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Journal of Virology, May 2005, p. 6544-6550, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6544-6550.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the Genomic Promoter of Borna Disease Virus (BDV): Implications of 3'-Terminal Sequence Heterogeneity for BDV Persistence

Debralee Rosario, Mar Perez, and Juan Carlos de la Torre*

The Scripps Research Institute, La Jolla, California

Received 27 August 2004/ Accepted 28 December 2004


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ABSTRACT
 
Borna disease virus (BDV) is an enveloped virus with a genome organization characteristic of Mononegavirales. However, based on its unique features, BDV is considered the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. We have described the establishment of a reverse genetics system for the rescue of BDV RNA analogues, or minigenomes, that is based on the use of polymerase I/polymerase II. Using this BDV minigenome rescue system, we have examined the functional implications of the reported sequence heterogeneity found at the 5' and 3' termini of the BDV genome and also defined the minimal BDV genomic promoter within the 3'-terminal 25 nucleotides. Our results suggest that the accumulation of RNA genome species containing truncations of one to three nucleotides at their 3' termini may contribute to modulate BDV RNA replication and gene expression during long-term persistence.


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TEXT
 
Borna disease virus (BDV) causes central nervous system disease in a variety of vertebrate species, which is frequently manifested by behavioral abnormalities (20, 33, 37). However, both viral and host factors can influence symptoms and pathology associated with BDV infection (18-20, 35, 37, 43). Serologic and molecular epidemiological data indicate that the natural host range of BDV as well as its prevalence and geographic distribution are very broad (19, 20, 35-37, 43). Moreover, there is evidence that BDV can infect humans, being possibly associated with certain neuropsychiatric disorders (2, 5, 31, 35-37, 43).

BDV is an enveloped virus with a nonsegmented, negative-strand RNA genome. Its genome (ca. 8.9 kb), the smallest among known nonsegmented, negative-strand RNA viruses, has an organization similar to that of other mononegaviruses (12, 40). Six major open reading frames are found in the BDV genome sequence (3'-N-p10/P-M-G-L-5') (12, 40). BDV has the property, unique among known animal nonsegmented, negative-strand RNA viruses, of a nuclear site for the replication and transcription of its genome (3, 9). Moreover, BDV uses a remarkable diversity of strategies, including RNA splicing, for the regulation of its genome expression (10, 12, 40, 41, 45). Based on its distinct features among known mononegaviruses, BDV is considered the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales.

We have established an RNA polymerase I/polymerase II system for intracellular reconstitution of BDV RNA replication and transcription (30). In this system, a BDV RNA analog, or minigenome, is intracellularly synthesized by the cellular RNA polymerase I. This BDV minigenome RNA contains the BDV 5' and 3' untranslated regions cis-acting sequences required for RNA synthesis mediated by the BDV polymerase (30, 39, 40, 42). Encapsidation of the polymerase I-derived BDV minigenome RNA by plasmid-supplied BDV N and P generates a template that is recognized by the intracellularly reconstituted BDV polymerase to direct synthesis of full-length antiminigenome RNA (replicate) and a subgenomic mRNA (transcript) that codes for the chloramphenicol acetyltransferase (CAT) reporter gene (30). Using this system, we have documented that BDV L, N, and P are the minimal viral trans-acting factors required for efficient RNA synthesis mediated by the BDV polymerase (30). Interestingly, among the two N isoforms, p40 and p38, present in BDV-infected cells, only p40 was strictly required for virus polymerase activity (30). Moreover, the BDV p10 polypeptide, coded by the P cistron, exhibited a strong inhibitory effect on BDV minigenome expression (30). Similar findings have been reported by others using a system based on the bacteriophage T7 RNA polymerase to direct the intracellular synthesis of BDV RNA analogs (42).

As with other mononegaviruses, the genome of BDV exhibits partial inverted complementarity at its termini. In the case of other prototypic mononegaviruses such as Rous sarcoma virus, vesicular stomatitis virus, and rabies virus, this terminal complementarity includes the very first nucleotides at the 5' and 3' ends of their genomes and antigenomes (25, 46, 49). However, for BDV the best-fit alignment of the published sequences showed that the first three or four nucleotides remained unpaired (32). In addition, the 3' and 5' termini of the BDV genome RNA appeared to exhibit some degree of heterogeneity (4, 11, 32). These findings could reflect features unique to BDV or technical difficulties in determining the authentic BDV genomic termini.

BDV is noncytolytic, and only very low infectivity is associated with BDV infection (37). These features determined that cell-free BDV virions and BDV RNP preparations that provided the source of RNA to determine published BDV genomic sequence termini were obtained from long-term persistently infected cells. Heterogeneity, predominantly short deletions, has been documented at the genomic termini during persistence with other negative-strand RNA viruses (22-24). It is therefore plausible that the originally determined terminal sequences of the BDV genome corresponded to molecules with altered termini. Here, we describe studies aimed at the molecular and functional characterization of the sequence heterogeneity exhibited by the 5' and 3' termini of the BDV genomic RNA species.

We first conducted a comprehensive analysis of the BDV 5' and 3' end genomic sequences to confirm or revisit previously documented termini of the BDV genome. We reasoned that it seemed highly unlikely that nonfunctional genome molecules would accumulate at high levels during the first 96 h of infection, which corresponds to a linear phase of increased virus RNA replication and transcription (Fig. 1). On the other hand, during its persistence in cell culture, BDV might accumulate molecules with altered termini. Therefore, we sought to compare BDV genomic terminal sequences determined using viral genome RNA isolated at early stages (96 h postinfection) of infection, and during long-term persistence (passage 20 [P20]). For this purpose we isolated nuclear encapsidated RNA from BDV-infected and mock-infected control Vero cells at 96 h postinfection as described (9). Encapsidated RNA was selected based on its resistance to treatment with micrococcal nuclease. Likewise, nuclear encapsidated RNA was also isolated from persistently BDV infected Vero cells at passage 20 (Vero/BDVp20).



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  		FIG. 1. Linear increase in levels of BDV RNA replication and transcription during the first 96 h of infection. (A) Vero cells were infected with BDV (multiplicity of infection of 0.5 FFU/cell). At the indicated h postinfection, RNA was isolated and analyzed by Northern blot using a 32P-labeled double-stranded DNA probe for the virus N. This probe detects the genome RNA as well as N mRNA and dicistronic N+P mRNA species. (B) Quantitation of levels of virus RNA replication and transcription. The blot from section A was subjected to phosphorimager analysis. Bands corresponding to BDV genome RNA and N mRNA were quantified. Levels of genome RNA (replication) and N mRNA (transcription) detected at 24 h postinfection were assigned arbitrary values of 1. Levels of genome RNA and N mRNA at times 48, 72, and 96 h postinfection were normalized with respect to those determined at 24 h postinfection.

We used three approaches to determine BDV terminal genomic sequences. The first was based on the use of standard 3' rapid amplification of cDNA ends (RACE) methods (17), by tailing the genomic and antigenomic (also contained in RNP) 3' ends with A's using poly(A) polymerase and reverse transcription (RT)-PCR to amplify regions containing genomic and antigenomic 3' end regions. For this purpose, the reverse transcription step was done using an adapter primer containing a dT tract and additional sequences to facilitate subsequent PCR and cloning steps. For the PCR, we used a primer complementary to sequences present in the adapter primer together with either an antisense N primer or a sense L primer, which allowed the amplification of the genome and antigenome 3' ends, respectively.

The second approach was based on the use of T4 RNA ligase treatment of RNA isolated from BDV ribonucleoprotein under conditions that favored the formation of RNA circular monomers over linear dimer products via ligation of their 5' and 3' ends (4, 11, 32). This RNA ligation was followed by RT-PCR, using PCR primers oriented across the ligation bridge. PCR products containing the specific RNA bridge sequences were cloned and sequenced.

The third approach was based on intermolecular ligation of the RNA and an oligonucleotide of known sequence, followed by RT-PCR using procedures described for the identification of the correct 3' end of the GB virus B genome (38), and characterization of the termini of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) (23). Briefly, an RNA oligonucleotide of specific sequence (38) was ligated to the 3'or 5' end of the BDV genomic RNA, and the corresponding junction was subsequently amplified by RT-PCR, cloned, and sequenced. Ligation of the RNA oligonucleotide to the genomic 3' end was favored by using an RNA oligonucleotide that was 5'-phosphorylated and 3'-blocked (NaIO4 oxidized). Ligation of the same RNA oligonucleotide to the genomic 5' end was facilitated by using a 5'-OH/3'-OH RNA oligonucleotide and treatment of the BDV genomic RNA with tobacco acid pyrophosphatase to eliminate a possible blockade of the 5' end, followed by phosphorylation of the 5' end by kinase treatment.

PCR products were resolved by agarose gel electrophoresis using optimized conditions to separate fragments with possible small size differences with respect to the predicted size based on previously determined 5' and 3' end BDV genomic sequences. All detected PCR products with the predicted sizes, or higher, were isolated and cloned. For each PCR product we sequenced several independent clones. RNA obtained from mock-infected cells did not yield specific RT-PCR products with any of these methods. As an additional control, we subjected to the same experimental approaches an in vitro transcribed BDV RNA containing predetermined known 5' and 3' end sequences of the BDV genome. This RNA (T7-5'gggA/3'C) corresponded to the sequence of minigenome 5'A/3'C but containing three additional G's at its 5' end. These extra G residues allowed for optimal transcriptional activity of the T7 RNA polymerase promoter. All the corresponding RT-PCR clones derived from using T7-ggg5'A/3'C as template RNA had the predicted 5' and 3' end sequences (not shown), indicating that these experimental procedures could be used to obtain reliable 5'- and 3'-terminal sequences of a given RNA species.

We observed a significant degree of heterogeneity at the 5'- and 3'-terminal sequences involving small (1 to 4 nucleotides) deletions with respect to the longest determined sequences (Fig. 2A). Based on the frequencies of these newly determined (Fig. 2B) and previously reported (4, 11, 32) sequences, we derived several potential consensus termini sequences for the BDV genome RNA (Fig. 2C). These results revealed three distinct features: the difficulty to determine unequivocally whether an A residue is located at the 5' or 3' end of the genome, consistently with previous findings (4, 11, 32), we observed a lack of perfect terminal complementarity, and genomic terminal sequences derived from BDV RNA obtained from long-term persistently infected cells (P20) exhibited a higher degree of heterogeneity compared to those determined using viral RNA isolated at 96 h postinfection Statistical analysis (Student t test) revealed that 3'-terminal genomic sequences corresponding to groups a, b, and c were predominant at 96 h postinfection, whereas those corresponding to groups d, e and f accumulated during viral persistence and become dominant at P20 (P < 0.01). This statistical analysis also revealed that during BDV persistence there was a trend towards the accumulation of 5' termini genomic sequences of groups e, d, and f. However, the differences between 96 h postinfection and P20 samples concerning their 5' termini genomic sequences were only marginally statistically significant.



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  		FIG. 2. Sequence analysis of the 3' and 5' termini of the BDV genome RNA. (A) Summary of the different 3'- and 5'-terminal sequences obtained using three experimental approaches: 3' RACE, RNA self-ligation, and oligonucleotide-RNA ligation (38). In the case of the 3'RACE approach, the 5' end genomic sequences were inferred from the 3' end antigenome sequences determined experimentally. (B) Frequency of the different types of 3'- and 5'-terminal sequences obtained by each of the indicated methods. N, indicates the total number of sequences determined by each method using RNA isolated either at 96 h postinfection or from Vero/BDV-Pi at P20. (C) Predicted 3'/5' structures derived from the data in A and B.

We then examined the possible functional significance of terminal heterogeneity of the BDV genome RNA. For this we assessed the expression of a variety of BDV minigenome containing the different types of 5' and 3' termini identified most commonly by us (Fig. 2) and others (4, 11, 32). In addition, based on published data with many other nonsegmented RNA viruses (8, 27), we considered the possibility that our experimental approaches failed to uncover existing molecules exhibiting a perfect terminal complementarity (5'/3'Pan) (Fig. 2C).

Differences in levels or stability of minigenome RNA synthesized by polymerase I, as well as in the efficiency of N-mediated encapsidation among the minigenome RNAs would affect the amount of template initially available to the intracellularly reconstituted BDV polymerase. This, in turn, would influence the assessment of the minigenome activity. To address this issue we first determined the ability of the different minigenome RNAs to be encapsidated by N and P, using as reference minigenome 5'A/3'C (Fig. 3A). For this, extracts of cells expressing the different minigenome RNAs together with BDV N and P were treated with micrococcal nuclease. Only the correctly encapsidated RNAs were expected to be resistant to this treatment (9).



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  		FIG. 3. (A) Levels of encapsidated polymerase I-derived minigenome RNAs. Cells (2 x 105 cells/4.5 cm2) were transfected using Lipofectamine with the indicated polymerase I minigenome (0.5 µg), together with pC-N (0.5 µg) and pC-P (0.04 µg). Sixty hours after transfection, cell extracts (CE) were prepared and treated with micrococcal nuclease. After inactivation of micrococcal nuclease by addition of EGTA, RNA was isolated and analyzed by semiquantitative RT-PCR using specific primers to amplify a 280-bp segment of the CAT open reading frame present within the minigenome RNAs. For each sample the same dilutions of the cDNA product were used for PCR as described (28). Equal amounts of PCR products obtained from the same amount of cDNA and corresponding to the exponential phase of PCR minigenome amplification were analyzed by agarose gel electrophoresis. (B) Levels of minigenome-derived CAT activity. Cells (2 x 105 cells/4.5 cm2) were transfected as in A with the indicated polymerase I minigenome (0.5 µg), together with pC-N (0.5 µg), pC-P (0.04 µg), and pC-L (0.5 µg). Sixty hours after transfection, cell extracts (CE) were prepared and assayed for CAT activity (29). (C) Detection of BDV CAT mRNA and antiminigenome RNA species. Cells were transfected with the indicated plasmid combinations as in B, and sixty hours later total cellular RNA was isolated. Ci. Total RNA (7 µg) was analyzed by Northern blot hybridization using a CAT antisense riboprobe. Cii. Equal amounts of total RNA (30 µg) from each sample were fractionated by oligo(dT) chromatography, and the corresponding polyadenylated RNA fractions were analyzed by Northern blot hybridization as in Ci.

After micrococcal nuclease treatment, RNA samples were isolated and analyzed by semiquantitative RT-PCR using specific primers to amplify a 280-bp segment of the CAT open reading frame contained within the minigenome RNA. The use of RT-PCR, instead of the less sensitive Northern blot analysis, was needed because only a minor fraction of polymerase I-derived minigenome RNA is initially encapsidated by plasmid-supplied N and P viral products. For each sample we first subjected to PCR the same series of RT-derived cDNA dilutions to determine conditions corresponding to the exponential phase of amplification. All the minigenomes examined were encapsidated with similar efficiencies (Fig. 3A). As predicted, minigenome-derived RT-PCR products were not obtained using micrococcal nuclease-treated samples prepared from cells transfected with minigenome constructs in the absence of N (not shown).

We then determined levels of CAT activity (Fig. 3B), as well as CAT mRNA and antiminigenome RNA species (Fig. 3C) associated with each minigenome construct. Both minigenome 5'A/3'C and minigenome 5'G/3'A exhibited similar levels of CAT activity as well as synthesis of CAT mRNA and antiminigenome RNA species mediated by the BDV polymerase. Thus, the presence of a purine (A) or pyrimidine (C) at the 3' end of the minigenome RNA did not appear to have a significant effect on genome promoter activity measured by the minigenome rescue assay. Evidence indicates that polymerases of nonsegmented RNA viruses usually cannot initiate synthesis with a pyrimidine triphosphate (1). The BDV polymerase might therefore be unique in this respect. Alternatively, the BDV polymerase could also, at least under certain conditions, initiate RNA synthesis internally, as has been documented for the Sendai virus (47) and vesicular stomatitis virus (6) polymerases. Consistent with this view, minigenomes containing deletions of one and three nucleotides at their 3' ends, with respect to minigenome 5'A/3'C, still exhibited 70% and 10%, respectively, of reporter gene activity.

The structure of a predicted panhandle formed between the 3' and 5' terminal genomic sequences has been proved to be strictly required for promoter function of several viruses. This is the case for orthomyxoviruses, like influenza virus (15, 16, 26) and Thogoto virus (48), where both 3'- and 5'-terminal sequences have been involved in the initiation of transcription. Likewise, for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), a minimal disruption of the terminal complementarity between the 3' and 5' ends of an LCMV minigenome abolished RNA synthesis by the virus polymerase (29). Terminal complementarity has also been shown to affect transcription and replication by vesicular stomatitis virus (49, 50). In contrast to these examples, formation of a panhandle does not appear to play any role in the replication of respiratory syncytial virus (13) or Sendai virus (44). Our results revealed that the 5'/3'Pan minigenome containing a reconstituted panhandle structure by addition of four nucleotides (UGUU) to the 3' end of minigenome 5'G/3'A exhibited 15% of the CAT activity of its parental minigenome 5'G/3'A.

We next examined whether minigenome promoter replication activities paralleled their corresponding promoter transcriptional activities that were initially assessed based on CAT assays (Fig. 3B). For this we determined levels of minigenome-derived CAT mRNA and antiminigenome RNA species by Northern blot using a CAT antisense riboprobe (Fig. 3C). The similar sizes of the CAT mRNA and antiminigenome RNA species made it difficult to clearly distinguish them by Northern blot (Fig. 3Ci).

To overcome this problem, we subjected RNA samples to oligo(dT) chromatography and analyzed the polyadenylated RNA by Northern blot to determine levels of CAT mRNA associated with each minigenome construct (Fig. 3Cii). These results showed that the 5'/3'Pan and 3'{Delta}3 minigenomes produced about 10-fold less CAT mRNA compared to minigenome 5'A/3'C, which correlated well with the corresponding levels of CAT activity determined for each of these minigenome constructs (Fig. 3B). Thus, BDV perfect terminal complementarity does not appear to be required for high levels of promoter activity. Moreover, unlike the findings with vesicular stomatitis virus (49) and rabies virus (14), but similar to the situation reported for respiratory syncytial virus (13), increased terminal complementarity did not appear to promote replication versus transcription by the BDV polymerase.

Finally, we attempted to define within the 3' untranslated region of the BDV genome the minimal cis-acting sequences required for significant levels of genome promoter activity. For this we generated a series of minigenomes containing deletions within the 3'-terminal 35 nucleotides (Fig. 4A). We first verified that these deletions did not affect levels of minigenome RNA that were synthesized by polymerase I and encapsidated by plasmid supplied N and P (Fig. 4B). Results obtained with minigenome-containing 3'-terminal deletions revealed that the 25 3'-terminal nucleotides were sufficient to provide significant levels of promoter activity (Fig. 4C). However, downstream sequences (nucleotides 25 to 33) appeared to be required for optimal promoter activity. Deletion of nucleotides 34 to 35 resulted in abrogation of CAT gene reporter activity, a finding consistent with the mapped boundaries of the gene start signal (GS1) for the BDV N present in our minigenome constructs (29, 39). Results from Northern blot analysis of RNA species synthesized by the reconstituted BDV polymerase in cells transfected with the different minigenome deletion mutants (Fig. 4D) were very consistent with the CAT activity data (Fig. 4C).



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  		FIG. 4. (A) Sequence of the 3'-untranslated region of the BDV genome RNA, including the deletion mutants examined in the minigenome rescue system. The boundaries of the first (GS1) transcription initiation signal are indicated by a box. The first nucleotide to be transcribed by the BDV polymerase is indicated by an arrow. The sequence UAC complementary to the first AUG (antigenome polarity) initiation codon is underlined. The symbol – corresponds to deleted nucleotide positions in the corresponding mutant minigenome construct. (B) Levels of encapsidated polymerase I-derived minigenome RNAs were determined as described for Fig. 3A. (C) Levels of CAT activity associated with the indicated BDV minigenome. (D) Total RNA (7 µg) was analyzed by Northern blot hybridization using a CAT antisense riboprobe.

It should be emphasized that the use for our studies of a BDV minigenome competent for amplification by the reconstituted BDV polymerase leads to situation where the antiminigenome RNA produced by the BDV polymerase can itself be used as a template to produce progeny minigenome RNA, which would amplify the amount of template available for transcription. Mutations affecting the synthesis of antiminigenome RNA (replication) could then dramatically affect the level of minigenome template, thus complicating the assessment of the transcriptional activity of the genomic promoter. Therefore, our results do not allow us to rule out the possibility that some of the mutations examined might have mainly an effect on the replication activity of the genomic promoter, which could also be responsible for the observed effect on the transcriptional activity of the promoter.

To solve this issue, future studies would be required to use a minigenome containing mutations in the trailer region that abrogate the activity of the antigenomic promoter, thus preventing synthesis of progeny minigenome RNA. Thus, the amount of template minigenome RNA is limited to that supplied directly from the plasmid and independent of the efficiency of replication. This results in uncoupled transcriptional and replication events, so that the effects of mutations on the transcriptional activity of the genomic promoter can be examined without confounding effects of the replication process. Such approach has been successfully used with the paramyxovirus respiratory syncytial virus (13) and the prototypic arenavirus LCMV (29).

Our results (Fig. 2), together with those reported by others (4, 32), strongly support the view that the 5' and 3' termini of the BDV genome and antigenome, RNA species present in infected cells have a significant degree of sequence heterogeneity. The development of a reverse genetic system for BDV (30, 42) provided us with a new powerful tool to examine the potential functional implications of this observation. Using a BDV minigenome rescue assay, we have obtained evidence that genome RNA species containing truncations of one to three nucleotides at their 3' termini retained significant levels of promoter activity, a finding similar to that reported for several other nonsegmented RNA viruses. Thus, deletion of the first 3'-terminal three nucleotides of the genome promoter of respiratory syncytial virus had no effect on promoter activity (7), whereas influenza A virus genome promoter lacking the first 3' nucleotides retained about 60% of wild-type promoter activity (21). Moreover, the 3'-terminal first two nucleotides of the S RNA of the phlebovirus Rift Valley Fever virus were not required for the transcriptional activity of the S genome promoter (34).

In contrast, deletion of a single nucleotide within the 3'-terminal 19 nucleotides of the LCMV minigenome resulted in nondetectable levels of genome promoter activity (29). Although the BDV 3'{Delta}1 and 3'{Delta}3 minigenomes were still active in RNA synthesis mediated by the virus polymerase, both their transcriptional and replication activities were diminished with respect to the 5'A/3'C or 5'G/3'A minigenomes. It should be noted that genome and antigenome RNA species containing truncations of one to three nucleotides at their 3' termini appear to accumulate during BDV persistence (Fig. 2), which might contribute to modulate BDV RNA replication and gene expression during long-term persistence.

Genomic and antigenomic viral RNA species with terminal deletions have been documented for several persistent viral infections. In the case of the arenavirus LCMV, these truncated RNAs have been proposed to be a new type of defective interfering genome that contributes to the establishment and maintenance of LCMV persistence (23, 24). In this case it was also proposed that dynamic changes in the ratio of truncated to full-length RNAs would result in fluctuating levels of virus gene expression. This hypothesis would be compatible with the observations that arenavirus persistence is usually characterized by cyclical changes in the numbers of antigen-positive cells and production of infectious virus. Whether this applies to BDV persistence remains to be determined. Nevertheless, circumstantial evidence would suggest that BDV persistence is characterized by stable expression of viral antigen in the majority of the cells within the population. However, production of infectious virus is known to diminish at late passages in BDV persistently infected cell lines, which could be related to the accumulation of RNA species with truncated termini. On the other hand, we cannot rule out the possibility that cellular factors induced or modified by BDV persistence could influence the activity of the virus polymerase in ways that are not recreated by our minigenome rescue assay.

The development of reverse genetics approaches for BDV has opened new and powerful experimental avenues for future detailed analysis of the role of the 5' and 3' untranslated regions in the control of replication and expression of the BDV genome.


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ACKNOWLEDGMENTS
 
This work was entirely supported by NIH grant RO1 NS32355 to J.C.T.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Neuropharmacology, IMM-6, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, 92037. Phone: 858.784.9462. Fax: 858.784.9981. E-mail: juanct{at}scripps.edu. Back


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Journal of Virology, May 2005, p. 6544-6550, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6544-6550.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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