-171.[CrossRef][Medline]
    6. 6
  6. Breiner, K. M., S. Urban, B. Glass, and H. Schaller. 2001. Envelope protein-mediated down-regulation of hepatitis B virus receptor in infected hepatocytes. J. Virol. 75:143-150.[Abstract/Free Full Text]
    7. 7
  7. Carbone, K. M. 2001. Borna disease virus and human disease. Clin. Microbiol. Rev. 14:513-527.[Abstract/Free Full Text]
    8. 8
  8. Conzelmann, K. K. 1998. Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes. Annu. Rev. Genet. 32:123-162.[CrossRef][Medline]
    9. 9
  9. Delwart, E. L., and A. T. Panganiban. 1989. Role of reticuloendotheliosis virus envelope glycoprotein in superinfection interference. J. Virol. 63:273-280.[Abstract/Free Full Text]
    10. 10
  10. Englert, C., X. Hou, S. Maheswaran, P. Bennett, C. Ngwu, G. G. Re, A. J. Garvin, M. R. Rosner, and D. A. Haber. 1995. WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis. EMBO J. 14:4662-4675.[Medline]
    11. 11
  11. Formella, S., C. Jehle, C. Sauder, P. Staeheli, and M. Schwemmle. 2000. Sequence variability of Borna disease virus: resistance to superinfection may contribute to high genome stability in persistently infected cells. J. Virol. 74:7878-7883.[Abstract/Free Full Text]
    12. 12
  12. Frese, M., G. Kochs, U. Meier-Dieter, J. Siebler, and O. Haller. 1995. Human MxA protein inhibits tick-borne Thogoto virus but not Dhori virus. J. Virol. 69:3904-3909.[Abstract]
    13. 13
  13. Gonzalez-Dunia, D., B. Cubitt, and J. C. De la Torre. 1998. Mechanism of Borna disease virus entry into cells. J. Virol. 72:783-788.[Abstract/Free Full Text]
    14. 14
  14. Gosztonyi, G., and H. Ludwig. 1995. Borna disease—neuropathology and pathogenesis. Curr. Top. Microbiol. Immunol. 190:39-73.[Medline]
    15. 15
  15. Haas, B., H. Becht, and R. Rott. 1986. Purification and properties of an intranuclear virus-specific antigen from tissue infected with Borna disease virus. J. Gen. Virol. 67:235-241.[Abstract/Free Full Text]
    16. 16
  16. Hallensleben, W., and P. Staeheli. 1999. Inhibition of Borna disease virus multiplication by interferon: cell line differences in susceptibility. Arch. Virol. 144:1209-1216.[CrossRef][Medline]
    17. 17
  17. Hornig, M., M. Solbrig, N. Horscroft, H. Weissenbock, and W. I. Lipkin. 2001. Borna disease virus infection of adult and neonatal rats: models for neuropsychiatric disease. Curr. Top. Microbiol. Immunol. 253:157-177.[Medline]
    18. 18
  18. Hutvagner, G., and P. D. Zamore. 2002. RNAi: nature abhors a double-strand. Curr. Opin. Genet. Dev. 12:225-232.[CrossRef][Medline]
    19. 19
  19. Jordan, I., and W. I. Lipkin. 2001. Borna disease virus. Rev. Med. Virol. 11:37-57.[CrossRef][Medline]
    20. 20
  20. Karpf, A. R., E. Lenches, E. G. Strauss, J. H. Strauss, and D. T. Brown. 1997. Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus. J. Virol. 71:7119-7123.[Abstract]
    21. 21
  21. Lieb, K., and P. Staeheli. 2001. Borna disease virus—does it infect humans and cause psychiatric disorders? J. Clin. Virol. 21:119-127.[CrossRef][Medline]
    22. 22
  22. Meier, E., G. G. Harmison, and M. Schubert. 1987. Homotypic and heterotypic exclusion of vesicular stomatitis virus replication by high levels of recombinant polymerase protein L. J. Virol. 61:3133-3142.[Abstract/Free Full Text]
    23. 23
  23. Mittelholzer, C., C. Moser, J. D. Tratschin, and M. A. Hofmann. 1998. Porcine cells persistently infected with classical swine fever virus protected from pestivirus-induced cytopathic effect. J. Gen. Virol. 79:2981-2987.[Abstract]
    24. 24
  24. Morris, M. C., J. Depollier, J. Mery, F. Heitz, and G. Divita. 2001. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19:1173-1176.[CrossRef][Medline]
    25. 25
  25. Muhlberger, E., B. Lotfering, H. D. Klenk, and S. Becker. 1998. Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virus-specific monocistronic minigenomes. J. Virol. 72:8756-8764.[Abstract/Free Full Text]
    26. 26
  26. Nowotny, N., J. Kolodziejek, C. O. Jehle, A. Suchy, P. Staeheli, and M. Schwemmle. 2000. Isolation and characterization of a new subtype of Borna disease virus. J. Virol. 74:5655-5658.[Abstract/Free Full Text]
    27. 27
  27. Pleschka, S., P. Staeheli, J. Kolodziejek, J. A. Richt, N. Nowotny, and M. Schwemmle. 2001. Conservation of coding potential and terminal sequences in four different isolates of Borna disease virus. J. Gen. Virol. 82:2681-2690.[Abstract/Free Full Text]
    28. 28
  28. Pletnikov, M. V., T. H. Moran, and K. M. Carbone. 2002. Borna disease virus infection of the neonatal rat: developmental brain injury model of autism spectrum disorders. Front. Biosci. 7:D593-D607.
    29. 29
  29. Sauder, C., W. Hallensleben, A. Pagenstecher, S. Schneckenburger, L. Biro, D. Pertlik, J. Hausmann, M. Suter, and P. Staeheli. 2000. Chemokine gene expression in astrocytes of Borna disease virus-infected rats and mice in the absence of inflammation. J. Virol. 74:9267-9280.[Abstract/Free Full Text]
    30. 30
  30. Sauder, C., D. P. Wolfer, H. P. Lipp, P. Staeheli, and J. Hausmann. 2001. Learning deficits in mice with persistent Borna disease virus infection of the CNS associated with elevated chemokine expression. Behav. Brain Res. 120:189-201.[CrossRef][Medline]
    31. 31
  31. Schneemann, A., P. A. Schneider, R. A. Lamb, and W. I. Lipkin. 1995. The remarkable coding strategy of Borna disease virus: a new member of the nonsegmented negative strand RNA viruses. Virology 210:1-8.[CrossRef][Medline]
    32. 32
  32. Schwemmle, M. 2001. Borna disease virus infection in psychiatric patients: are we on the right track? Lancet Infect. Dis. 1:46-52.[CrossRef][Medline]
    33. 33
  33. Schwemmle, M., C. Jehle, S. Formella, and P. Staeheli. 1999. Sequence similarities between human bornavirus isolates and laboratory strains question human origin. Lancet 354:1973-1974.[CrossRef][Medline]
    34. 34
  34. Staeheli, P., C. Sauder, J. Hausmann, F. Ehrensperger, and M. Schwemmle. 2000. Epidemiology of Borna disease virus. J. Gen. Virol. 81:2123-2135.[Free Full Text]
    35. 35
  35. Staeheli, P., M. Sentandreu, A. Pagenstecher, and J. Hausmann. 2001. Alpha/beta interferon promotes transcription and inhibits replication of Borna disease virus in persistently infected cells. J. Virol. 75:8216-8223.[Abstract/Free Full Text]
    36. 36
  36. Von Rheinbaben, F., L. Stitz, and R. Rott. 1985. Influence of interferon on persistent infection caused by Borna disease virus in vitro. J. Gen. Virol. 66:2777-2780.[Abstract/Free Full Text]
    37. 37
  37. Watanabe, M., Q. Zhong, T. Kobayashi, W. Kamitani, K. Tomonaga, and K. Ikuta. 2000. Molecular ratio between Borna disease viral-p40 and -p24 proteins in infected cells determined by quantitative antigen capture ELISA. Microbiol. Immunol. 44:765-772.[Medline]
    38. 38
  38. Weber, F., O. Haller, and G. Kochs. 1996. Nucleoprotein viral RNA and mRNA of Thogoto virus: a novel "cap-stealing" mechanism in tick-borne orthomyxoviruses? J. Virol. 70:8361-8367.[Abstract]

Journal of Virology, April 2003, p. 4283-4290, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4283-4290.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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