Phylogenetic and codon usage analysis for replicase and capsid genes of porcine circovirus 3.

Porcine circovirus type 3 (PCV3) is a highly contagious virus belonging to the family Circoviridae that causes the severe dermatitis and nephropathy syndrome. To date, PCV3 has a worldwide distribution and bring huge economic losses to swine industry. Replicase (Rep) and capsid (Cap) are two major coded proteins of PCV3. Considering the large number of new PCV3 isolates were reported in the past few years and the research for the codon usage pattern of Rep and Cap genes was still a gap, phylogenetic and codon usage analysis of these two genes was performed. Phylogenetic analyses showed that Rep genes in PCV3a were dispersed with no clear clusters while corresponding sequences in PCV3b clustered into two groups and Cap genes clustered into distinct clades according to different genotypes. Relative synonymous codon usage (RSCU) analysis revealed that the codon usage bias existed and effective number of codon (ENC) analysis showed that the bias was slight low. ENC-GC3s plot indicated that mutational pressure and other factors both played a role in PCV3 codon usage and neutrality plot analysis showed that natural selection was the main force influencing the codon usage pattern. The results presented here provided the important basic data on codon usage pattern of Rep and Cap genes, and a better understanding of the evolution and potential origin of PCV3.

Mechanism of DNA Interaction and Translocation by the Replicase of a Circular Rep-Encoding Single-Stranded DNA Virus.

Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses infect members from all three domains of life (Archaea, Prokarya, and Eukarya). The replicase (Rep) from these viruses is responsible for initiating rolling circle replication (RCR) of their genomes. Rep is a multifunctional enzyme responsible for nicking and ligating ssDNA and unwinding double-stranded DNA (dsDNA). We report the structure of porcine circovirus 2 (PCV2) Rep bound to ADP and single-stranded DNA (ssDNA), and Rep bound to ADP and double-stranded DNA (dsDNA). The structures demonstrate Rep to be a member of the superfamily 3 (SF3) of ATPases Associated with diverse cellular Activities (AAA+) superfamily clade 4. At the Rep N terminus is an endonuclease domain (ED) that is responsible for ssDNA nicking and ligation, in the center of Rep is an oligomerization domain (OD) responsible for hexamerization, and at the C terminus is an ATPase domain (AD) responsible for ssDNA/dsDNA interaction and translocation. The Rep AD binds to DNA such that the ED faces the replication fork. The six AD spiral around the DNA to interact with the backbone phosphates from four consecutive nucleotides. Three of the six AD are able to sense the backbone phosphates from the second strand of dsDNA. Heterogeneous classification of the data demonstrates the ED and AD to be mobile. Furthermore, we demonstrate that Rep exhibits basal nucleoside triphosphatase (NTPase) activity. IMPORTANCE CRESS-DNA viruses encompass a significant portion of the biosphere's virome. However, little is known about the structure of Rep responsible for initiating the RCR of CRESS-DNA viruses. We use cryo-electron microscopy (cryo-EM) to determine the structure of PCV2 Rep in complex with ADP and ss/dsDNA. Our structures demonstrate CRESS-DNA Reps to be SF3 members (clade 4) of the AAA+ superfamily. The structures further provide the mechanism by which CRESS-DNA virus Reps recognize DNA and translocate DNA for genome replication. Our structures also demonstrate the ED and AD of PCV2 Rep to be highly mobile. We propose the mobile nature of these domains to be necessary for proper functioning of Reps. We further demonstrate that Reps exhibit basal NTPase activity. Our studies also provide initial insight into the mechanism of RCR.

Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein.

OBJECTIVE: We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep). RESULTS: For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface. CONCLUSION: We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.

Crystal Structure of the Dimerized N Terminus of Porcine Circovirus Type 2 Replicase Protein Reveals a Novel Antiviral Interface.

Two replicase (Rep) proteins, Rep and Rep', are encoded by porcine circovirus (PCV) ORF1; Rep is a full ORF1 transcript, and Rep' is a truncated transcript generated by splicing. These two proteins are crucial for the rolling-circle replication (RCR) of PCV. The N-terminal sequences of Rep and Rep' are identical and interact to form homo- or heterodimers. The three types of dimers perform different functions during replication. A structural examination of the interfacing termini has not been performed. In this study, a crystal structure of dimerized Rep protein N termini was resolved at 2.7 Å. The dimerized protein was maintained by nine intermolecular hydrogen bonds and 15 pairs of hydrophobic interactions. The amino acid residue Ile37 participates in 11 of the hydrophobic interactions, mostly with its side chain. To find the predominant sites for protein dimerization and virus replication, a series of mutant proteins and virus replicons were generated by alanine substitution. Of all the single amino acid substitutions, the mutation at Ile37 showed the greatest effect on protein dimerization and virus replication. A double mutation at Leu35 and Ile37 almost eliminated protein dimerization and had the greatest negative effect on virus replication. These studies demonstrate that Leu35 and Ile37 are the most important residues for protein dimerization and are crucial for virus replication. Our results also show that PCV replication can be decreased by disrupting the dimerization of Rep or Rep' at the N terminus, suggesting that the structural interface responsible for dimerization offers a promising antiviral target.IMPORTANCE Porcine circovirus type 2 (PCV2) is one of the most economically damaging pathogens affecting the swine industry. Although vaccines have been available for more than 10 years, the virus still remains prevalent. More effective strategies for disease prevention are clearly required. The Rep and Rep' proteins of the virus have identical N-terminal regions that interact with each other, allowing the formation of homo- or heterodimers. The heterodimer has crucial functions during different stages of viral replication. Here, we resolved the crystal structure of the Rep (Rep') dimerization domain. The individual residues involved in the intermolecular interaction were visualized in the protein structure, and several interactions were verified by mutant analysis. Our studies show that disrupting the interaction decreases viral replication, thus revealing a new target for the design of antiviral agents.

A comparison of PCR assays for beak and feather disease virus and high resolution melt (HRM) curve analysis of replicase associated protein and capsid genes.

Beak and feather disease virus (BFDV) threatens a wide range of endangered psittacine birds worldwide. In this study, we assessed a novel PCR assay and genetic screening method using high-resolution melt (HRM) curve analysis for BFDV targeting the capsid (Cap) gene (HRM-Cap) alongside conventional PCR detection as well as a PCR method that targets a much smaller fragment of the virus genome in the replicase initiator protein (Rep) gene (HRM-Rep). Limits of detection, sensitivity, specificity and discriminatory power for differentiating BFDV sequences were compared. HRM-Cap had a high positive predictive value and could readily differentiate between a reference genotype and 17 other diverse BFDV genomes with more discriminatory power (genotype confidence percentage) than HRM-Rep. Melt curve profiles generated by HRM-Cap correlated with unique DNA sequence profiles for each individual test genome. The limit of detection of HRM-Cap was lower (2×10-5ng/reaction or 48 viral copies) than that for both HRM-Rep and conventional BFDV PCR which had similar sensitivity (2×10-6ng or 13 viral copies/reaction). However, when used in a diagnostic setting with 348 clinical samples there was strong agreement between HRM-Cap and conventional PCR (kappa=0.87, P<0.01, 98% specificity) and HRM-Cap demonstrated higher specificity (99.9%) than HRM-Rep (80.3%).

Dual ATPase and GTPase activity of the replication-associated protein (Rep) of beak and feather disease virus.

BACKGROUND: Psittacine beak and feather disease affects parrots resulting in an immunosuppressive disease that is often characterized by an abnormal shape and growth of the animal's beak, feathers, and claws. Beak and feather disease virus (BFDV) is a single-stranded circular DNA virus and is classified as a member of the Circoviridae family. Two major open reading frames (ORFs) are known to encode the replication-associated (Rep) protein and the capsid-associated (Cap) protein. METHODS: The Rep and Cap genes of BFDV were fused with tags and then expressed and purified, respectively. Both the ATPase and GTPase activities of the recombinant Rep protein are measured. The substrate and ion preference, the optimal conditions, the effects of ATPase and GTPase inhibitors and the presence of Cap protein on the ATPase and GTPase activity of the Rep protein are examined. Finally, the effects of the Walker A motif, the Walker B motif, and a novel GYDG motif of the Rep protein on the ATPase and GTPase activities are studied by various mutants. RESULTS: The recombinant Rep protein could display ATPase activity and GTPase activity. The Rep protein was able to hydrolyze both deoxyribonucleotides and ribonucleotides. Among nucleoside triphosphates and deoxynucleoside triphosphates, the substrate preference orders were found to be ATP>GTP>CTP≥UTP and dATP>dGTP>dTTP>dCTP, respectively. Both the ATPase and GTPase activity of the BFDV Rep protein required magnesium ions and the presence of calcium ions significantly inhibited the ATPase and GTPase activity of the Rep protein. The optimal temperatures for ATPase activity and GTPase activity were both 56 °C, while their optimal pH values were both pH 7.5. Both the ATPase activity and GTPase activity of the BFDV Rep protein were significantly down-regulated by polynucleotides and the dsDNA of 36 bp (located in origin of replication) of BFDV genome. The ATPase activity of the BFDV Rep protein was found to be more sensitive to sodium azide than sodium orthovanadate and N-ethylmaleimide. Linoleic acid more strongly inhibited the GTPase activity of the Rep protein than dynasore. This suggested the Rep protein of BFDV should be classified as an F-type ATPase and polyunsaturated fatty acid-sensitive GTPase. In the presence of 10 ng of the Cap protein, the ATPase activity and GTPase activity of the BFDV Rep protein were significantly increased. Furthermore, the BFDV Rep protein contains the Walker A motif, the Walker B motif and a novel GYDG motif. Both the ATPase activity and the GTPase activity of various deletion and site-directed mutants of the Rep protein affecting these motifs were significantly reduced, suggesting all the three motifs contribute to the ATPase and GTPase activities; specifically. In addition, the ATPase activity and GTPase activity of the deletion mutants of the Rep protein were reversed in the presence of the Cap protein. This is the first example of dual ATPase and GTPase activity being reported in the Rep protein of BFDV.

Characterization of the nuclear localization signals of duck circovirus replication proteins.

Duck circovirus (DuCV) possess a circular, single-stranded DNA genome that requires the replication protein (Rep) for its replication. Based on the viral genotype, there are two categories of Rep proteins: Rep1 and Rep2. To characterize the nuclear localization signals (NLSs) conferring the nuclear localization of the Rep proteins, defined coding regions of the rep gene of two genotypes of DuCV were cloned and co-expressed with the red fluorescent protein DsRed2. The results showed that deleting the putative N-terminal NLS located at amino acid residues 10-37 of Rep1 and Rep2 abrogated nuclear translocation, while deleting the putative C-terminal NLS located at residues 244-274 of Rep1 did not significantly alter its subcellular localization, confirming that only the NLS located at residues 10-37 in the N-termini of the Rep proteins had nuclear targeting activity.

Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3).

Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy.

Porcine circovirus Cap protein production by P. pastoris with strong AOX promoter suffered with the problems with traditional pure methanol induction: (1) inefficient methanol metabolism; (2) extensive oxygen supply load; (3) difficulty in stable DO control; (4) low protein titer. In this study, based on the difference of DO change patterns in response to methanol and sorbitol additions, a novel fuzzy control system was proposed to automatically regulate the co-feeding rates of methanol and sorbitol for efficient Cap protein induction. With aid of the proposed control system when setting DO control level at 10%, overall fermentation performance was significantly improved: (1) DO could be stably controlled under mild aeration condition; (2) methanol consumption rate could be restricted at moderate level and the major enzymes involved with methanol metabolism were largely activated; (3) Cap protein concentration reached a highest level of 198mg/L, which was about 64% increase over the best one using the pure methanol induction strategies.

Characterization and epitope mapping of monoclonal antibodies recognizing N-terminus of Rep of porcine circovirus type 2.

Two genotypes of porcine circovirus (PCV) have been described, the non-pathogenic PCV1 and the pathogenic PCV2 in pigs. PCV-ORF1 encodes Rep and Rep' proteins which have identical N-terminal sequence (RepN) within each PCV strain, but RepN has only 88% similarity between PCV1 and PCV2. Purified RepN of PCV2 was used as an immunogen to produce monoclonal antibodies (mAbs). 11 mAbs were screened out and established, and they were divided into two groups according to Western blot and IFA results. One group, including 1C1, bound only PCV2-RepN, while the other, including 3D10, had cross reactivity with RepN of both PCV1 and PCV2. Epitope mapping indicated that 1C1 and 3D10 recognized the linear epitopes L(39)FDYFIVG(46) and K(99)EGNLLIE(106) in PCV2-RepN, respectively. Protein sequence alignment of RepN indicated L(39)FDYFIVG(46) is conserved in all PCV2 in NCBI database, whereas the PCV1 has amino acid substitutions V(41)C(42) in this region. mAb 3D10 could recognize all PCV because all natural mutations in its epitope did not affect its binding. The information about characteristics and epitope of monoclonal antibodies may be useful for the development of diagnostic methods for PCV2 and for analyzing the function of Rep and Rep' of PCV.

[Biological character of porcine circovirus type 2 with site mutation at interferon stimulation reaction element (ISRE) like region in Rep gene promoter].

OBJECTIVE: To explore the biological function of the interferon stimulation reaction element (ISRE) like motif CTGAAAACGAAAGA within porcine circovirus type 2 (PCV2) Rep promoter. METHODS: Two recombinant PCV2 strains, namely PCV2 1740(G-C) and PCV2 1741(A-T), were constructed by transfecting PK15 cells with site-mutated infectious clone of PCV2 strain Denta. Replication character, genetic stability and reactive character to porcine interferon alpha (poIFN-alpha) were compared among parental PCV2 and the two mutant viruses. RESULTS: The ISRE like motif in Rep promoter was not necessary for the replication of PCV2 because two site-mutated viral genome clones both produced infectious virus. In contrast to parental PCV2, the viral antigen positive PK15 cells of the two site-mutated PCV2 were decreased. PCV2 1740(G-C) was genetically stable in the PK15 cell while PCV2 1741(A-T) was found to have another two nucleotide mutated from 1744AC1745 to 1744TT1745 between 3rd and 7th passage in the PK15 cell. After treated with 100 U/mL porcine interferon alpha, the viral antigen positive PK15 cells and virus genomes of parental PCV2 and two site-mutated viruses were both increased. But the enhancement rate of the two site-mutated PCV2 was significantly lower than parental PCV2. CONCLUSION: Site-mutation of ISRE like motif in Rep promoter decreased the replication and poIFN-alpha induced enhancement of PCV2 in PK15 cells. According to these above results, it maybe speculated that ISRE like motif in PCV2 Rep gene promoter contain a functional element and it may contribute to the interferon inducible enhancement of virus replication in PK15 cells.

PCV2 replicase transcripts in infected porcine kidney (PK15) cells.

The Circoviridae family includes small viruses containing circular single strand DNA. There are two circoviruses described in pigs. Porcine Circovirus 2 (PCV2) has been linked to a disease termed Postweaning Multisystemic Wasting Syndrome (PMWS) whereas a close relative of this virus, termed PCV type 1 (PCV1) has been considered a non-pathogenic contaminant of the PK15 cell line. Rolling circle replication of small DNA viruses like circoviruses is mediated by a replicase (Rep) gene that is transcribed into different mRNAs by an alternative splicing mechanism. In PCV1, two transcripts Rep and Rep' implicated in DNA replication, have been identified from infected cells. These transcripts are the product of a single gene with Rep' having a different carboxyl end than Rep. Sequence comparison shows that there is a high degree of homology for the Rep gene in both viruses. In this article, we have identified three different transcripts by RT-PCR in PCV2 infected PK15 cells. Two of them have homology with their PCV1 counterparts and the third transcript, termed Rep", corresponds to another spliced version of the Rep gene. This transcript, unique to PCV2, encodes for a protein of 80 aminoacids in frame with the Rep sequence.