Giant virus news
Rossmann's laboratory published a preliminary cryo-electron microscopy study of Mimivirus with its capsid reconstructed at a resolution of 75 angstroms (PMID: 16185710). The study confirms the icosahedral symmetry of the particle that establishes a new world-record breaking triangulation number of 1179 (thus each of the 20 faces of the icosahedral particle is made of about 3540 capsid protein molecules). The core particle has a diameter of 0.750 micrometer, including a 0.125 micrometer-thick layer of densely packed fibers, that might consist of cross-linked glycosylated-collagen-repeat containing proteins, as predicted in the original genome analysis (PMID: 15486256). This unique highly reticulated "virus-wall" is most likely responsible for the retention of the Gram stain that caused the virus to be originally misidentified as a Gram-positive cocci (PMID: 12663918).
Plenty of new large phycodnaviruses
Brussaard laboratory from the Royal Netherlands Institute for Sea Research, published the preliminary characterization of several new viruses infecting the algal bloom species Phaeocystis globosa (isolated from the southern North Sea in 2000-2001). These includes some with giant genome sizes of about 466 kb (PMID: 16081120). In the meantime, Bubeck and Pfitzner in Stuttgart (Germany) isolated a new virus named Acanthocystis turfacea Chlorella virus (ATCV) that infect the endosymbiotic Chlorella algae of the heliozoon Acanthocystis turfacea (PMID: 16186243)(this is another strange and beautiful protist).
We cannot wait to see the corresponding genome sequences!
Mimivirus: one more exceptional genomic feature
Suhre, Audic and Claverie discovered that 50% of the 911 Mimivirus predicted genes are preceded by the exactly conserved motif AAAATTGA (PMID:16203998). This "word" most likely constitutes the main promoter signal for the early or late/early gene of the virus, and is not prevalent in the promoter regions of other known dsDNA viruses, or known eukaryotes, including amoebal protists. Such a level of promoter conservation is also unheard among all organisms, in particular eukaryotic ones, where locating promoter has remained an unmet challenge. The AAAATTGA motif may correspond to an ancestral promoter structure predating the radiation of the eukaryotic kingdoms. Its presence in front of half Mimivirus genes strongly supports the notion that Mimivirus genome is not a "bag of genes" laterally transferred from random host encounters, but that it is the result of an evolutionary process that preserved a remarkable genome-wide homogeneity, not yet seen in other microorganisms.
Confusion in Microbiology
In an insightful review article, Ward and Fraser (PMID: 16125442) discuss how recent genomic breakthroughs are increasingly affecting key microbiological concepts thought to be established for ever. Mimivirus is, indeed, cited as one of the key player in this game of mass destruction.
Following the publication of the 407 kb-genome sequence of EhV-86, a dsDNA virus that infects the marine coccolithophorid Emiliania huxleyi, Willie Wilson's team at Plymouth Marine Laboratory has been very quick in producing two follow up studies, one describing the various repeat families found in the genome (PMID: 16195784), the other determining the phylogenetic relationship between EhV-86 and other large dsDNA viruses (PMID:16151186). EhV-86 does cluster with the other known Phycodnaviridae, although the unique presence of a complete set of RNA polymerase subunits in its genome suggests a somewhat different evolutionary history and life style for this virus.
dUTPase (deoxy-UTP pyrophosphatase), an "essential" enzyme of DNA metabolism curiously lacking from Mimivirus genome, has been enzymatically characterized in two more giant viruses: Phycodnavirus classic PBCV-1 (PMID: 16014955) and the unique of its kind Nimaviridae White spot syndrome virus (PMID: 15845252), where it performs as predicted. Mimivirus might still be endowed with a dUTPase too divergent in sequence to be identified by current programs. In a different study using DNA micro-array (PMID: 15958687), 79% of the open reading frames identified in the White spot syndrome virus (a large majority without any similarity in current databases) where found to correspond to bona fide transcripts detected in the gills of WSSV-infected shrimps. Although they don't resemble anything, these many giant virus "ORFans" of unknown origin, are indeed actual genes.
It's official: there are viral-encoded microRNAs
Willie Wilson and his team at Plymouth Marine Laboratory, in collaboration with scientists from the Sanger Institute published the 407,339-base pair genome sequence of a Coccolithovirus that infects the calcifying microalga Emiliania huxleyi (Science, 2005, 309:1090, PMID: 16099989). Coccolithovirus HeV-86 genome is now third in size behind Acanthamoeba polyphaga Mimivirus and Phage G (see http://giantvirus.org/top.html ). In addition to finding a number of original enzymes (including the first RNA polymerase found in a phycodnavirus), the authors used a microarray transcriptomic analysis to show that 65% of the predicted virus-encoded genes are expressed during lytic infection. Thus, the many genes of this large virus (and presumably of others) are not just for show, and the paradoxical complexity of giant viruses remains to be explained.
Elodie Ghedin (from TIGR) and Jean-Michel Claverie reported the likely presence of Mimivirus relative in the Sargasso Sea, using bioinformatic and phylogenetic analyses of the environmental sequence data previously gathered by Venter's sampling expedition (Virology J. 2005, 2:62, PMID: 16105173). The race is now open among several grous to isolate one of them for real†! In the meantime, new giant viruses (with genome sizes up to 466 kb) infecting the marine unicellular eukaryotic algal bloom species Phaeocystis globosa have been described by Baudoux and Brussaard (Virology, 2005, PMID: 16081120).
Following Mimivirus tyrosyl-tRNA synthetase (Abergel et al. Acta Cryst. 2005, F61: 212-215), Chantal Abergel's team is now getting closer to the 3D structure of Mimivirus Nucleoside Diphosphate kinase (NDK), the first viral example of such an enzyme (Jeudy et al., Acta Cryst. 2005, F61: 212-215).
We forgot to mention last month's interesting publication by Nagasaki et al. (Appl Environ Microbiol. 2005, 71: 3599-607, PMID: 16000767) , on the intein found in the DNA polymerase gene of various strains of Heterosigma akashiwo virus (HaV), a large double-stranded DNA virus infecting the single-cell bloom-forming raphidophyte (golden brown alga) H. akashiwo. The HaV intein is closely related to the mimivirus intein, and both are apparently monophyletic to the archaeal inteins. One more good reason to look for Mimivirus relatives in the ocean!
Finally, we posted a manuscript with some reflexions on the paradoxical complexity of giant viruses, and the rationale behind some of mimivirus properties on the preprint public archive site at http://arxiv.org, in preparation for a future review/opinion article. Comments are welcome, in particular on our proposal to use the term "Girus" for giant viruses of high genomic complexity (Claverie et al., Mimivirus and the emerging concept of "giant" virus, q-bio.PE/0506007). On the same arxiv.org site, you may want to read other preprints of incoming mimivirus-related articles †: Suhre et al., Mimivirus Gene Promoters Exhibit an Unprecedented Conservation among all Eukaryotes, q-bio.GN/0504012†; and Suhre, Gene & Genome Duplication in Acanthamoeba Polyphaga Mimivirus, q-bio.GN/0505049).
A short report on giant viruses in the ocean (Virology Journal 2005, 2: 52) made it to the Top 10 most accessed list of BMC Virology journal articles: Read it before going to the beach!
Following Paramecium bursaria chlorella virus 1 (PBCV-1, 330,744 bp) infecting the fresh water chlorella species NC64A, two more of viruses infecting NC64A have had their genome sequenced: AR158 (344,690 bp) and NY2A (368,683 bp). In addition the genome sequence of MT325 (314,335 bp), infecting chlorella species Pbi, is now also available. All sequences can be downloaded from Michael Graves' site (http://greengene.uml.edu/research.html). They also have been incorporated to our GiantVirus blast server. These sequences have been produced at TIGR through a NSF/Microbial Genome Sequencing Award (EF-0333197) to Michael Graves and Jim van Etten. There are now 10 available viral genome sequences larger than 300 kb, including 6 from phycodnaviruses.
The 407,339 bp genome sequence of Coccolithovirus EhV-86 and the corresponding translated amino-acid ORF sequences are now available for blasting separately from the other large DNA viruses. The data was downloaded from the Wellcome Trust Sanger Center Institute ftp site. The Sanger Institute sequenced the genome of Emiliania huxleyi virus 86 in collaboration with Dr. Declan Schroeder of the The Marine Biological Association, Plymouth, UK, and Dr. William Wilson of the The Plymouth Marine Laboratory, UK. Please refer to the Sanger Center Data release policy and Guidelines on use of this data in publications.
The 4th Algal Virus Workshop organized by Corina Brussaard and Herman Gons, and hosted by the Royal Netherlands Institute for Sea Research, was held in Amsterdam 17-21 april 2005. Though marine ecology rather than basic virology was the main focus of this meeting, exciting new results on the genomics of large/giant viruses kept turning up in many talks. In collaboration with DoE, Corina Brussaard is herself sequencing a variety of dsDNA Phaeocystis globosa and Micromonas pusilla viruses for comparative analysis, of which some of them are estimated to have a genome size of 460 kb.
In his overview, Curtiss Suttle (University of British Columbia, Vancouver, Canada) pointed out that viruses (including RNA-, DNA-, prokaryotic and eukaryotic viruses) constitute a significant part of the biomass in ocean coastal waters (with up to 50 millions particles/ml, for a total estimate of 25 to 270 Megatons in the oceans) where they play a dominant role in the control of phyto- and bacterio-plankton populations, and hence on the production of oxygen and atmospheric dimethylsulphide, an important factor in climate regulation. Most of these viruses are uncharacterized.
Ironically, this is from a freshwater unicellular green alga that the best characterized large DNA virus Paramecium bursaria chlorella virus (PBCV-1), the prototype of the Phycodnaviridae, was isolated more than 20 years ago in Jim van Etten's laboratory (University of Nebraska, Lincoln). Lisa Fitzgerald (J. van Etten's laboratory) reported on the ongoing annotation of the finished genomic sequences of two new species of Paramecium bursaria chlorella viruses: NY-2A (infecting PBCV-1 host Chlorella species NC64A) and Chlorella Pbi virus MT325. NY-2A genome contains 368,683 bp, making it the largest phycodnavirus sequenced to date (although the competition is fierce, see below). Despite a 10% difference in size, the NY-2A genome and PBCV-1 genome (330 kb) exhibits a near perfect colinearity. With 314,335 bp the MT325 genome is slightly smaller and does not exhibit long range colinearity with the PBCV-1 and NY-2A genomes. As in previously sequenced phycodnaviridae, new unexpected functions turned up to be encoded is these two new genomes, such as the first aquaglycerolporin the activity of which has been experimentally verified. A detailed comparative proteomics of the three viral particles (each exhibiting about 120 virus-encoded polypeptides) is also under way (D. Dunigan et al.). It was known for some time that filamentous marine brown alga of genus Feldmannia were infected by large dsDNA viruses (phaeovirus) coming in two genome sizes: 158kbp and 178kbp (Ivey et al., Virology 220; 267-273, 1996). Prof. T-J. Choi, (Pukyong National University, Busan, Korea) reported on the completion of the genome sequencing of the short form of FsV infecting Feldmannia sp. The final sequence size is 153,259 bp (51.8 G+C). About 50% of the 161 predicted ORFs have their best matching homologues in Feldmannia irregularis virus (FirrV-1) or Ectocarpus Siliculosus virus (EsV-1). Nicolas Delaroque (Max Plank Institute for Chemical Ecology, Jena, Germany) reported on the difficulty to reach full closure in sequencing the genome of Feldmannia irregularis virus (FirrV-1), most probably due to the presence of long repeats. The current FirrV genome sequence data consist of 191,667 bp (in 16 contigs), encoding 156 putative proteins. His more recent experiments indicate that FirrV infection may lead to the release of a mixture of virus forms associated with a wide range of genome sizes (from 192kbp to 10 kbp).
Dr Keizo Nagasaki (National Research Institute of Fisheries, Hiroshima, Japan) reported that the sequencing the 356 kbp-genome of dinoflagelate Heterocapsa circularisquama infecting virus (HcV01 and the 294 kbp-genome sequence of Heterosigma akashivo infecting virus (HaV01), and the 145 kb-genome of T4-like looking cyanophage Ma-LMM01 is now under way. The same laboratory also recently completed the sequencing of the 145 kb-genome of T4-like looking cyanophage Ma-LMM01 is now under way. The same laboratory also recently completed the sequencing of the 145 kb-genome of T4-like looking cyanophage Ma-LMM01, infecting the toxic cyanobacterium Microcystis aeruginosa.
Coccolithovirus EhV-86¬†: a new bronze medal in the viral genome size conte
During the meeting, Willie Wilson (Plymouth Marine Laboratory) announced the completion by the Sanger center of the genome sequencing of EhV-86, a coccolithovirus infecting Emiliania huxleyi, an alien-looking calcarous nanoplankton. The genome is made of 407,339 bp (40.2% G+C) and encodes 472 putative protein coding regions. Only 66 (14%) of them have recognizable homologues in the public databases. As other giant viruses, the EhV-86 exhibits its share of unexpected genes and functions, most notably a number of enzymes involved in the biosynthesis of sphingolipids. Albeit phylogenetically branching at the root of the Phycodnaviridae (e.g. PBCV-1 or EsV), EhV-86 does encode it own DNA-dependent RNA polymerase complex, thus filling the gap with the other Nucleo-Cytoplasmic Large DNA virus families (Irido-, Asfar-, Pox-, and Mimi-viridae) that all exhibit virally-encoded RNA-polymerases. Pending approval by ICTV, EhV-86 might become the prototype of the coccolithovirinae, a new subfamily of phycodnaviridae.
In his closing lecture, Jim Van Etten, reminded the new comers in the field of algal viruses that reports of very large icosahedral virus -like particles in various aquatic and marine organisms can be traced back to the 50's, but failed to elicit much interest outside of community of marine biologists. The discovery and genome characterization of the large freshwater chlorella viruses, and more recently of giant amoeba infecting Mimivirus (remotely related to phycodnaviruses but not an algal virus) elicited a renewed interest in the genomics of these large marine viruses, as they may provide new insight on the early evolution of eukaryotes. Not unexpectedly, close relatives of Mimivirus appear to exist in the marine environment, as suggested by the numerous homologous sequences found by J.-M. Claverie and E. Ghedin, (The Institute for Genomic Research, Rockville, USA) in their exhaustive analysis of the Sargasso Sea environmental data set. The 4th Algal Virus Workshop made it clear that these giant algal viruses are now entering the genomic era at full speed. The amount of surprises that we can expect while deciphering their genomes will be as big as their diversity, and more dogma on what a virus should look like will probably be shattered along the way.
According to the International Committee on Taxonomy of Viruses, Mimivirus now becomes the first identified member of the genus Mimivirus from the family Mimiviridae . Given its known host, Mimivirus official species name becomes Acanthamoeba polyphaga mimivirus (APMV), and the name of this first isolate is Acanthamoeba polyphaga mimivirus.
Mimivirus associated with pneumonia patients in ICU¬†?
Is Acanthamoeba polyphaga mimivirus also a human pathogen¬†? In a first attempt to answer this question, La Scola et al. (Emerg. Infect. Dis. 11: 449-452, 2005) have performed a serological survey of pneumonia patients in ICU. They found that up to 20% of them exhibited antibody cross-reacting with Mimivirus. These antibodies appear to recognize the capsular material surrounding the viral particle, leaving open the possibility of a cross-reaction with a polysaccharidic moiety of bacterial origin. Mimivirus DNA was also found in respiratory samples of a patient with hospital-acquired pneumonia. The authors insist that their results should be interpreted with caution, and that the formal demonstration that Acanthamoeba polyphaga mimivirus is a bona fide human pathogen -that would make it the virus with the broadest host range - is not in yet.
Thanks to the work of F. Enault (a PhD student here), the display of viral proteins phylogenetic profiles is now available for representatives of all NCLDV¬†: Arfar-, Pox-, Irido, Phycodna- , Baculo-, Herpes-, Nima- & Mimi- viridae. This tool can now be used to help annotate genes from their co-evolution patterns (in particular unknown ones), or get a one-glance picture of the conservation pattern of a given gene across all NCLDV families (e.g. use PBCV-1 A10R and see how capsid proteins exhibit a clear relationship in Irido, Phycodna, and Mimi-viridae). The analysis takes advantage of two unpublished phycodnavirus genomes (NY-2A, and MT-325, thanks to Prof. van Etten) and (Phage G, thanks to Dr. Hendrix). A manuscript describing the details of this analysis is in preparation (F. Enault et coll.).
More Inteins in Nucleo-Cytoplasmic Large DNA viruses
A detailed report describing the intein found in Mimivirus DNA polymerase is now available on line (Ogata et al., Virol. J. 2(1):8, PMID: 15707490). Dr. Nagasaki, Pietrokovski and coll., also have a paper in press (Appl. Environ. Microbiol. in press) reporting on the presence of an intein in the DNA polymerase of Heterosigma akashiwo viruses (HaV). This intein is unexpectedly similar to the one found in Mimivirus.
Thanks to Dr. Roger W. Hendrix from the Pittsburgh Bacteriophage Institute, the largest known phage genome sequence is now available for similarity search on this site (select the relevant option in ¬† Comprehensive double-stranded DNA virus genome Blast search (nt)). Depending on your query type (nt, aa, or nt) use Blastn, Blastx, or Tblastn. A database of translated ORFs (>300nt) is also provided for blastp searches.
According to Dr. Hendrix, phage G appears to encode 682 proteins, 26% of which have a match in Genbank. His team has also recognized 17 tRNAs, one tmRNA and many genes not normally associated with viral genomes. Find out by yourself which ones they are
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