Viral Rhodopsins Research Articles

Eukaryotic viruses encode the ribosomal protein eL40

Viruses in the phylum Nucleocytoviricota are large, complex and have an exceptionally diverse metabolic repertoire. Some encode hundreds of products involved in the translation of mRNA into protein, but none was known to encode any of the proteins in ribosomes, the central engines of translation. With the discovery of the eL40 gene in FloV-SA2, we report the first example of a eukaryotic virus encoding a ribosomal protein and show that this gene is also present and expressed in other uncultivated marine giant viruses. FloV-SA2 also encodes a “group II” viral rhodopsin, a viral light-activated protein of unknown function previously only reported in metagenomes. FloV-SA2 is thus a valuable model system for investigating new mechanisms by which viruses manipulate eukaryotic cell metabolism.

Calcium-Sensitive Microbial Rhodopsin VirChR1: A Femtosecond to Second Photocycle Study.

Viral rhodopsins are light-gated cation channels representing a novel class of microbial rhodopsins. For viral rhodopsin 1 subfamily members VirChR1 and OLPVR1, channel activity is abolished above a certain calcium concentration. Here we present a calcium-dependent spectroscopic analysis of VirChR1 on the femtosecond to second time scale. Unlike channelrhodopsin-2, VirChR1 possesses two intermediate states P1 and P2 on the ultrafast time scale, similar to J and K in ion-pumping rhodopsins. Subsequently, we observe multifaceted photocycle kinetics with up to seven intermediate states. Calcium predominantly affects the last photocycle steps, including the appearance of additional intermediates P6Ca and P7 representing the blocked channel. Furthermore, the photocycle of the counterion variant D80N is drastically altered, yielding intermediates with different spectra and kinetics compared to those of the wt. These findings demonstrate the central role of the counterion within the defined reaction sequence of microbial rhodopsins that ultimately defines the protein function.

Hijacking of internal calcium dynamics by intracellularly residing viral rhodopsins.

Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.

E. coli Expression and Purification of Microbial and Viral Rhodopsins.

Microbial rhodopsins have become an indispensable tool for neurobiology. Of thousands of identified microbial rhodopsins, a minute fraction has been studied so far and they have shown remarkable functional diversity suggesting more great promises that this large family holds. Effective production of recombinant microbial and viral rhodopsins is a prerequisite for the success of functional and structural studies of these proteins. Escherichia coli (E. coli) are suitable for high yield expression of many of microbial and viral rhodopsins and they facilitate rapid exploration of this large protein family.

Viral rhodopsins 1 are\xa0an unique family of light-gated cation channels

Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability.

Unique structure and function of viral rhodopsins

Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists.

Targeted metagenomic recovery of four divergent viruses reveals shared and distinctive characteristics of giant viruses of marine eukaryotes.

Giant viruses have remarkable genomic repertoires—blurring the line with cellular life—and act as top–down controls of eukaryotic plankton. However, to date only six cultured giant virus genomes are available from the pelagic ocean. We used at-sea flow cytometry with staining and sorting designed to target wild predatory eukaryotes, followed by DNA sequencing and assembly, to recover novel giant viruses from the Pacific Ocean. We retrieved four ‘PacV’ partial genomes that range from 421 to 1605 Kb, with 13 contigs on average, including the largest marine viral genomic assembly reported to date. Phylogenetic analyses indicate that three of the new viruses span a clade with deep-branching members of giant Mimiviridae, incorporating the Cafeteria roenbergensis virus, the uncultivated terrestrial Faunusvirus, one PacV from a choanoflagellate and two PacV with unclear hosts. The fourth virus, oPacV-421, is phylogenetically related to viruses that infect haptophyte algae. About half the predicted proteins in each PacV have no matches in NCBI nr (e-value < 10−5), totalling 1735 previously unknown proteins; the closest affiliations of the other proteins were evenly distributed across eukaryotes, prokaryotes and viruses of eukaryotes. The PacVs encode many translational proteins and two encode eukaryotic-like proteins from the Rh family of the ammonium transporter superfamily, likely influencing the uptake of nitrogen during infection. cPacV-1605 encodes a microbial viral rhodopsin (VirR) and the biosynthesis pathway for the required chromophore, the second finding of a choanoflagellate-associated virus that encodes these genes. In co-collected metatranscriptomes, 85% of cPacV-1605 genes were expressed, with capsids, heat shock proteins and proteases among the most highly expressed. Based on orthologue presence–absence patterns across the PacVs and other eukaryotic viruses, we posit the observed viral groupings are connected to host lifestyles as heterotrophs or phototrophs.This article is part of a discussion meeting issue ‘Single cell ecology’.

Microbial and viral-like rhodopsins present in coastal marine sediments from four polar and subpolar regions.

Rhodopsins are broadly distributed. In this work, we analyzed 23 metagenomes corresponding to marine sediment samples from four regions that share cold climate conditions (Norway; Sweden; Argentina and Antarctica). In order to investigate the genes evolution of viral rhodopsins, an initial set of 6224 bacterial rhodopsin sequences according to COG5524 were retrieved from the 23 metagenomes. After selection by the presence of transmembrane domains and alignment, 123 viral (51) and non-viral (72) sequences (>50 amino acids) were finally included in further analysis. Viral rhodopsin genes were homologs of Phaeocystis globosa virus and Organic lake Phycodnavirus. Non-viral microbial rhodopsin genes were ascribed to Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Proteobacteria, Deinococcus-Thermus and Cryptophyta and Fungi. A rescreening using Blastp, using as queries the viral sequences previously described, retrieved 30 sequences (>100 amino acids). Phylogeographic analysis revealed a geographical clustering of the sequences affiliated to the viral group. This clustering was not observed for the microbial non-viral sequences. The phylogenetic reconstruction allowed us to propose the existence of a putative ancestor of viral rhodopsin genes related to Actinobacteria and Chloroflexi. This is the first report about the existence of a phylogeographic association of the viral rhodopsin sequences from marine sediments.

Proteorhodopsin genes in giant viruses

Viruses with large genomes encode numerous proteins that do not directly participate in virus biogenesis but rather modify key functional systems of infected cells. We report that a distinct group of giant viruses infecting unicellular eukaryotes that includes Organic Lake Phycodnaviruses and Phaeocystis globosa virus encode predicted proteorhodopsins that have not been previously detected in viruses. Search of metagenomic sequence data shows that putative viral proteorhodopsins are extremely abundant in marine environments. Phylogenetic analysis suggests that giant viruses acquired proteorhodopsins via horizontal gene transfer from proteorhodopsin-encoding protists although the actual donor(s) could not be presently identified. The pattern of conservation of the predicted functionally important amino acid residues suggests that viral proteorhodopsin homologs function as sensory rhodopsins. We hypothesize that viral rhodopsins modulate light-dependent signaling, in particular phototaxis, in infected protists.This article was reviewed by Igor B. Zhulin and Laksminarayan M. Iyer. For the full reviews, see the Reviewers’ reports section.