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1. Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

   https://doi.org/10.3390/v12060678  

   Murry, Carter R. ; Agarkova, Irina V. ; Ghosh, Jayadri S. ; Fitzgerald, Fiona C. ; Carlson, Roger M. ; Hertel, Brigitte ; Kukovetz, Kerri ; Rauh, Oliver ; Thiel, Gerhard ; Van Etten, James L. ( June 2020 , Viruses)

   null (Ed.)

   Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels. 

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2. Chloroviruses

   https://doi.org/10.3390/v12010020  

   Van Etten, James L. ; Agarkova, Irina V. ; Dunigan, David D. ( January 2020 , Viruses)

   Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation. 

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3. Viral DNA Accumulation Regulates Replication Efficiency of Chlorovirus OSy-NE5 in Two Closely Related Chlorella variabilis Strains

   https://doi.org/10.3390/v15061341  

   Esmael, Ahmed ; Agarkova, Irina V. ; Dunigan, David D. ; Zhou, You ; Van Etten, James L. ( June 2023 , Viruses)

   Many chloroviruses replicate in Chlorella variabilis algal strains that are ex-endosymbionts isolated from the protozoan Paramecium bursaria, including the NC64A and Syngen 2-3 strains. We noticed that indigenous water samples produced a higher number of plaque-forming viruses on C. variabilis Syngen 2-3 lawns than on C. variabilis NC64A lawns. These observed differences led to the discovery of viruses that replicate exclusively in Syngen 2-3 cells, named Only Syngen (OSy) viruses. Here, we demonstrate that OSy viruses initiate infection in the restricted host NC64A by synthesizing some early virus gene products and that approximately 20% of the cells produce a small number of empty virus capsids. However, the infected cells did not produce infectious viruses because the cells were unable to replicate the viral genome. This is interesting because all previous attempts to isolate host cells resistant to chlorovirus infection were due to changes in the host receptor for the virus. 

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4. Diversity of tRNA Clusters in the Chloroviruses

   https://doi.org/10.3390/v12101173  

   Duncan, Garry A. ; Dunigan, David D. ; Van Etten, James L. ( October 2020 , Viruses)

   null (Ed.)

   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 due to differences in the GC content between the host and virus genomes. Here, we examine 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, each infecting a different algal host, have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. While differences were observed between clades and even within clades, seven tRNA genes were common to all three clades of chloroviruses, including the tRNAArg gene, which was found in all 41 chloroviruses. By comparing the codon usage of one chlorovirus algal host, in which the 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. Evidence presented herein supports the hypothesis that a chlorovirus tRNA cluster was present in the most recent common ancestor (MRCA) prior to divergence into three clades. In addition, the MRCA encoded a putative isoleucine lysidine synthase (TilS) that remains in 39/41 chloroviruses examined herein, suggesting a strong evolutionary pressure to retain the gene. TilS alters the anticodon of tRNAMet that normally recognizes AUG to then recognize AUA, a codon for isoleucine. This is advantageous to the chloroviruses because the AUA codon is 12–13 times more common in the chloroviruses than their host, further helping the chloroviruses to overcome CUB. Among large DNA viruses infecting eukaryotes, the presence of tRNA genes and tRNA clusters appear to be most common in the Phycodnaviridae and, to a lesser extent, in the Mimiviridae. 

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5. Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans

   https://doi.org/10.1073/pnas.2016626117  

   Speciale, Immacolata ; Laugieri, Maria Elena ; Noel, Eric ; Lin, Sicheng ; Lowary, Todd L. ; Molinaro, Antonio ; Duncan, Garry A. ; Agarkova, Irina V. ; Garozzo, Domenico ; Tonetti, Michela G. ; et al ( November 2020 , Proceedings of the National Academy of Sciences)

   null (Ed.)

   Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a β- l -rhamnosyltransferase; domain 2 is an α- l -rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α- l -rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α- l -Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N -glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α- l -Rha-(1→2)-β- l -Rha fragment. Remarkably, this fragment can be attached to any xylose unit. 

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