Biology subjects

Van Etten, J. L.

Publications and source records attributed to Van Etten, J. L..

7 recordsLinked to original sources

bioRxiv2022

Life history diversity and signals of trade-offs in a large group of chloroviruses

Phytoplankton viruses are key players in aquatic ecosystems, where they control algal populations and affect nutrient flow. The ecological impact and evolution of these viruses can be understood by studying their life history traits, but very little is known about the life history diversity of related viruses. We quantified the life cycle of 34 strains in the genus Chlorovirus, which infects freshwater green algae. All chloroviral life history traits varied 5-to 75-fold across strains, in some cases rivaling the known trait range for all phytoplankton viruses. The trait variation affected viral growth rates but was not detectably constrained by life history trade-offs. This study represents the most in-depth characterization of algal viruses to date and raises the question whether all phytoplankton virus genera are equally diverse.

evolutionary biology↗

bioRxiv2021

Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

The ability to carry out genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae and are present in freshwater environments throughout the world. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. In an effort to develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we first tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). Agrobacterium -mediated transfection of chlorovirus host Chlorella variabilis NC64A with a binary vector containing a chlorovirus-encoded glycosyltransferase mutant gene was also examined. Attempts at developing a reliable chlorovirus transformation system were unsuccessful. However, in one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting CA-4B-encoded gene 034r, which encodes a putative glycosyltransferase. Selection of these mutants using antibodies was dependent on a specific change in the pattern of glycans attached to the virus major capsid protein (MCP). Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

molecular biology↗

bioRxiv2020

Diversity of tRNA Clusters in the Chloroviruses

Viruses rely on their host's translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus with differences in the GC content between the host/virus genome. Here, we evaluate the hypothesis that chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 chloroviruses comprising three clades of three different algal hosts have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. One tRNA gene was common to all three clades of chloroviruses, while differences were observed between clades and even within clades. By comparing the codon usage of one chlorovirus algal host, whose genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host's CUB by encoding tRNAs that recognize AU-rich codons. In addition, 39/41 chloroviruses encode a putative lysidine synthase, which alters the anticodon of tRNAmet that normally recognizes AUG to recognize the codon AUA, a codon for isoleucine. This is advantageous to the viruses because the AU-rich codon AUA is 12-13 times more common in the chloroviruses than their host. Evidence is presented that supports the concept that chlorovirus tRNA clusters were acquired prior to events that separated them into the three clades.View Full Text

genomics↗

bioRxiv2020

Codon bias determines sorting of ion channel protein

The choice of codons can influence local translation kinetics during protein synthesis. The question of whether the modulation of polypeptide folding and binding to chaperons influences sorting of nascent membrane proteins remains unclear. Here, we use two similar K+ channels as model systems to examine the impact of codon choice on protein sorting. By monitoring transient expression of GFP tagged proteins in mammalian cells we find that targeting of one channel to the secretory pathway is insensitive to codon optimization. In contrast, sorting of the second channel to the mitochondria is very sensitive to codon choice. The protein with an identical amino acid sequence is sorted in a codon and cell cycle dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves together with a cell-state depending decoding mechanism as a secondary code for sorting intracellular proteins.

cell biology↗

bioRxiv2020

Glacier ice archives fifteen-thousand-year-old viruses

While glacier ice cores provide climate information over tens to hundreds of thousands of years, study of microbes is challenged by ultra-low-biomass conditions, and virtually nothing is known about co-occurring viruses. Here we establish ultra-clean microbial and viral sampling procedures and apply them to two ice cores from the Guliya ice cap (northwestern Tibetan Plateau, China) to study these archived communities. This method reduced intentionally contaminating bacterial, viral, and free DNA to background levels in artificial-ice-core control experiments, and was then applied to two authentic ice cores to profile their microbes and viruses. The microbes differed significantly across the two ice cores, presumably representing the very different climate conditions at the time of deposition that is similar to findings in other cores. Separately, viral particle enrichment and ultra-low-input quantitative viral metagenomic sequencing from [~]520 and [~]15,000 years old ice revealed 33 viral populations (i.e., species-level designations) that represented four known genera and likely 28 novel viral genera (assessed by gene-sharing networks). In silico host predictions linked 18 of the 33 viral populations to co-occurring abundant bacteria, including Methylobacterium, Sphingomonas, and Janthinobacterium, indicating that viruses infected several abundant microbial groups. Depth-specific viral communities were observed, presumably reflecting differences in the environmental conditions among the ice samples at the time of deposition. Together, these experiments establish a clean procedure for studying microbial and viral communities in low-biomass glacier ice and provide baseline information for glacier viruses, some of which appear to be associated with the dominant microbes in these ecosystems. ImportanceThis study establishes ultra-clean microbial and viral sampling procedures for glacier ice, which complements prior in silico decontamination methods and expands, for the first time, the clean procedures to viruses. Application of these methods to glacier ice confirmed prior common microbiological findings for a new ice core climate record, and provides a first window into viral genomes and their ecology from glacier ice across two time horizons, and emphasizes their likely impact on abundant microbial groups. Together these efforts provide clean sampling approaches and foundational datasets that should enable simultaneous access to an archived virosphere in glacier ice.

ecology↗

bioRxiv2019

A small viral potassium ion channel with an inherent inward rectification

Some algal viruses have coding sequences for K+ channels with structural and functional characteristics of pore modules of complex K+ channels. Here we exploit the immense structural diversity of natural channel orthologs to discover new basic principles of structure/function correlates in K+ channels. The comparative analysis of three similar K+ channels with monomer sizes [≤] 86 amino acids (AA) shows that one channel (Kmpv1) generates an ohmic conductance in HEK293 cells while the other two channels (KmpvSP1, KmpvPL1) exhibit typical features of canonical Kir channels. Like Kir channels, the rectification of the viral channels is a function of the K+ driving force. Reconstitution of KmpvSP1 and KmpvPL1 in planar lipid bilayers showed rapid channel fluctuations only at voltages negative of the K+ reversal voltage. This rectification was maintained in KCl buffer with 1 mM EDTA, which excludes blocking cations as the source of rectification. This means that rectification of the viral channels must be, unlike Kir channels, an inherent property of the channel proteins. The structural basis for rectification was investigated by a chimera between rectifying and non-rectifying channels as well as point mutations, which made the rectifying channels similar to the ohmic conducting channel. The results of these experiments exclude the domain, which connects the two transmembrane helixes and which includes the pore helix and the selectivity filter, as playing a major role in rectification; inward rectification must be conferred by the transmembrane domains. The finding that a swapping of the AA, which is typical for the two inward rectifiers, with the respective AA from Kmpv1 did not compromise rectification suggests that tertiary or quaternary structural interactions are responsible for this type of gating.

biophysics↗

bioRxiv2019

Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host

Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilis NC64A host, and found that virus survivorship was highest (69.5 {+/-} 16.5 %) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9 - 30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts.

microbiology↗