Analysis and characterization of the complete genome of tupaia (tree shrew) herpesvirus

The tupaia herpesvirus (THV) was isolated from spontaneously degenerating tissue cultures of malignant lymphoma, lung, and spleen cell cultures of tree shrews (Tupaia spp.). The determination of the complete nucleotide sequence of the THV strain 2 genome resulted in a 195,857-bp-long, linear DNA molecule with a G+C content of 66.5%. The terminal regions of the THV genome and the loci of conserved viral genes were found to be G+C richer. Furthermore, no large repetitive DNA sequences could be identified. This is in agreement with the previous classification of THV as the prototype species of herpesvirus genome group F. The search for potential coding regions resulted in the identification of 158 open reading frames (ORFs) regularly distributed on both DNA strands. Seventy-six out of the 158 ORFs code for proteins that are significantly homologous to known herpesvirus proteins. The highest homologies found were to primate and rodent cytomegaloviruses. Biological properties, protein homologies, the arrangement of conserved viral genes, and phylogenetic analysis revealed that THV is a member of the subfamily Betaherpesvirinae. The evolutionary lineages of THV and the cytomegaloviruses seem to have branched off from a common ancestor. In addition, it was found that the arrangements of conserved genes of THV and murine cytomegalovirus strain Smith, both of which are not able to form genomic isomers, are colinear with two different human cytomegalovirus (HCMV) strain AD169 genomic isomers that differ from each other in the orientation of the long unique region. The biological properties and the high degree of relatedness of THV to the mammalian cytomegaloviruses allow the consideration of THV as a model system for investigation of HCMV pathogenicity.     

Identification of functional open reading frames in chloroplast genomes

We have used a rapid computer dot-matrix comparison method to identify all DNA regions which have been evolutionarily conserved between the completely sequenced chloroplast genomes of tobacco and a liverwort. Analysis of these regions reveals 74 homologous open reading frames (ORFs) which have been conserved as to length and amino acid sequence; these ORFs also have an excess of nucleotide substitutions at silent sites of codons. Since the nonfunctional parts of these genomes have become saturated with mutations and show no sequence similarity whatsoever, the homologous ORFs are almost certainly functional. A further four pairs of ORFs show homology limited to only a short part of their putative gene products. Amino acid sequence identities range between 50 and 99%; some chloroplast proteins are seen to be among the most slowly evolving of all known proteins. A search of the nucleotide and amino acid sequence databanks has revealed several previously unidentified genes in chloroplast sequences from other species, but no new homologies to prokaryotic genes.     

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.     

Genome comparison of Pseudomonas aeruginosa large phages

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.     

树鼩神经肽Y的分子克隆及其灵长类类似物的同源性比较

树鼩由于与灵长类动物有较密切的亲缘关系和其个体小,以及繁殖周期短等特性而倍受关注,尤其是作为医用实验动物的研究,近年来已受到越来越多的重视,但树鼩的分类地位还一直有所争论。该研究从树鼩脑cDNA文库中克隆得到编码树鼩神经肽Y(neuropeptide Y,NPY)前体序列,序列比对发现该序列与灵长类NPY序列同源性高达96.9%。将该序列与GenBank数据库中其他物种的NPY序列构建系统进化树,发现树鼩与灵长类处于同一分支。该研究结果揭示了树鼩与灵长类较近的亲缘关系。     

Sulfolobus tengchongensis spindle-shaped virus STSV1: virus-host interactions and genomic features

A virus infecting the hyperthermophilic archaeon Sulfolobus tengchongensis has been isolated from a field sample from Tengchong, China, and characterized. The virus, denoted STSV1 (Sulfolobus tengchongensis spindle-shaped virus 1), has the morphology of a spindle (230 by 107 nm) with a tail of variable length (68 nm on average) at one end and is the largest of the known spindle-shaped viruses. After infecting its host, the virus multiplied rapidly to high titers (>10(10) PFU/ml). Replication of the virus retarded host growth but did not cause lysis of the host cells. STSV1 did not integrate into the host chromosome and existed in a carrier state. The STSV1 DNA was modified in an unusual fashion, presumably by virally encoded modification systems. STSV1 harbors a double-stranded DNA genome of 75,294 bp, which shares no significant sequence similarity to those of fuselloviruses. The viral genome contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs encode viral structural proteins, including a putative coat protein of high abundance. The products of the other nine ORFs are probably involved in polysaccharide biosynthesis, nucleotide metabolism, and DNA modification. The viral genome divides into two nearly equal halves of opposite gene orientation. This observation as well as a GC-skew analysis point to the presence of a putative viral origin of replication in the 1.4-kb intergenic region between ORF1 and ORF74. Both morphological and genomic features identify STSV1 as a novel virus infecting the genus Sulfolobus.     

Genetic analysis of the tomato golden mosaic virus. II. The product of the AL1 coding sequence is required for replication.

Tomato golden mosaic virus (TGMV) belongs to the geminivirus subgroup that is characterized by a split genome consisting of two single-stranded circular DNAs. The TGMV A genome component encodes the virus coat protein as well as all of the functions necessary for viral DNA replication. Analysis of the nucleotide sequence indicates that the TGMV A component has, in addition to the coat protein encoding ORF, four overlapping open reading frames (ORFs) with the potential to encode proteins of greater than 10 kD. We have investigated the functions of these putative proteins in both symptom formation and DNA replication by creating mutations in each of the ORFs. Our results show that the AL4 ORF, which is encoded within the N-terminal region of ORF AL1, is not essential for normal virus infection. In contrast, we find that disruption of the AL3 ORF results in delay and attenuation of symptom formation. We also report that the products of the AL1 and AL2 ORFs are absolutely required for symptom formation. Studies of DNA replication show that only the AL1 open reading frame is essential for viral DNA synthesis. The significance of these results for the development of vectors from the geminiviruses is discussed.     

Life-cycle and genome of OtV5, a large DNA virus of the pelagic marine unicellular green alga Ostreococcus tauri

Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon.     

Complete genome sequence of the shrimp white spot bacilliform virus.

We report the first complete genome sequence of a marine invertebrate virus. White spot bacilliform virus (WSBV; or white spot syndrome virus) is a major shrimp pathogen with a high mortality rate and a wide host range. Its double-stranded circular DNA genome of 305,107 bp contains 181 open reading frames (ORFs). Nine homologous regions containing 47 repeated minifragments that include direct repeats, atypical inverted repeat sequences, and imperfect palindromes were identified. This is the largest animal virus that has been completely sequenced. Although WSBV is morphologically similar to insect baculovirus, the two viruses are not detectably related at the amino acid level. Rather, some WSBV genes are more homologous to eukaryotic genes than viral genes. In fact, sequence analysis indicates that WSBV differs from all known viruses, although a few genes display a weak homology to herpesvirus genes. Most of the ORFs encode proteins that bear no homology to any known proteins, either suggesting that WSBV represents a novel class of viruses or perhaps implying a significant evolutionary distance between marine and terrestrial viruses. The most unique feature of WSBV is the presence of an intact collagen gene, a gene encoding an extracellular matrix protein of animal cells that has never been found in any viruses. Determination of the genome of WSBV will facilitate a better understanding of the molecular mechanism underlying the pathogenesis of the WSBV virus and will also provide useful information concerning the evolution and divergence of marine and terrestrial animal viruses at the molecular level.     

Complete nucleotide sequence of the Eastern equine encephalomyelitis virus genome

The complete nucleotide sequence of the genomic 42S RNA of Eastern equine encephalitis virus has been defined for the first time. The strategy of this viral genome occurred analogous to the ones of the other alfa viruses. The comparison of amino acid sequences of E1 and E2 proteins from the two strains of the virus has revealed a number of differences. Partially, they are localized in the hydrophilic regions of the protein molecules and evidently participate in organization of the specific antigenic structures. The amino acid sequences of all viral proteins have been comparatively analysed with the sequences of the analogous proteins of other known alfa viruses.     

Determination and analysis of the primary structure of NM.BstSEI operone from Bacillus stearothermophilus SE-589 which produces N.BstSEI site-specific nickase

Nucleotide sequence of Bacillus stearothermophilus SE-589 DNA fragment which includes an operone for site-specific NM-system with a gene for BstSEI nickase has been determined. Analysis of the regions adjacent to nickase gene has revealed two genes encoding DNA methyltransferases, which belong to different classes. Three genes which form system operone are separated with short open reading frames (ORFs). Analysis of these ORFs has shown that they encode polypeptides which are homologous to different parts of BstSEI nickase, NatB protein and arginase. A difference in GC-content of the beginning and ending regions of the cloned DNA fragment as well as presence of short ORFs similar to genes for known proteins may indicate that NM.BstSEI system operone has evolved by horizonthal DNA transfer.     

Nucleotide sequence analysis of the 3'-terminal region of two Korean isolates of lily symptomless Carlavirus and expression of the coat protein in E. coli.

The 3'-terminal regions of the genomic RNAs of two Korean isolates of the lily symptomless Carlavirus (LSV), LSV-Ko and LSV-KII, were cloned and their nucleotide sequences were determined. The nucleotide sequence analysis and protein analysis by the Western blot revealed that E. coli expressed a 32-kDa protein that is the viral coat protein (CP) for the LSV. The two Korean strains share 98.4% and 98.3% sequence identities at the nucleotide and amino acid levels, respectively. The CP gene of LSV-Ko showed 99.1% and 87.0% nucleotide sequence identities, and 99.0% and 96.6% amino acid sequence identities with those of the Netherlands and the Japanese LSV strains, respectively. A pairwise amino acid sequence comparison revealed a sequence similarity of 29.6% to 69.8% between LSV-Ko and other species of the carlavirus. The 16 kDa protein of LSV-Ko shares 17.6% to 42.7% amino acid similarity with those of 8 other the carlaviruses, and they are variable in the N-terminal region. The Cys repeated zinc finger nucleic acid binding domain was found in the 16 kDa protein for all of the LSV strains. Sequence comparisons of the 7 kDa protein of LSV in the strain level showed significant identities from 100.0% to 98.4%. LSV-Ko shares 21.9% to 42.2% amino acid similarity with those of 8 other carlaviruses, 4 members of the potexviruses, and a closterovirus. LSV is closely related to blueberry scorch virus (BISV) based upon the phylogenetic tree analyses of the three proteins, indicating LSV to be a quite distinct member of the genus Carlavirus.     

Nucleotide sequence and molecular characterization of the structural glycoprotein gp41 gene homologue of Spodoptera litura nucleopolyhedrosis virus (spltNPV-I)

The structural glycoprotein gene gp41 homologue of Spodoptera litura nucleopolyhedrosis virus (SpltNPV-I *) was identified in the 4.0 kb EcoRI-L fragment of the viral genome. The nucleotide sequence of 2063 bp of this fragment revealed an open reading frame of 1014 nucleotides to encode a polypeptide of 337 amino acids. Analysis of nucleotide and deduced amino acid sequences of the putative ORF indicated its identity with gp41 protein of other baculoviruses sharing maximum homology with that of Spodoptera frugiperda nucleopolyhedrosis virus (SfNPV). The coding sequence was preceded by an AT-rich region containing the consensus baculoviral late promoter motif RTAAG. The putative SpltNPV gp41 ORF was abundantly expressed as a 37 kDa apoprotein in E. coli and as a 50 kDa glycoprotein in Sf9 cells. The recombinant protein expressed in insect cells was glycosylated (20%) and has GlcNAc as the terminal sugar. The gene is conserved among baculoviruses and places SpltNPV-I close to Spodoptera frugiperda and Spodoptera exigua NPVs in phylogenetic tree.