Can Replication of Beak and Feather Disease Virus be Controlled? Two Proposals
Frank D. Niagro
Clinical Investigation Division, Dwight D. Eisenhower Army Medical Center, Fort Gordon, GA 30905-5650 (USA)
Abstract: The unique properties of the beak and feather disease virus presents a potential for therapeutic intervention in affected animals. The probable rolling-circle mode of viral DNA replication and features of the viral replication protein suggest that either conventional drug intervention or gene therapy-based approaches might be developed. Although inhibitors of rolling-circle replication have not been described, a search for such compounds might yield a drug which will selectively inhibit the viral replication cycle with minimal affect on the host. Alternatively, a nucleic acid decoy molecule could possibly be developed to irreversibly block the viral replication protein within infected cells. While entirely conceptual, this alternative approach could be developed to prevent disease progression and lessen the morbidity associated with this devastating disease.
Key Words: Beak and feather disease, Rolling circle replication, ssDNA virus, Circovirus, Topoisomerase, DNA decoy.
Introduction
The circovirus responsible for beak and feather disease of psittacine birds belongs to a group of emerging pathogens affecting both plant and animal species.1 Inferred phylogenetic relationships, based on genome sequence analyses, suggest that beak and feather disease virus (BFDV) and the closely related porcine circovirus (PCV) are related to both the banana bunchy top virus-like viruses and geminiviruses of plants.2,3 Detailed sequence analysis of the viral genomes and of the putative viral replication proteins suggest that these viruses synthesize progeny viral DNAs via a rolling circle mode of replication.3-5
Rolling circle replication (RCR) of DNA is characteristic of viruses possessing circular, single-stranded DNA genomes or single-stranded DNA (ssDNA) intermediates in their replication cycles.6 This includes numerous bacteriophages, the plant geminiviruses, and probably the circoviruses. The virus-encoded replication proteins involved in RCR of ssDNA perform ATP-dependent strand nicking-closing and topoisomerase I activities.6,7 Eukaryotic topoisomerase I is ubiquitous and is an essential enzyme in normal DNA metabolism of the host animal.8
Inhibitors of topoisomerase I have been identified and developed for various experimental therapies.8 Characterization of both naturally occurring and experimentally derived drug-resistant mutants have identified the amino acids involved in enzyme catalysis and drug resistance.9-10 While there are similarities between the eukaryotic enzymes and those involved in RCR, the proteins are quite different in sequence content and mechanism of action. Might these differences be exploitable in the development of therapies for beak and feather disease?
Materials and Methods
Published sequences for circoviruses were obtained from the GenBank sequence database through the WWW server at the NCBI (http://www.ncbi.nlm.nih.gov). Sequence alignments were performed the PILEUP program in the GCG suite.11 Sequence similarity searches were performed using the BLAST E-mail server at the National Center for Biotechnology Information (blast@ncbi.nlm.nih.gov).12 Protein structure prediction and multiple sequence alignment were performed using the PredictProtein server at the European Molecular Biology Laboratory home page (URL:www.embl-heidelberg.de/predictprotein/predictprotein.html).13
Results
A comparison of inferred amino acid sequences for the circovirus replication proteins has previously been utilized for phylogeny inference. 3 Detailed comparisons reveal that the circovirus replication proteins are quite divergent from the bacterial and geminivirus forms. Figure 1 is a cartoon representation of the predicted functional and structural domains for the circovirus replication proteins and the geminivirus proteins, to which they appear to be distantly related. Four regions of high sequence similarity are shared by BFDV and PCV. These include the three sequence motifs characteristic of RCR proteins and the ATP binding domain (p-loop).6 Analysis of circovirus replication protein sequences by the PredictProtein server failed to identify structural similarities to any known proteins. However, helices were predicted in both the BFDV and PCV replication proteins. The first is located between motifs 1 and 2, similar to that found in the geminivirus AL1 proteins.14 The second is located within the fourth region of shared sequence similarity.
 |
| Fig. 1. Schematic diagram of predicted circovirus replication protein sequence motifs and structural domains. Blue regions are highly conserved among the circoviruses, red regions are the 3 motifs found in rolling circle replication proteins and the ATP binding (p-loop) motif, conserved predicted helices are underlined, and the putative active site tyrosine (Y) is shown below. |
The intergenic stem-loop structure in the circovirus DNA molecule bears striking similarity to that of the geminiviruses and the ori hairpin of single-stranded bacterial plasmids.3, 15, 16 This conserved loop sequence (Figure 2) probably contains the nick site where the replicating molecule becomes covalently attached to the active site tyrosine in motif 3 of the replication protein (Figure 1).
 |
| Fig 2. Intergenic region of beak and feather disease virus genomic DNA containing the stem-loop structure. A. Cartoon representation of the stem-loop and adjacent region showing the positional relationship to the putative capsid protein and replication protein coding regions. Numbering of nucleotide bases is according to Niagro et al.3 B. Nucleotide sequence of the viral and inferred complementary strand of the stem-loop and adjacent region. Inverted repeats involved in stem base pairing are indicated by parallel lines. |
Discussion
The crystal structure for human topoisomerase I has been solved and a model has been proposed for its mechanism of action.17,18 The amino acid sequence is highly conserved in the chicken enzyme (the only avian gene sequenced to date), possessing a 92% sequence similarity (89% identity) to the human form. The conserved topoisomerase I amino acid sequence motif, [DE]x(6)[GS]xSKx(2)Y[LIVM]x(3)[LIVM], is not found in the circovirus replication proteins. In addition, the amino acid motif involved in camptothecin resistance and enzyme catalysis is strictly conserved in topoisomerase I, but not found in the circovirus proteins.9 These differences and the virus-unique replication mechanism make it unlikely that inhibitors of topoisomerase I will be effective against circovirus replication. However, this also suggests that virus-specific agents might be developed to selectively inhibit rolling circle replication with minimal or no effect on the host. Interestingly, there appear to be no reported inhibitors of rolling circle replication, even in the bacterial systems where the mechanics of DNA synthesis have been elucidated. Perhaps a pharmacological approach to this problem is now warranted and might prove to be successful in this system.
Alternatively, the covalent attachment of the replication protein to the end of the nicked DNA might present an opportunity for a gene therapy approach. While the use of RNA decoys for the selective inhibition of HIV replication has been explored, these attempts rely on overexpression of molecules which competitively compete for binding to viral or cellular trans-acting factors.19 However, a report of the replication-specific inactivation of a rolling-circle plasmid initiator protein suggests that a DNA decoy might inhibit circovirus DNA replication.16 Such a decoy might consist of a molecule mimicking the viral stem loop structure to such an extent that the strand nicking reaction takes place. The result would be the covalent attachment of a nonreplicable DNA to the replication protein at the active site tyrosine (Figure 3).
 |
| Fig 3. Idealized representation of possible circovirus replication scheme and mechanism of action for DNA decoy. A. Replication protein (checkered oval) and viral DNA are present following viral uncoating and expression of replication protein gene. B. In the usual infectious cycle, the replication protein covalently attaches to the viral DNA within the loop sequence through an ATP-dependent strand breakage reaction. Replication proceeds with the help of host-derived DNA polymerase. C. In the inhibited cycle, the DNA decoy provides an alternate substrate for the strand cleavage reaction, in which the replication protein becomes covalently attached to the decoy. Because the decoy cannot serve as a functional template for the complete synthesis cycle, it remains irreversibly attached to the replication protein and prevents its participation in subsequent rounds of replication. |
In order for this scheme to work, several obvious technical problems must be solved. Among these are the delivery method and improvements in the stability (half life) of single-stranded DNA molecules once delivered to the target cell.19 However, these obstacles are not insurmountable. Optimization of the sequence and resulting secondary structure of the DNA decoy will be required and can potentially provide an irreversible inhibitor with a higher affinity for the viral replication protein than the uncoated viral DNA. Engineered sequence and structural modifications or even alternate structural chemistries could also make such molecules less susceptible to endonucleolytic degradation.20
A more detailed characterization of the BFDV replication cycle and a suitable in vitro culture system are needed before most rational drug design can take place. Knowledge being gained with the PCV culture system and characterization of that circovirus replication protein may provide a vehicle for novel drug discovery.21 This might already be a suitable screening system for identifying conventional therapeutics usable against BFDV and could possibly provide an in vitro test system for development of a DNA decoy. I present these ideas for those still involved in psittacine disease research, in an effort to stimulate future work based on what we have recently learned about the circoviruses.
Acknowledgements
This study is based on work previously performed by the author while at the University of Georgia College of Veterinary Medicine, Athens, GA, USA. This work and the views expressed here are those of the author and are neither supported nor endorsed by the United States Army .
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