Biological Sciences, School of


First Advisor

James L. Van Etten

Date of this Version

Spring 4-8-2021


A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biological Sciences (Genetics, Cellular & Molecular Biology), Under the Supervision of Professor James L. Van Etten. Lincoln, Nebraska: April 2021

Copyright (c) 2021 Eric A. Noel


Giant dsDNA chloroviruses encode a diverse repertoire of glycosyltransferases (GTases) and methyltransferases (MTases) that biosynthesize unusual, methylated sugars independent of their host chlorella-like green algae prompting a reexamination of glycobiology systems. Unlike most other viruses, the prototype chlorovirus PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans do not resemble any other glycans in the three domains of life. Here, we investigated the potential involvement of chlorovirus- encoded putative GTases and MTases in glycosylation of the viral MCP. First, we aimed to generate site-directed virus mutants by targeting associated viral genes. We tested transformation methods using cell wall-degrading enzymes, electroporation, SiC whiskers, cell-penetrating peptides, and Agrobacterium to generate GT-gene mutations in the chlorovirus CA-4B. We successfully delivered preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs) to macerozyme-treated NC64A cells that resulted in a frameshift mutation in the CA-4B-encoded gene 034r, a homolog of PBCV-1 GT gene a064r. Unable to duplicate these results, we shifted our focus to characterize PBCV-1- encoded proteins involved in glycan synthesis. Here, we demonstrated that protein A064R has three functional domains: domain 1 is a β-L-rhamnosyltransferase, domain 2 is an α-L-rhamnosyltransferase, and domain 3 is a MT that methylates the C-2 hydroxyl group of the terminal α-L-rhamnose unit (α-L-Rha). We also established that methylation of the C-3 hydroxyl group of the terminal α-L-Rha is achieved by A061L. Moreover, genetic and structural analyses indicate the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a GT with three putative domains: galactosyltransferase (domain 1), xylosyltransferase (domain 2), and fucosyltransferase (domain 3). Hydrolytic assays supported these predictions suggesting that A111/114R is likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan. These findings provide additional support that the chloroviruses do not use the canonical host ER-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus- encoded enzymes.

Advisor: James L. Van Etten