Phycodnaviridae

Phycodnaviridae is a family of large double-stranded DNA viruses that infect marine or freshwater eukaryotic algae. Viruses within this family have a similar morphology, with an icosahedral capsid. As of 2014, there were 33 species in this family, divided among 6 genera. This family belongs to a super-group of large viruses known as nucleocytoplasmic large DNA viruses. Evidence was published in 2014 suggesting that specific strains of Phycodnaviridae might infect humans rather than just algal species, as was previously believed. Most genera under this family enter the host cell by cell receptor endocytosis and replicate in the nucleus. Phycodnaviridae play important ecological roles by regulating the growth and productivity of their algal hosts. Algal species such Heterosigma akashiwo and the genus Chrysochromulina can form dense blooms which can be damaging to fisheries, resulting in losses in the aquaculture industry. Heterosigma akashiwo virus has been suggested for use as a microbial agent to prevent the recurrence of toxic red tides produced by this algal species. Phycodnaviridae cause death and lysis of freshwater and marine algal species, liberating organic carbon, nitrogen and phosphorus into the water, providing nutrients for the microbial loop.

Taxonomy

Group: double-stranded DNA
The taxonomy of this family was initially based on host range: chloroviruses infect chlorella-like green algae from freshwaters; whereas, members of the other five genera infect marine microalgae and a some species of brown macroalgae. This was subsequently confirmed by analysis of their B-family DNA polymerases, which indicated that members of the Phycodnaviridae are more closely related to one another, in comparison to other double stranded DNA viruses, forming a monophyletic group. The phycodnaviruses contain six genera: Coccolithovirus, Chlorovirus, Phaeovirus, Prasinovirus, ''Prymnesiovirus and Raphidovirus''. The genera can be distinguished from one another by, for example, differences in life cycle and gene content.

Structure

All six genera in the family Phycodnaviridae have similar virion structure and morphology. They are large virions that can range between 100 and 220 nm in diameter. They have a double-stranded DNA genome, and a protein core surrounded by a lipid bilayer and an icosahedral capsid. The capsid has 2, 3 and 5 fold axis of symmetry with 20 equilateral triangle faces composing of protein subunits. In all known members of the Phycodnaviridae the capsid is composed of ordered substructures with 20 trisymmetrons and 12 pentasymmetrons made up of donut-shaped trimeric capsomers, where each capsomer is made up of three monomers of the major capsid protein. If all the trimeric capsomers are identical in structure, the virion capsid contains 5040 copies of the major capsid protein in total with a triangulation number of 169. At the five-fold vertices there are 12 pentamer-capsomers consist of different proteins. The protein that can be found below the axial channel of each pentamer may be responsible for digesting the host cell wall during viral infection. The species Phaeocystis puchetii virus from the genus Prymnesiovirus has the largest capsid structure in the Phycodnaviridae family.
The lipid bilayer membrane in phycodnaviruses is not well understood or researched. Some studies suggested that the membrane originates from the endoplasmic reticulum and may also be directly acquired from the host cell membrane during viral assembly. Although members of the family Phycodnaviridae are highly diverse, they share very conserved genes involved with virion morphology or structure.
Despite the similarity of the capsid structure of phycodnaviruses, recent experiments have identified morphological differences among members in this family. Emiliania huxleyi virus 86, a coccolithovirus strain, differs from its algal virus counterparts in that its capsid is enveloped by a lipid membrane. In addition, recent 3D reconstruction experiments revealed that the chlorella virus PBCV-1 has a 250A-long cylindrical spike extending from one of its vertices. EhV-86 may also possess a spike or tail structure.

Genome

Phycodnaviruses are known for their large double-stranded DNA genomes ranging from 100kb to over 550 kb with 40% to 50% GC content. Currently, complete genome sequences are available for several members of the family Phycodnaviridae and there are also some partial sequences available for a different coccolithovirus.
The genome structures of phycodnaviruses have considerable variation. The chlorovirus PBCV-1 has a linear 330 kb genome with non-permuted double-stranded DNA that is covalently closed by hairpin termini. Similarly, the EsV-1 phaeovirus has a linear double-stranded DNA genome with inverted repeats that have almost perfect homology. These inverted repeats could facilitate effective circularization of the genome and for a period of time it has been suspected that EsV-1 has a circular genome. The EhV-86 coccolithovirus is suggested to have both linear and circular genomes at different phases during DNA packaging. PCR amplification reveals random A/T overhangs, detection of DNA ligases and endonucleases hinting that a linear genome may be packaged and circularizes during DNA replication. The phycodnaviruses have compact genomes for replication efficiency with approximately one gene per 900 to 1000 bp of genome sequences. The EsV-1 phaeovirus is an exception with 231 protein encoding genes, which means it has one gene per approximately 1450 bp. In spite of the compact genomes typically found in viruses, Phycodnaviridae genomes have repetitive regions usually near the terminal ends and certain tandem repeats located throughout the genome. It is suggested that these repetitive sequences may play a role in gene recombination that allows the virus to exchange genetic information with other viruses or the host cell.

Phylogeny

Viruses belonging to Phycodnaviridae harbor double-stranded DNA genomes with sizes of several 100kbp, which together with other Megavirales are named nucleocytoplasmic large DNA viruses. Because of their large genome sizes and various proteins that are encoded, viruses of Phycodnaviridae are challenging the traditional concepts that viruses are small and simple "organisms at the edge of life". Phylogenetic analyses of core genes based on gene concatenation, individual phylogenies of the DNA polymerase, and the major capsid protein, indicate the close evolutionary relationships among members of Phycodnaviridae and between Phycodnaviridae and other families of nucleocytoplasmic large DNA viruses.

Life cycle

Genus	Host details	Tissue tropism	Entry details	Release details	Replication site	Assembly site	Transmission
Raphidovirus	Alga	-	Cell receptor endocytosis	Lysis	Nucleus	Cytoplasm	Passive diffusion
Coccolithovirus	Alga	-	Cell receptor endocytosis	Budding	Nucleus	Cytoplasm	Passive diffusion
Phaeovirus	Alga	-	Cell receptor endocytosis	Lysis	Nucleus	Cytoplasm	Passive diffusion
Chlorovirus	Alga	-	Cell receptor endocytosis	Lysis	Nucleus	Cytoplasm	Unknown
Prymnesiovirus	Alga	-	Cell receptor endocytosis	Lysis	Nucleus	Cytoplasm	Passive diffusion
Prasinovirus	Alga	-	Cell receptor endocytosis	Lysis and budding	Nucleus	Cytoplasm	Passive diffusion

Raphidovirus

In Raphidovirus, there is only one species, Heterosigma akashiwo virus, which infects the unicellular alga, Heterosigma akashiwo. H. akashiwo is a member of the class Raphidophyceae, a bloom forming species and is widely distributed in temperate and neritic waters. Several other types of viruses infecting H. akashiwo have been isolated and are not to be confused with HaV, such as the H. akashiwo RNA virus. and H. akashiwo nuclear inclusion virus. As HaV was first isolated and characterized in 1997, information about the life cycle is limited.
HaV specifically infects H. akashiwo and does not infect other marine phytoplankton species tested. The mechanisms determining the virus-host specificity is not well understood. Tomaru et al. suggest that virus-host specificity maybe caused by unique interactions between a viral ligand and a host receptor. In a study by Nagaski et al., virus particles were found inside the host cytoplasm at 24 hours post-infection. The latent period or lysogenic cycle was estimated to be 30–33 h with an average burst size of 770 per cell. Virus particles were found in the subsurface area and in the viroplasm area

Coccolithovirus

In 2009, MacKinder et al. elucidated the entry mechanism of the genera Coccolithovirus. Using confocal and electron microscopy, the researchers demonstrated that the virus strain EhV-86 uses a unique infection mechanism, which differs from other algal viruses, and shows a greater similarity to the entry and exit strategies seen in animal-like nucleocytoplasmic large double stranded DNA viruses. EhV-86 differs from its algal counterparts in that its capsid is enveloped by a lipid membrane. EhV-86 enters cells by endocytosis, or direct fusion. EhV-86 entry by endocytosis results in an additional membrane coat surrounding the capsid encapsulated genome. Regardless of the mechanism of entry, the capsid enters the cytoplasm intact. After entering the cell, the viral capsid disassembles and the DNA is released into the host cytoplasm or directly into the nucleus. EhV-86 is unique to other phycodnaviruses as it encodes six RNA polymerase subunits. Neither PBCV-1 nor ESV-1, for example encodes RNA polymerase components. Viral RNA polymerase genes are not transcribed until at least 2 hours post infection. At 3–4 p.i, virions are assembled in the cytoplasm, with the help of ATPase and transported to the plasma membrane where they are released from the host via a budding mechanism. In this budding mechanism, EhV-86 gains an outer membrane from the host membrane. Burst size ranges from 400 to 1000 particles per cell.
A cluster of sphingolipid-producing genes have been identified in EhV-86. Researchers have found that the production of viral sphingolipids produced during the lytic stage are involved in programmed cell death in coccolithophore populations. A high correlation was found between glycosphingolipid production and caspase activity during the lytic stage in infected cells. Caspases are a family of protease enzymes involved in programmed cell death. The researchers also found that a critical concentration of GSLs is required to initiate cell lysis. Thus, the authors suggest that the production of GSLs to a critical concentration may be part of a timing mechanism for the lytic cycle. The authors also suggest that these biomolecules may be able to induce programmed cell death in other unaffected cells, thus serving as an algal bloom termination signal.