While viruses with distinct phylogenetic origins and different nucleic acid types can infect and lyse eukaryotic phytoplankton, “giant” dsDNA viruses have been found to be associated with important ecological processes, including the collapse of algal blooms. However, the molecular aspects of giant virus–host interactions remain largely unknown. Aureococcus anophagefferens virus (AaV), a giant virus in the Mimiviridae clade, is known to play a critical role in regulating the fate of brown tide blooms caused by the pelagophyte Aureococcus anophagefferens. To understand the physiological response of A. anophagefferens CCMP1984 upon AaV infection, we studied the transcriptomic landscape of this host–virus pair over an entire infection cycle using a RNA-sequencing approach. A massive transcriptional response of the host was evident as early as 5 min post-infection, with modulation of specific processes likely related to both host defense mechanism(s) and viral takeover of the cell. Infected Aureococcus showed a relative suppression of host-cell transcripts associated with photosynthesis, cytoskeleton formation, fatty acid, and carbohydrate biosynthesis. In contrast, host cell processes related to protein synthesis, polyamine biosynthesis, cellular respiration, transcription, and RNA processing were overrepresented compared to the healthy cultures at different stages of the infection cycle. A large number of redox active host-selenoproteins were overexpressed, which suggested that viral replication and assembly progresses in a highly oxidative environment. The majority (99.2%) of annotated AaV genes were expressed at some point during the infection cycle and demonstrated a clear temporal–expression pattern and an increasing relative expression for the majority of the genes through the time course. We detected a putative early promoter motif for AaV, which was highly similar to the early promoter elements of two other Mimiviridae members, indicating some degree of evolutionary conservation of gene regulation within this clade. This large-scale transcriptome study provides insights into the Aureococcus cells infected by a giant virus and establishes a foundation to test hypotheses regarding metabolic and regulatory processes critical for AaV and other Mimiviridae members.

Some giant viruses are ecological agents that are predicted to be involved in the top-down control of single-celled eukaryotic algae populations in aquatic ecosystems. Despite an increased interest in giant viruses since the discovery and characterization of Mimivirus and other viral giants, little is known about their physiology and ecology. In this study, we characterized the genome and functional potential of a giant virus that infects the freshwater haptophyte Chrysochromulina parva , originally isolated from Lake Ontario. This virus, CpV-BQ2, is a member of the nucleo-cytoplasmic large DNA virus (NCLDV) group and possesses a 437 kb genome encoding 503 ORFs with a GC content of 25%. Phylogenetic analyses of core NCLDV genes place CpV-BQ2 amongst the emerging group of algae-infecting Mimiviruses informally referred to as the “extended Mimiviridae ,” making it the first virus of this group to be isolated from a freshwater ecosystem. During genome analyses, we also captured and described the genomes of three distinct virophages that co-occurred with CpV-BQ2 and likely exploit CpV for their own replication. These virophages belong to the polinton-like viruses (PLV) group and encompass 19–23 predicted genes, including all of the core PLV genes as well as several genes implicated in genome modifications. We used the CpV-BQ2 and virophage reference sequences to recruit reads from available environmental metatranscriptomic data to estimate their activity in fresh waters. We observed moderate recruitment of both virus and virophage transcripts in samples obtained during Microcystis aeruginosa blooms in Lake Erie and Lake Tai, China in 2013, with a spike in activity in one sample. Virophage transcript abundance for two of the three isolates strongly correlated with that of the CpV-BQ2. Together, the results highlight the importance of giant viruses in the environment and establish a foundation for future research on the physiology and ecology CpV-BQ2 as a model system for algal Mimivirus dynamics in freshwaters.

Candida albicans is among the most prevalent opportunistic human fungal pathogens. The ability to mask the immunogenic polysaccharide β (1,3)-glucan from immune detection via a layer of mannosylated proteins is a key virulence factor of C. albicans. We previously reported that hyperactivation of the Cek1 mitogen-activated protein (MAP) kinase pathway promotes β (1,3)-glucan exposure. In this communication, we report a novel upstream regulator of Cek1 activation and characterize the impact of Cek1 activity on fungal virulence. Lrg1 encodes a GTPase-activating protein (GAP) that has been suggested to inhibit the GTPase Rho1. We found that disruption of LRG1 causes Cek1 hyperactivation and β (1,3)-glucan unmasking. However, when GTPase activation was measured for a panel of GTPases, the lrg1ΔΔ mutant exhibited increased activation of Cdc42 and Ras1 but not Rho1 or Rac1. Unmasking and Cek1 activation in the lrg1ΔΔ mutant can be blocked by inhibition of the Ste11 MAP kinase kinase kinase (MAPKKK), indicating that the lrg1ΔΔ mutant acts through the canonical Cek1 MAP kinase cascade. In order to determine how Cek1 hyperactivation specifically impacts virulence, a doxycycline-repressible hyperactive STE11ΔN467 allele was expressed in C. albicans. In the absence of doxycycline, this allele overexpressed STE11ΔN467, which induced production of proinflammatory tumor necrosis factor alpha (TNF-α) from murine macrophages. This in vitro phenotype correlates with decreased colonization and virulence in a mouse model of systemic infection. The mechanism by which Ste11ΔN467 causes unmasking was explored with RNA sequencing (RNA-Seq) analysis. Overexpression of Ste11ΔN467 caused upregulation of the Cph1 transcription factor and of a group of cell wall-modifying proteins which are predicted to impact cell wall architecture. IMPORTANCE Candida albicans is an important source of systemic infections in humans. The ability to mask the immunogenic cell wall polymer β (1,3)-glucan from host immune surveillance contributes to fungal virulence. We previously reported that the hyperactivation of the Cek1 MAP kinase cascade promotes cell wall unmasking, thus increasing strain immunogenicity. In this study, we identified a novel regulator of the Cek1 pathway called Lrg1. Lrg1 is a predicted GTPase-activating protein (GAP) that represses Cek1 activity by downregulating the GTPase Cdc42 and its downstream MAPKKK, Ste11. Upregulation of Cek1 activity diminished fungal virulence in the mouse model of infection, and this correlates with increased cytokine responses from macrophages. We also analyzed the transcriptional profile determined during β (1,3)-glucan exposure driven by Cek1 hyperactivation. Our report provides a model where Cek1 hyperactivation causes β (1,3)-glucan exposure by upregulation of cell wall proteins and leads to more robust immune detection in vivo, promoting more effective clearance.

The discovery of infectious particles that challenge conventional thoughts concerning “what is a virus” has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning “giant viruses”, with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host–virus systems that fall into this “giant” category, demonstrating that this field of inquiry presents great opportunities for future research.

The scope for ecological studies of eukaryotic algal viruses has greatly improved with the development of molecular and bioinformatic approaches that do not require algal cultures. Here, we review the history and perceived future opportunities for research on eukaryotic algal viruses. We begin with a summary of the 65 eukaryotic algal viruses that are presently in culture collections, with emphasis on shared evolutionary traits (e.g., conserved core genes) of each known viral type. We then describe how core genes have been used to enable molecular detection of viruses in the environment, ranging from PCR-based amplification to community scale “-omics” approaches. Special attention is given to recent studies that have employed network-analyses of -omics data to predict virus-host relationships, from which a general bioinformatics pipeline is described for this type of approach. Finally, we conclude with acknowledgement of how the field of aquatic virology is adapting to these advances, and highlight the need to properly characterize new virus-host systems that may be isolated using preliminary molecular surveys. Researchers can approach this work using lessons learned from the Chlorella virus system, which is not only the best characterized algal-virus system, but is also responsible for much of the foundation in the field of aquatic virology.