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1. Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans

   https://doi.org/10.3390/v13010087  

   Noel, Eric ; Notaro, Anna ; Speciale, Immacolata ; Duncan, Garry A. ; De Castro, Cristina ; Van Etten, James L. ( January 2021 , Viruses)

   The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosylation pathway to glycosylate their glycoproteins; instead,more »they perform glycosylation independent of cellular organelles using virus-encoded enzymes.« less
2. 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)

   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-1more »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.« less
3. 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)

   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 thatmore »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.« less
4. 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)

   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 newmore »predicted channels were shown to be functional K+ channels.« less
5. Near-atomic, non-icosahedrally averaged structure of giant virus Paramecium bursaria chlorella virus 1

   https://doi.org/10.1038/s41467-022-34218-4  

   Shao, Qianqian ; Agarkova, Irina V. ; Noel, Eric A. ; Dunigan, David D. ; Liu, Yunshu ; Wang, Aohan ; Guo, Mingcheng ; Xie, Linlin ; Zhao, Xinyue ; Rossmann, Michael G. ; et al ( October 2022 , Nature Communications)

   Giant viruses are a large group of viruses that infect many eukaryotes. Although components that do not obey the overall icosahedral symmetry of their capsids have been observed and found to play critical roles in the viral life cycles, identities and high-resolution structures of these components remain unknown. Here, by determining a near-atomic-resolution, five-fold averaged structure of Paramecium bursaria chlorella virus 1, we unexpectedly found the viral capsid possesses up to five major capsid protein variants and a penton protein variant. These variants create varied capsid microenvironments for the associations of fibers, a vesicle, and previously unresolved minor capsid proteins. Our structure reveals the identities and atomic models of the capsid components that do not obey the overall icosahedral symmetry and leads to a model for how these components are assembled and initiate capsid assembly, and this model might be applicable to many other giant viruses.