Replication and transcription machinery for ranaviruses: components, correlation, and functional architecture

Background Ranaviruses (family Iridoviridae) are promiscuous pathogens that can infect across species barriers in poikilotherms and can replicate in amphibian and fish cells and even in cultured mammalian cells. However, as nucleocytoplasmic large DNA viruses (NCLDVs), their replication and transcription mechanisms remain largely unknown. Here, we screened and uncovered the replication and transcription machinery of two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), by a combination of methods, including the isolation of proteins on nascent DNA, recombinant virus-based affinity, and NanoLuc complementation assay. Results The ranavirus replication and transcription machinery was deeply dissected and identified as a complicated apparatus containing at least 30 viral and 6 host proteins. The viral proteins ADRV-47L/RGV-63R (DNA polymerase, vDPOL), ADRV-23L/RGV-91R (proliferating cell nuclear antigen, vPCNA), ADRV-85L/RGV-27R (single-stranded DNA binding protein, vSSB), ADRV-88L/RGV-24R (vhelicase/primase), etc., constitute the core replisome. Specifically, the core of the transcription complex, the viral RNA polymerase, contain the host RNAPII subunits Rpb3, Rpb6, and Rpb11, which was a first report in NCLDVs. Furthermore, correlations and interactions among these factors in the machinery were described. Significantly, the replisome core protein vDPOL (ADRV-47L) can interact with numerous viral and host proteins and could act as a linker and regulation center in viral DNA replication and transcription. Thus, these results depicted an architecture for ranavirus replication and transcription. Conclusions Up to 36 components from ranavirus and their host were found to form viral replisomes and transcription complexes using a series of precise methods, which further constructed an architecture for ranavirus replication and transcription in which vDPOL was a key central factor and various components correlated and cooperated. Therefore, it provides a cornerstone for further understanding the mechanisms of the replication and transcription of ranaviruses which can ensure the efficient production of progeny virus and adaptation to cross-species infection.

Virus-Host Interactions Reveal Potential Roles Towards Phosphorus Cycling in South Atlantic Ocean Euphotic Zones

BackgroundDue to their role as obligate parasites of marine microorganisms, viruses are primary mediators of marine biogeochemical cycles. Recent studies have provided irrevocable evidence showing that viruses augment the metabolisms of bacteria and archaea through expression of auxiliary metabolic genes (AMGs). Several studies have shown that AMGs affect the biogeochemical recycling of sulphur and nitrogen but comparatively less is known regarding their influence on phosphorus recycling.ResultsHere, we provide the first insights regarding the potential effects of phosphorus limitation and AMGs on putative prokaryotic hosts in the euphotic zone of the South Atlantic Ocean (SAO). We identified 7,176 viral contigs that were clustered into 5,999 viral operational taxonomic units (vOTUs, >5kb). These SAO viral communities appear to be unique, as over 89% had no taxonomic assignment, possibly due to the genetic endemism in this ocean. Three phosphatases, phoN, gmhB and rnhA-cobC, were identified as P-cycle AMGs in both prokaryotic double-stranded DNA viruses and eukaryotic Nucleocytoplasmic Large DNA viruses. These genes are associated with the acquisition of inorganic phosphate from phosphate esters, the largest reservoir of P-containing compounds in the marine environment. AMGs were identified in both uncultured and unclassified prokaryotic double-stranded DNA viruses predicted to infect Bacteriodetes, Proteobacteria, Chloroflexota and Poseidonales lineages. ConclusionTogether, these results suggest that viruses modulate P-cycling in euphotic zones of the ocean and that the acquisition of these phosphatase genes may be cues of P-ester stress.

A capsid with a twist: Assembly mechanism of the pleomorphic immature poxvirus scaffold

In Vaccinia virus (VACV), the prototype poxvirus, scaffold protein D13 forms a honeycomb-like lattice on the viral membrane that results in formation of the pleomorphic immature virion (IV). The structure of D13 is similar to those of major capsid proteins that readily form icosahedral capsids in nucleocytoplasmic large DNA viruses (NCLDVs). However, the detailed assembly mechanism of the non-icosahedral poxvirus scaffold has never been understood. Here we show the cryo-EM structures of D13 trimer and scaffold intermediates produced in vitro. The structures reveal that the short N-terminal α-helix is critical for initiation of D13 self-assembly. The continuous curvature of the IV is mediated by electrostatic interactions that induce torsion between trimers. The assembly mechanism explains the semi-ordered capsid-like arrangement of D13 that is distinct from icosahedral NCLDVs. Our structures explain how a single protein can self-assemble into different capsid morphologies, and represents a local exception to the universal Caspar-Klug theory of quasi-equivalence.

Unique viruses that infect Archaea related to eukaryotes

Asgard archaea are globally distributed, newly described microbes related to eukaryotes. Despite their importance, Asgard viruses have not been described. Here we characterize seven double-stranded DNA (dsDNA) viral genomes that infected Lokiarchaeota, Helarchaeota, and Thorarchaeota in deep-sea hydrothermal sediments. These viruses code for Caudovirales-like structural proteins, as well as proteins distinct from those described in archaeal viruses. They contain genes common in eukaryotic nucleocytoplasmic large DNA viruses (NCLDVs), and appear to be capable of semi-autonomous genome replication, repair, epigenetic modifications, and transcriptional regulation. Moreover, Helarchaeota viruses may hijack host ubiquitin systems similar to eukaryotic viruses. Recovery of these Asgard viral genomes reveals they contain features of both prokaryotic and eukaryotic viruses, and provides insights into their roles in the ecology and evolution of their hosts.

The genomes of nucleocytoplasmic large DNA viruses: viral evolution writ large

The nucleocytoplasmic large DNA viruses (NCLDVs) are a diverse group that currently contain the largest known virions and genomes, also called giant viruses. The first giant virus was isolated and described nearly 20 years ago. Their genome sizes were larger than for any other known virus at the time and it contained a number of genes that had not been previously described in any virus. The origin and evolution of these unusually complex viruses has been puzzling, and various mechanisms have been put forward to explain how some NCLDVs could have reached genome sizes and coding capacity overlapping with those of cellular microbes. Here we critically discuss the evidence and arguments on this topic. We have also updated and systematically reanalysed protein families of the NCLDVs to further study their origin and evolution. Our analyses further highlight the small number of widely shared genes and extreme genomic plasticity among NCLDVs that are shaped via combinations of gene duplications, deletions, lateral gene transfers and de novo creation of protein-coding genes. The dramatic expansions of the genome size and protein-coding gene capacity characteristic of some NCLDVs is now increasingly understood to be driven by environmental factors rather than reflecting relationships to an ancient common ancestor among a hypothetical cellular lineage. Thus, the evolution of NCLDVs is writ large viral, and their origin, like all other viral lineages, remains unknown.

The Mimivirus L375 Nudix enzyme hydrolyzes the 5’ mRNA cap

The giant Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group of diverse viruses that contain double-stranded DNA (dsDNA) genomes that replicate primarily in eukaryotic hosts. Two members of the NCLDV, Vaccinia Virus (VACV) and African Swine Fever Virus (ASFV), both synthesize Nudix enzymes that have been shown to decap mRNA, a process thought to accelerate viral and host mRNA turnover and promote the shutoff of host protein synthesis. Mimivirus encodes two Nudix enzymes in its genome, denoted as L375 and L534. Importantly, L375 exhibits sequence similarity to ASFV-DP and eukaryotic Dcp2, two Nudix enzymes shown to possess mRNA decapping activity. In this work, we demonstrate that recombinant Mimivirus L375 cleaves the 5’ m7GpppN mRNA cap, releasing m7GDP as a product. L375 did not significantly cleave mRNAs containing an unmethylated 5’GpppN cap, indicating that this enzyme specifically hydrolyzes methylated-capped transcripts. A point mutation in the L375 Nudix motif completely eliminated cap hydrolysis, showing that decapping activity is dependent on this motif. Addition of uncapped RNA significantly reduced L375 decapping activity, suggesting that L375 may recognize its substrate through interaction with the RNA body.

Morphological and Genomic Features of the New Klosneuvirinae Isolate Fadolivirus IHUMI-VV54

Since the discovery of Mimivirus, viruses with large genomes encoding components of the translation machinery and other cellular processes have been described as belonging to the nucleocytoplasmic large DNA viruses. Recently, genome-resolved metagenomics led to the discovery of more than 40 viruses that have been grouped together in a proposed viral subfamily named Klosneuvirinae. Members of this group had genomes of up to 2.4Mb in size and featured an expanded array of translation system genes. Yet, despite the large diversity of the Klosneuvirinae in metagenomic data, there are currently only two isolates available. Here, we report the isolation of a novel giant virus known as Fadolivirus from an Algerian sewage site and provide morphological data throughout its replication cycle in amoeba and a detailed genomic characterization. The Fadolivirus genome, which is more than 1.5Mb in size, encodes 1,452 predicted proteins and phylogenetic analyses place this viral isolate as a near relative of the metagenome assembled Klosneuvirus and Indivirus. The genome encodes for 66 tRNAs, 23 aminoacyl-tRNA synthetases and a wide range of transcription factors, surpassing Klosneuvirus and other giant viruses. The Fadolivirus genome also encodes putative vacuolar-type proton pumps with the domains D and A, potentially constituting a virus-derived system for energy generation. The successful isolation of Fadolivirus will enable future hypothesis-driven experimental studies providing deeper insights into the biology of the Klosneuvirinae.

Unique viruses that infect Archaea related to eukaryotes

Asgard archaea are newly described microbes that are related to eukaryotes. Asgards are diverse and globally distributed, however, their viruses have not been described. Here we characterize seven viral genomes that infected Lokiarchaeota, Helarchaeota, and Thorarchaeota in deep-sea hydrothermal sediments. These viruses code for structural proteins similar to those in Caudovirales, as well as proteins distinct from those described in archaeal viruses. They also have genes common in eukaryotic nucleocytoplasmic large DNA viruses (NCLDVs), and are predicted to be capable of semi-autonomous genome replication, repair, epigenetic modifications, and transcriptional regulation. Moreover, Helarchaeota viruses may hijack host ubiquitin systems similar to eukaryotic viruses. This first glimpse of Asgard viruses reveals they have features of both prokaryotic and eukaryotic viruses, and provides insights into their roles in the ecology and evolution of these globally distributed microbes.

Emerging Viral Pathogens in Sturgeon Aquaculture in Poland: Focus on Herpesviruses and Mimivirus Detection

Recently, Poland has become a leading producer of sturgeon meat and caviar in Europe and is one of the largest in the world. The growing importance of this branch of aquaculture means that diseases of these fish, especially viral ones, are becoming the object of interest for ichthyopathologists. In recent years, there have been increasing reports of health problems in the dynamically developing sturgeon farming. The greatest risk appears to be emerging infectious diseases that are caused by viruses and that can become a serious threat to the development of the aquaculture industry and the success of sturgeon restitution programs undertaken in many European countries, including Poland. In this paper, an attempt was made to determine the spread of the two most important groups of viruses in Polish sturgeon farming: These include the herpesviruses and sturgeon nucleocytoplasmic large DNA viruses (sNCLDV), in particular, mimiviruses. In the years 2016–2020, 136 samples from nine farms were collected and tested by using the WSSK-1 cell line, PCR and Real Time PCR methods. All results were negative for herpesviruses. Out of the samples, 26% of the samples have been tested positive for mimiviruses. Sanger sequencing of mimiviruses demonstrated their affiliation with AciV-E. The sequence characterization confirmed the presence of both V1 and V2 lineages in Polish fish facilities, but variant V2 seems to be more widespread, as is observed in other European countries.

Comparative Analysis of Transcriptional Regulation Patterns: Understanding the Gene Expression Profile in Nucleocytoviricota

The nucleocytoplasmic large DNA viruses (NCLDV) possess unique characteristics that have drawn the attention of the scientific community, and they are now classified in the phylum Nucleocytoviricota. They are characterized by sharing many genes and have their own transcriptional apparatus, which provides certain independence from their host’s machinery. Thus, the presence of a robust transcriptional apparatus has raised much discussion about the evolutionary aspects of these viruses and their genomes. Understanding the transcriptional process in NCLDV would provide information regarding their evolutionary history and a better comprehension of the biology of these viruses and their interaction with hosts. In this work, we reviewed NCLDV transcription and performed a comparative functional analysis of the groups of genes expressed at different times of infection of representatives of six different viral families of giant viruses. With this analysis, it was possible to observe a temporal profile of their gene expression and set of genes activated in specific phases throughout the multiplication cycle as a common characteristic of this group. Due to the lack of information regarding the transcriptional regulation process of this group of pathogens, we sought to provide information that contributes to and opens up the field for transcriptional studies of other viruses belonging to Nucleocytoviricota.