Putative Replication-associated Proteins Research Articles

A New Circular Single-Stranded DNA Virus Related with Howler Monkey Associated Porprismacovirus 1 Detected in Children with Acute Gastroenteritis.

Putative replication-associated protein (REP) and capsid-like (CAP) proteins are encoded by circular single-stranded DNA viruses (CRESS DNA), which have been found in samples from most eukaryotic groups. However, the details of these viruses’ life cycles and their significance in diseases have yet to be established. We presented and analyzed two full-length CRESS DNA genomes acquired from two children diagnosed with acute gastroenteritis (GI) in the northeast state of Tocantins, Brazil, using next-generation sequencing and a virus-like filtration approach. Both sequences (named SmaCV3BR08 and SmaCV3BR291) are closely similar to a prior CRESS DNA sequence discovered in the feces of a new world monkey (Alouatta caraya) from the United States in 2009 and termed Howler monkey-associated porprismacovirus 1 (Genbank ID: NC 026317). According to our comparative study, these porprismacovirus genomes deviate by 10% at the nucleotide level. For comparative reasons, the divergence between our sequences (SmaCV3BR08 and SmaCV3BR291) and a porprismacovirus recently identified in a human fecal sample from Peru is 37%. These data suggest that there is a great diversity of porprismacoviruses in South America, perhaps more than two species. In addition, the finding of closely related sequences of porprismacoviruses in humans and native monkeys highlights the zoonotic potential of these viruses.

Novel Cyclovirus Species in Dogs with Hemorrhagic Gastroenteritis.

Nested PCRs with circovirus/cyclovirus pan-rep (replicase gene) primers detected eukaryotic circular Rep-encoding single-stranded DNA (CRESS DNA) viruses in three (samples CN9E, CN16E and CN34) of 18 canine parvovirus-2-positive fecal samples from household dogs with hemorrhagic gastroenteritis on the Caribbean island of Nevis. The complete genomes of CRESS DNA virus CN9E, CN16E and CN34 were determined by inverse nested PCRs. Based on (i) genome organization, (ii) location of the putative origin of replication, (iii) pairwise genome-wide sequence identities, (iv) the presence of conserved motifs in the putative replication-associated protein (Rep) and the arginine-rich region in the amino terminus of the putative capsid protein (Cp) and (v) a phylogenetic analysis, CN9E, CN16E and CN34 were classified as cycloviruses. Canine-associated cycloviruses CN16E and CN34 were closely related to each other and shared low genome-wide nucleotide (59.642–59.704%), deduced Rep (35.018–35.379%) and Cp (26.601%) amino acid sequence identities with CN9E. All the three canine-associated cycloviruses shared < 80% genome-wide pairwise nucleotide sequence identities with cycloviruses from other animals/environmental samples, constituting two novel species (CN9E and CN16E/34) within the genus Cyclovirus. Considering the feeding habits of dogs, we could not determine whether the cycloviruses were of dietary origin or infected the host. Interestingly, the CN9E putative Rep-encoding open reading frame was found to use the invertebrate mitochondrial genetic code with an alternative initiation codon (ATA) for translation, corroborating the hypothesis that cycloviruses are actually arthropod-infecting viruses. To our knowledge, this is the first report on the detection and complete genome analysis of cycloviruses from domestic dogs.

Detection and Complete Genome Analysis of Circoviruses and Cycloviruses in the Small Indian Mongoose (Urva auropunctata): Identification of Novel Species

Fecal samples from 76 of 83 apparently healthy small Indian mongooses (Urva auropunctata) were PCR positive with circovirus/cyclovirus pan-rep (replicase gene) primers. In this case, 30 samples yielded high quality partial rep sequences (~400 bp), of which 26 sequences shared maximum homology with cycloviruses from an arthropod, bats, humans or a sheep. Three sequences exhibited maximum identities with a bat circovirus, whilst a single sequence could not be assigned to either genus. Using inverse nested PCRs, the complete genomes of mongoose associated circoviruses (Mon-1, -29 and -66) and cycloviruses (Mon-20, -24, -32, -58, -60 and -62) were determined. Mon-1, -20, -24, -29, -32 and -66 shared <80% maximum genome-wide pairwise nucleotide sequence identities with circoviruses/cycloviruses from other animals/sources, and were assigned to novel circovirus, or cyclovirus species. Mon-58, -60 and -62 shared maximum pairwise identities of 79.90–80.20% with human and bat cycloviruses, which were borderline to the cut-off identity value for assigning novel cycloviral species. Despite high genetic diversity, the mongoose associated circoviruses/cycloviruses retained the various features that are conserved among members of the family Circoviridae, such as presence of the putative origin of replication (ori) in the 5′-intergenic region, conserved motifs in the putative replication-associated protein and an arginine rich region in the amino terminus of the putative capsid protein. Since only fecal samples were tested, and mongooses are polyphagous predators, we could not determine whether the mongoose associated circoviruses/cycloviruses were of dietary origin, or actually infected the host. To our knowledge, this is the first report on detection and complete genome analysis of circoviruses/cycloviruses in the small Indian mongoose, warranting further studies in other species of mongooses.

Molecular-based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms.

Porcine circovirus 4 (PCV4), a novel and unclassified member of the genus Circovirus, was first reported in China in 2019. Aiming to provide more evidence about the active circulation of PCV4, this study screened 335 pooled internal organs and detected the virus (i) at a rate of 3.28%, (ii) from both clinically healthy and clinically sick pigs of various age groups, and (iii) in six out of nine provinces of Korea. The complete genomic sequence of the Korean PCV4 strain (E115) was 1,770 nucleotides in length and had 98.5%-98.9% identity to three PCV4 strains currently available at GenBank. Utilizing a set of bioinformatic programs, it was revealed that the Korean PCV4 strain contained several genomic features of (i) a palindrome stem-loop structure with a conserved nonanucleotide, (ii) packed overlapping ORFs oriented in different directions and (iii) two intergenic regions in between genes encoding the putative replication-associated protein (Rep) and capsid (Cap) proteins. This study also predicted the presence of essential elements for the replication of circoviruses in all PCV4 strains, for example the origin of DNA replication, endonuclease and helicase domains of Rep, and the nuclear localization signal on the putative Cap protein. Finally, based on the phylogeny inferred from sequences of the putative Rep protein, this study further clarified the genetic relationships between PCV4 and other CRESS DNA viruses in general and circoviruses in particular.

Genomic characterization of circoviruses associated with acute gastroenteritis in minks in northeastern China.

Mink circovirus (MiCV), a virus that was newly discovered in 2013, has been associated with enteric disease. However, its etiological role in acute gastroenteritis is unclear, and its genetic characteristics are poorly described. In this study, the role of circoviruses (CVs) in mink acute gastroenteritis was investigated, and the MiCV genome was molecularly characterized through sequence analysis. Detection results demonstrated that MiCV was the only pathogen found in this infection. MiCVs and previously characterized CVs shared genome organizational features, including the presence of (i) a potential stem-loop/nonanucleotide motif that is considered to be the origin of virus DNA replication; (ii) two major inversely arranged open reading frames encoding putative replication-associated proteins (Rep) and a capsid protein; (iii) direct and inverse repeated sequences within the putative 5ʹ region; and (iv) motifs in Rep. Pairwise comparisons showed that the capsid proteins of MiCV shared the highest amino acid sequence identity with those of porcine CV (PCV) 2 (45.4%) and bat CV (BatCV) 1 (45.4%). The amino acid sequence identity levels of Rep shared by MiCV with BatCV 1 (79.7%) and dog CV (dogCV) (54.5%) were broadly similar to those with starling CV (51.1%) and PCVs (46.5%). Phylogenetic analysis indicated that MiCVs were more closely related to mammalian CVs, such as BatCV, PCV, and dogCV, than to other animal CVs. Among mammalian CVs, MiCV and BatCV 1 were the most closely related. This study could contribute to understanding the potential pathogenicity of MiCV and the evolutionary and pathogenic characteristics of mammalian CVs.

Cloning of a novel endogenous promoter for foreign gene expression in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a model diatom, and its genomic sequence data and expressed sequence tag databases are available. This study was to discover a new endogenous promoter that drives strong constitutive expression of a protein of interest in P. tricornutum. To find promoter candidates, the intracellular proteins of P. tricornutum grown to stationary phase were extracted and identified by LC–MS/MS. Glutamine synthetase (GLNA) was one of the most abundantly expressed proteins during the stationary phase. Promoter is usually located on 5′ upstream region of open reading frame of the gene. Thus, two fragments of 5′ upstream region of the GLNA gene as putative promoters, 501 and 997 bp long, were amplified and cloned into enhanced Green Fluorescent Protein (eGFP) reporter systems. The constructed reporter systems were transformed into P. tricornutum and the eGFP expression levels were compared to those of reporter systems using the promoters of fcpA (fucoxanthin chlorophyll a/c binding protein A) and CIP1 (putative replication-associated proteins of a Chaetoceros lorenzianus-infecting DNA virus) as controls. The expression of eGFP driven by either GLNA promoter (501 and 997 bp) was linearly related to cell density, and eGFP was expressed constitutively regardless of the cultivation phase. The eGFP expression level driven by the GLNA promoters was at least 4 times higher than the fcpA-driven eGFP expression level at the stationary phase. The 501 and 997 bp regions of the GLNA promoter had similar activity patterns for transcribing the downstream gene. These results indicate that at least 501-bp region of the GLNA promoter can be used as a strong constitutive promoter in genetic engineering of P. tricornutum.

A novel, highly divergent ssDNA virus identified in Brazil infecting apple, pear and grapevine

Fruit trees of temperate and tropical climates are of great economical importance worldwide and several viruses have been reported affecting their productivity and longevity. Fruit trees of different Brazilian regions displaying virus-like symptoms were evaluated for infection by circular DNA viruses. Seventy-four fruit trees were sampled and a novel, highly divergent, monopartite circular ssDNA virus was cloned from apple, pear and grapevine trees. Forty-five complete viral genomes were sequenced, with a size of approx. 3.4kb and organized into five ORFs. Deduced amino acid sequences showed identities in the range of 38% with unclassified circular ssDNA viruses, nanoviruses and alphasatellites (putative Replication-associated protein, Rep), and begomo-, curto- and mastreviruses (putative coat protein, CP, and movement protein, MP). A large intergenic region contains a short palindromic sequence capable of forming a hairpin-like structure with the loop sequence TAGTATTAC, identical to the conserved nonanucleotide of circoviruses, nanoviruses and alphasatellites. Recombination events were not detected and phylogenetic analysis showed a relationship with circo-, nano- and geminiviruses. PCR confirmed the presence of this novel ssDNA virus in field plants. Infectivity tests using the cloned viral genome confirmed its ability to infect apple and pear tree seedlings, but not Nicotiana benthamiana. The name “Temperate fruit decay-associated virus” (TFDaV) is proposed for this novel virus.

Unusual genome complexity in Lactobacillus salivarius JCM1046.

BackgroundLactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046.ResultsJCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM.ConclusionExperimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-771) contains supplementary material, which is available to authorized users.

Discovery of a novel circular single-stranded DNA virus from porcine faeces

A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (<30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.

Genomic Characterization of a Novel Virus of the Family Tymoviridae Isolated from Mosquitoes

BackgroundThe family Tymoviridae comprises three plant virus genera, including Tymovirus, Marafivirus, and Maculavirus, which are found in most parts of the world and cause severe agricultural losses. We describe a putatively novel member of the family Tymoviridae, which is isolated from mosquitoes (Culex spp.), referred to as CuTLV.Methods and ResultsThe CuTLV was isolated by cell culture, which replicates and causes cytopathic effects in Aedes albopictus C6/36 cells, but not in mammalian BHK-21 or Vero cells. The complete 6471 nucleotide sequence of CuTLV was determined. The genome of CuTLV is predicted to contain three open reading frames (ORFs). The largest ORF1 is 5307 nucleotides (nt) in length and encodes a putative polypeptide of 1769 amino acids (aa), which contains the conserved motifs for the methyltransferase (MTR), Tymovirus endopeptidase (PRO), helicase (HEL), and RNA-dependent RNA polymerase (RdRp) of the replication-associated proteins (RPs) of positive-stranded RNA viruses. In contrast, the ORF1 sequence does not contain the so-called “tymobox” or “marafibox”, the conserved subgenomic RNA promoter present in all tymoviruses or marafiviruses, respectively. ORF2 and ORF3 putatively encode a 248-aa coat protein (CP) and a proline-rich 149-aa polypeptide. The whole genomic nucleotide identity of CuTLV with other members of family Tymoviridae ranged from 46.2% (ChiYMV) to 52.4% (GFkV). Phylogenetic analysis based on the putative RP and CP genes of CuTLV demonstrated that the virus is most closely related to viruses in the genus Maculavirus.ConclusionsThe CuTLV is a novel virus related to members of the family Tymoviridae, with molecular characters that are distinct from those of tymoviruses, marafiviruses, and other maculaviruses or macula-like viruses. This is the first report of the isolation of a Tymoviridae-like virus from mosquitoes. Further investigations are required to clarify the origin, replication strategy, and the public health or agricultural importance of the CuTLV.

Molecular characterization and epidemiological investigation of Pigeon circovirus isolated in eastern China

To investigate the prevalence and genetic characterization of Pigeon circovirus (PiCV) circulating in Chinese flocks, the genomic DNA of 144 samples collected from pigeons in 6 different geographic regions of eastern China between 2009 and 2010 were amplified using previously published PiCV primers. The PiCV sequence was detected in 83 of 104 unhealthy pigeons (79.8%) and 25 of 40 healthy pigeons (62.5%). The overall positive rate was 75% for all samples. An inverse primer polymerase chain reaction (IP-PCR) assay was performed to amplify the full-length sequence from a random sample of each region, and 6 specific DNA fragments were gel-purified and sequenced. The 6 full-length sequences were designated as SHWH-AB4 (2,031 bp), NJPK-21 (2,035 bp), HBLF-E2 (2,031 bp), JSJN (2,039 bp), SDDZ (2,037 bp), and AHBZ (2,035 bp) after BLAST analyses. The phylogenic tree and amino acid comparison indicated that all the strains examined were derived from a common strain, but had undergone genetic mutations through time. Pairwise comparisons revealed 93.4%-100% amino acid identity for the putative replication-associated proteins and 67.5%-100% for the putative capsid proteins.

Geminivirus-related extrachromosomal DNAs of the X-clade phytoplasmas share high sequence similarity.

Southern blot hybridization analysis revealed that the extrachromosomal DNAs (EC-DNAs) associated with Vaccinium witches' broom (VAC) and walnut witches' broom phytoplasmas and various strains of the Italian clover phyllody phytoplasma (ICPh) were highly homologous among themselves but distinct from EC-DNAs of aster yellows related phytoplasmas occurring in the same insect and plant hosts and collected at the same site as the ICPh strains. The EC-DNAs of various strains of the ICPh differed significantly in number and size, more markedly among samples from different host plant species than among samples from the same host plant species. However, experiments on insect-mediated transmission suggested that the size variation is not associated with plant host specificity. Sequence analysis of cloned fragments revealed the presence of highly conserved ORFs (with substantially invariant putative translation products) but also the presence of regions rich in short direct and inverted repeats, which may be the cause of the size variations. The partial sequence of an EC-DNA associated with VAC encoding a putative replication-associated protein indicated their close phylogenetic relationship with geminiviruses.

Ten distinct circular ssDNA components, four of which encode putative replication-associated proteins, are associated with the faba bean necrotic yellows virus genome.

Four further circular ssDNA components (C7-C10), about 1 kb in size and structurally similar to the previously described components (C1-C6) found associated with a Syrian (Sy) isolate of faba bean necrotic yellows virus (FBNYV), have been identified. Similar to C1 and C2, two of the new components (C7 and C9) encode putative replication-associated (Rep) proteins of 33.2 and 32.7 kDa, respectively, the former of which is 90% identical to the C10 Rep protein of milk vetch dwarf virus (MDV). C8 encodes a putative protein (17.4 kDa) whose function is unknown, but which is highly conserved between FBNYV and the other nanoviruses MDV, subterranean clover stunt virus and banana bunchy top virus. The putative protein (19.7 kDa) encoded by C10 contains an LXCXE motif, which is also present in the homologues of its relatives, suggesting that they may all interact with plant retinoblastoma-like proteins. Sequence information for seven components of an Egyptian (Eg) FBNYV isolate indicated that six of them share > 96% identity with FBNYV-Sy. However, the C1 Rep protein of FBNYV-Eg was only 63.5% identical to that of FBNYV-Sy, but was 88.3 % identical to the MDV-C2 Rep protein. We conclude that the FBNYV genome consists of seven to ten components, six of which encode non-Rep proteins. All ten components of the FBNYV genome, except the Rep components C2 and C9, had closely related counterparts in the MDV genome. In spite of this similarity, FBNYV and MDV appear to be distinct virus species.

A novel subviral agent associated with a geminivirus: the first report of a DNA satellite.

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem-loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication.

Autocatalytic Processing of the 223-kDa Protein of Blueberry Scorch Carlavirus by a Papain-like Proteinase

The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine 994 and histidine 1075. A second possible proteinase domain, which contained cysteine 895 and histidine 984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative hellcase and the polymerase domains, possibly between or close to glycine 1472 and alanine 1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine 994, histidine 1075, or glycine 1472 abolished autoproteolysis in vitro , supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine 895 and histidine 984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine 994 or histidine 1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine 1472 significantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine 895 or histidine 984 also were infectious, but resulted in delayed symptom expression in plants.