Capsid Structure of a Marine Algal Virus of the Order Picornavirales.

The order Picornavirales includes viruses that infect different kinds of eukaryotes and that share similar properties. The capsid proteins (CPs) of viruses in the order that infect unicellular organisms, such as algae, presumably possess certain characteristics that have changed little over the course of evolution, and thus these viruses may resemble the Picornavirales ancestor in some respects. Herein, we present the capsid structure of Chaetoceros tenuissimus RNA virus type II (CtenRNAV-II) determined using cryo-electron microscopy at a resolution of 3.1 Å, the first alga virus belonging to the family Marnaviridae of the order Picornavirales A structural comparison to related invertebrate and vertebrate viruses revealed a unique surface loop of the major CP VP1 that had not been observed previously, and further, revealed that another VP1 loop obscures the so-called canyon, which is a host-receptor binding site for many of the mammalian Picornavirales viruses. VP2 has an N-terminal tail, which has previously been reported as a primordial feature of Picornavirales viruses. The above-mentioned and other critical structural features provide new insights on three long-standing theories about Picornavirales: (i) the canyon hypothesis, (ii) the primordial VP2 domain swap, and (iii) the hypothesis that alga Picornavirales viruses could share characteristics with the Picornavirales ancestor.IMPORTANCE Identifying the acquired structural traits in virus capsids is important for elucidating what functions are essential among viruses that infect different hosts. The Picornavirales viruses infect a broad spectrum of hosts, ranging from unicellular algae to insects and mammals and include many human pathogens. Those viruses that infect unicellular protists, such as algae, are likely to have undergone fewer structural changes during the course of evolution compared to those viruses that infect multicellular eukaryotes and thus still share some characteristics with the Picornavirales ancestor. This article describes the first atomic capsid structure of an alga Marnavirus, CtenRNAV-II. A comparison to capsid structures of the related invertebrate and vertebrate viruses identified a number of structural traits that have been functionally acquired or lost during the course of evolution. These observations provide new insights on past theories on the viability and evolution of Picornavirales viruses.

Can diatom girdle band pores act as a hydrodynamic viral defense mechanism?

Diatoms are microalgae encased in highly structured and regular frustules of porous silica. A long-standing biological question has been the function of these frustules, with hypotheses ranging from them acting as photonic light absorbers to being particle filters. While it has been observed that the girdle band pores of the frustule of Coscinodiscus sp. resemble those of a hydrodynamic drift ratchet, we show using scaling arguments and numerical simulations that they cannot act as effective drift ratchets. Instead, we present evidence that frustules are semi-active filters. We propose that frustule pores simultaneously repel viruses while promoting uptake of ionic nutrients via a recirculating, electroosmotic dead-end pore flow, a new mechanism of "hydrodynamic immunity".

RNA viruses as major contributors to Antarctic virioplankton.

Early work on marine algal viruses focused exclusively on those having DNA genomes, but recent studies suggest that RNA viruses, especially those with positive-sense, single-stranded RNA (+ssRNA) genomes, are abundant in tropical and temperate coastal seawater. To test whether this was also true of polar waters, we estimated the relative abundances of RNA and DNA viruses using a mass ratio approach and conducted shotgun metagenomics on purified viral samples collected from a coastal site near Palmer Station, Antarctica on six occasions throughout a summer phytoplankton bloom (November-March). Our data suggest that RNA viruses contributed up to 65% of the total virioplankton (8-65%), and that, as observed previously in warmer waters, the majority of RNA viruses in these Antarctic RNA virus metagenomes had +ssRNA genomes most closely related to viruses in the order Picornavirales. Assembly of the metagenomic reads resulted in five novel, nearly complete genomes, three of which had features similar to diatom-infecting viruses. Our data are consistent with the hypothesis that RNA viruses influence diatom bloom dynamics in Antarctic waters.

Discovery of two novel viruses expands the diversity of single-stranded DNA and single-stranded RNA viruses infecting a cosmopolitan marine diatom.

Recent studies have suggested that diatom viruses are an important factor affecting diatom population dynamics, which in turn are important in considering marine primary productivity. The marine planktonic diatom Chaetoceros tenuissimus Meunier is a cosmopolitan species and often causes blooms off the western coast of Japan. To date, two viruses, C. tenuissimus DNA virus (CtenDNAV) type I and CtenRNAV type I, have been identified that potentially affect C. tenuissimus population dynamics in the natural environment. In this study, we successfully isolated and characterized two additional novel viruses (CtenDNAV type II and CtenRNAV type II). This paper reports the basic characteristics of these new viruses isolated from surface water or sediment from the Hiroshima Bay, Japan. The physiological and morphological characteristics of the two new viruses were similar to those of the previously isolated viruses. However, the amino acid sequences of the structural proteins of CtenDNAV type II and CtenRNAV type II were clearly distinct from those of both type I viruses, with identity scores of 38.3% and 27.6%, respectively. Our results suggest that at least four genetically distinct viruses sharing the same diatom host are present in western Japan and affect the population dynamics of C. tenuissimus. Moreover, the result that CtenRNAV type II lysed multiple diatom species indicates that RNA viruses may affect various diatom populations in the natural environment.

Characterization of Chaetoceros lorenzianus-infecting DNA virus-derived promoters of genes from open reading frames of unknown function in Phaeodactylum tricornutum.

Promoters are key elements for the regulation of gene expression. Recently, we investigated the activity of promoters derived from marine diatom-infecting viruses (DIVs) in marine diatoms. Previously, we focused on potential promoter regions of the replication-associated protein gene and the capsid protein gene of the DIVs. In addition to these genes, two genes of unknown function (VP1 and VP4 genes) have been found in the DIV genomes. In this study, the promoter regions of the VP1 gene and VP4 gene derived from a Chaetoceros lorenzianus-infecting DNA virus (named ClP3 and ClP4, respectively) were newly isolated. ClP4 was found to be a constitutive promoter and displayed the highest activity. In particular, the 3' region of ClP4 (ClP4 3' region) showed a higher promoter activity than full-length ClP4. The ClP4 3' region might involve high-level promoter activity of ClP4. In addition, the ClP4 3' region may be useful for substance production and metabolic engineering of diatoms.

Isolation and characterization of a single-stranded DNA virus infecting Chaetoceros lorenzianus Grunow.

Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 10(4) infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus.

Growth Rate-dependent Cell Death of Diatoms due to Viral Infection and Their Subsequent Coexistence in a Semi-continuous Culture System.

Viral infections are a major factor in diatom cell death. However, the effects of viruses on diatom dynamics remain unclear. Based on laboratory studies, it is hypothesized that virus-induced diatom mortality is dependent on the diatom growth rate. The present study aimed to elucidate the relationship between the diatom growth rate and virus-induced mortality using model systems of the marine planktonic diatom, Chaetoceros tenuissimus and its infectious viruses. We also examined the fate of diatom populations in a semi-continuous dilution culture system, in which host growth rates were controlled at 0.69, 2.08, and 3.47 day-1. Diatom populations gradually decreased following the viral inoculation of each culture system, and virus-induced mortality inversely correlated with the diatom growth rate. Furthermore, the viral burst size was slightly higher in lower growth rate cultures. These results suggested that the host physiological status related to the growth rate affected viral infection and proliferation. Diatom populations were not completely lysed or washed out in any of the dilution systems; they showed steady growth in the presence of infectious viruses. This may be partially explained by defective interference particles from viruses and cell debris. The present results indicate that diatoms in dilution environments maintain their populations, even under viral pressure. Moreover, diatom populations with a low growth rate may partially sustain higher growth populations through nutrient recycling following virus-induced cell death. The results of the present study provide insights into diatom dynamics in natural environments in the presence of infectious viruses.

The genome of the diatom Chaetoceros tenuissimus carries an ancient integrated fragment of an extant virus.

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

Crystallization and preliminary X-ray analysis of a marine diatom-infecting single-stranded RNA virus.

Crystals of a diatom-infecting virus (CtenRNAV) that diffracted to a resolution of 4.0 Šwere grown in a mixture of 2-methyl-2,4-pentanediol (MPD), calcium chloride and sodium acetate. It was possible to freeze the crystals directly at liquid-nitrogen temperature as the reservoir solution, which included about 30% MPD, acted as a cryoprotectant during X-ray diffraction data collection. A data set was collected from a single frozen crystal obtained using this method. The crystals belonged to space group P6(3)22, with unit-cell parameters a = b = 448.67, c = 309.76 Šand two virus particles in the unit cell. The virus-particle orientation was determined using a rotation function and the virus-particle centre was estimated on the basis of crystallographic considerations. The packing of CtenRNAV in the crystal lattice was revealed by this preliminary crystallographic study.

Viral RNA Genomes Identified from Marine Macroalgae and a Diatom.

Protists provide insights into the diversity and function of RNA viruses in marine systems. Among them, marine macroalgae are good targets for RNA virome analyses because they have a sufficient biomass in nature. However, RNA viruses in macroalgae have not yet been examined in detail, and only partial genome sequences have been reported for the majority of RNA viruses. Therefore, to obtain further insights into the distribution and diversity of RNA viruses associated with marine protists, we herein examined RNA viruses in macroalgae and a diatom. We report the putative complete genome sequences of six novel RNA viruses from two marine macroalgae and one diatom holobiont. Four viruses were not classified into established viral genera or families. Furthermore, a virus classified into Totiviridae showed a genome structure that has not yet been reported in this family. These results suggest that a number of distinct RNA viruses are widespread in a broad range of protists.

The possibility of using marine diatom-infecting viral promoters for the engineering of marine diatoms.

Marine diatoms constitute a major group of unicellular photosynthetic eukaryotes. Diatoms are widely applicable for both basic studies and applied studies. Molecular tools and techniques have been developed for diatom research. Among these tools, several endogenous gene promoters (e.g., the fucoxanthin chlorophyll a/c-binding protein gene promoter) have become available for expressing transgenes in diatoms. Gene promoters that drive transgene expression at a high level are very important for the metabolic engineering of diatoms. Various marine diatom-infecting viruses (DIVs), including both DNA viruses and RNA viruses, have recently been isolated, and their genome sequences have been characterized. Promoters from viruses that infect plants and mammals are widely used as constitutive promoters to achieve high expression of transgenes. Thus, we recently investigated the activity of promoters derived from marine DIVs in the marine diatom, Phaeodactylum tricornutum. We discuss novel viral promoters that will be useful for the future metabolic engineering of diatoms.

Characterization of the Chaetoceros salsugineum nuclear inclusion virus coat protein gene.

Analysis of the genome of Chaetoceros salsugineum nuclear inclusion virus (CsNIV) revealed the presence of six putative open reading frames (ORFs) in the genome. We further characterized ORF3, which encodes a putative coat protein. Polymerase chain reaction (PCR) using ORF3 gene-specific primers amplified a single DNA band nearly 1.2kb. This amplified product was gel-purified, cloned, sequenced, and expressed in Escherichia coli. Specific antiserum was raised against the recombinant protein and used for Western blotting to test whether the ORF3 protein is the CsNIV coat protein. One major CsNIV protein of approximately 46kDa reacted positively with the antiserum, suggesting that this antiserum is specific for the CsNIV coat protein. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the 46kDa structural band revealed 14 peptide sequences that matched the ORF3 regions of CsNIV. The expression of ORF3 in host cells was examined by constructing a cDNA library of CsNIV-infected cells. Nucleotide sequences of the cDNA clones were complementary to various regions of both CsNIV ORF3 and ORF4; however, no clones containing only the ORF3 region were identified. Also, Northern blotting revealed a single 2.5-kb transcript, indicating that ORF3 could be transcribed together with ORF4.

Identification of freshwater Phycodnaviridae and their potential phytoplankton hosts, using DNA pol sequence fragments and a genetic-distance analysis.

Viruses that infect phytoplankton are an important component of aquatic ecosystems, yet in lakes they remain largely unstudied. In order to investigate viruses (Phycodnaviridae) infecting eukaryotic phytoplankton in lakes and to estimate the number of potential host species, samples were collected from four lakes at the Experimental Lakes Area in Ontario, Canada, during the ice-free period (mid-May to mid-October) of 2004. From each lake, Phycodnaviridae DNA polymerase (pol) gene fragments were amplified using algal-virus-specific primers and separated by denaturing gradient gel electrophoresis; 20 bands were extracted from the gels and sequenced. Phylogenetic analysis indicated that freshwater environmental phycodnavirus sequences belong to distinct phylogenetic groups. An analysis of the genetic distances "within" and "between" monophyletic groups of phycodnavirus isolates indicated that DNA pol sequences that differed by more than 7% at the inferred amino acid level were from viruses that infect different host species. Application of this threshold to phylogenies of environmental sequences indicated that the DNA pol sequences from these lakes came from viruses that infect at least nine different phytoplankton species. A multivariate statistical analysis suggested that potential freshwater hosts included Mallomonas sp., Monoraphidium sp., and Cyclotella sp. This approach should help to unravel the relationships between viruses in the environment and the phytoplankton hosts they infect.

Isolation and characterization of a single-stranded RNA virus infecting the bloom-forming diatom Chaetoceros socialis.

Diatoms are very significant primary producers in the world's oceans. Various environmental factors affect the depletion of diatom populations. The importance of viruses as a potential mortality source has recently been recognized. We isolated and characterized a new diatom virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) causing the lysis of the bloom-forming species Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko. The virus infectious to C. socialis f. radians was isolated from water samples collected in Hiroshima Bay. Here we show the physiology, morphology, and genome characteristics of the virus clone. Virions were 22 nm in diameter and accumulated in the cytoplasm of the host cells. The latent period and the burst size were estimated to be <48 h and 66 infectious units per host cell, respectively. CsfrRNAV harbors a single-stranded RNA (ssRNA) genome and encodes at least three polypeptides of 32.0, 28.5, and 25.0 kDa. Sequencing analysis shows the length of the genome is 9,467 bases, excluding a poly(A) tail. The monophyly of CsfrRNAV and other diatom-infecting RNA viruses, Rhizosolenia setigera RNA virus and Chaetoceros tenuissimus RNA virus, was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domains. This suggested a new ssRNA virus family, Bacillariornaviridae. This discovery of CsfrRNAV may aid in further understanding the ecological dynamics of the C. socialis f. radians population in nature and the relationships between ssRNA diatom viruses and their hosts.

Silicon limitation facilitates virus infection and mortality of marine diatoms.

Diatoms are among the most globally distributed and ecologically successful organisms in the modern ocean, contributing upwards of 40% of total marine primary productivity1,2. By converting dissolved silicon into biogenic silica, and photosynthetically fixing carbon dioxide into particulate organic carbon, diatoms effectively couple the silicon (Si) and carbon cycles and ballast substantial vertical flux of carbon out of the euphotic zone into the mesopelagic and deep ocean3-5. Viruses are key players in ocean biogeochemical cycles6,7, yet little is known about how viral infection specifically impacts diatom populations. Here, we show that Si limitation facilitates virus infection and mortality in diatoms in the highly productive coastal waters of the California Current Ecosystem. Using metatranscriptomic analysis of cell-associated diatom viruses and targeted quantification of extracellular viruses, we found a link between Si stress and the early, active and lytic stages of viral infection. This relationship was also observed in cultures of the bloom-forming diatom Chaetoceros tenuissimus, where Si stress accelerated virus-induced mortality. Together, these findings contextualize viruses within the ecophysiological framework of Si availability and diatom-mediated biogeochemical cycling.

Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 µm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Isolation and characterization of a single-stranded RNA virus infecting the marine planktonic diatom Chaetoceros tenuissimus Meunier.

Diatoms are important components of the biological community and food web in the aquatic environment. Here, we report the characteristics of a single-stranded RNA (ssRNA) virus (CtenRNAV01) that infects the marine diatom Chaetoceros tenuissimus Meunier (Bacillariophyceae). The ca. 31-nm virus particle is icosahedral and lacks a tail. CtenRNAV01 forms crystalline arrays occupying most of the infected host's cytoplasm. By growth experiments, the lytic cycle and the burst size were estimated to be <24 h and approximately 1 x 10(4) infectious units per host cell, respectively. Stationary-phase C. tenuissimus cultures were shown to be more sensitive to CtenRNAV01 than logarithmic-phase cultures. The most noticeable feature of this virus is its exceptionally high yields of approximately 10(10) infectious units ml(-1); this is much higher than those of any other algal viruses previously characterized. CtenRNAV01 has two molecules of ssRNA of approximately 8.9 and 4.3 kb and three major proteins (33.5, 31.5, and 30.0 kDa). Sequencing of the total viral genome has produced only one large contig [9,431 bases excluding the poly(A) tail], suggesting considerable overlapping between the two RNA molecules. The monophyly of CtenRNAV01 compared to another diatom-infecting virus, Rhizosolenia setigera RNA virus, was strongly supported in a maximum likelihood phylogenetic tree constructed based on the concatenated amino acid sequences of the RNA-dependent RNA polymerase domains. Although further analysis is required to determine the detailed classification and nomenclature of this virus, these data strongly suggest the existence of a diatom-infecting ssRNA virus group in natural waters.

Dinoflagellates, diatoms, and their viruses.

Since the first discovery of the very high virus abundance in marine environments, a number of researchers were fascinated with the world of "marine viruses", which had previously been mostly overlooked in studies on marine ecosystems. In the present paper, the possible role of viruses infecting marine eukaryotic microalgae is enlightened, especially summarizing the most up-to-the-minute information of marine viruses infecting bloom-forming dinoflagellates and diatoms. To author's knowledge, approximately 40 viruses infecting marine eukaryotic algae have been isolated and characterized to different extents. Among them, a double-stranded DNA (dsDNA) virus "HcV" and a single-stranded RNA (ssRNA) virus "HcRNAV" are the only dinoflagellate-infecting (lytic) viruses that were made into culture; their hosts are a bivalve-killing dinoflagellate Heterocapsa circularisquama. In this article, ecological relationship between H. circularisquama and its viruses is focused. On the other hand, several diatom-infecting viruses were recently isolated and partially characterized; among them, one is infectious to a pen-shaped bloom-forming diatom species Rhizosolenia setigera; some viruses are infectious to genus Chaetoceros which is one of the most abundant and diverse diatom group. Although the ecological relationships between diatoms and their viruses have not been sufficiently elucidated, viral infection is considered to be one of the significant factors affecting dynamics of diatoms in nature. Besides, both the dinoflagellate-infecting viruses and diatom-infecting viruses are so unique from the viewpoint of virus taxonomy; they are remarkably different from any other viruses ever reported. Studies on these viruses lead to an idea that ocean may be a treasury of novel viruses equipped with fascinating functions and ecological roles.

New insight into the RNA aquatic virosphere via viromics.

RNA viruses that infect microbes are now recognized as an active, persistent and important component of the aquatic microbial community. While some information about the diversity and dynamics of the RNA virioplankton has been derived from culture-based and single gene approaches, research based on viromic and metatransciptomic methods has generated unprecedented insight into this relatively understudied class of microbes. Here, the relevant literature is summarized and discussed, including viromic studies of extracellular aquatic RNA viral assemblages, and transcriptomic studies of active and associated RNA viruses from aquatic environments followed by commentary on the present challenges and future directions of this field of research.