Biology subjects

Cottee, M. A.

Publications and source records attributed to Cottee, M. A..

3 recordsLinked to original sources

bioRxiv2025

Capsid flexibility during Ty1 virus-like particle assembly

The Ty1/Copia family (Pseudoviridae) is a widely studied group of long terminal repeat (LTR) retrotransposons present in diverse eukaryotes that share a common ancestor with retroviruses. The founding member Ty1 of Saccharomyces assembles into heterogeneous virus-like particles (VLPs) that are composed of the Gag and Gag-Pol proteins. We used cryo-electron tomography (cryo-ET) of Ty1 VLPs purified from a Ty1-less yeast strain expressing a functional Ty1 element to determine the organization and interactions of Gag in Ty1 VLPs. Using sub-tomogram averaging (STA), we observed unique capsomere structures and obtained EM density maps for pentagonal (7.5 [A]) and hexagonal (7.9 [A]) capsomeres. Through iterative rounds of alignment and classification, we demonstrate that Ty1 VLPs are composed of diverse arrangements of capsomeres, unlike true icosahedral virions. We modeled the structure of the conserved Ty1 Gag capsid C-terminal domain (CA-CTD), previously determined using X-ray crystallography into the capsomeric arrangements, and identified roles for the Dimer-1 interface present in the asymmetric unit, as well as additional interfaces involved in intra- and inter-capsomere assembly. We show that pentagonal or hexagonal capsomere assembly results from flexibility of CA-CTD - CA-CTD interactions at Interface-2. Additionally, we determined the structure of the Ty1 Gag capsid N-terminal domain (CA-NTD) using solution NMR and fitted the CA-NTD into the cryo-ET density maps. Our results indicate that the heterogeneous population of VLP size and morphology arises, at least in part, from the ability of the capsomeres to assemble in diverse organizations. (238 words)

biophysics↗

bioRxiv2024

Structural and functional characterization of the KHNYN extended-diKH domain for mediating ZAP antiviral activity

Zinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAP-mediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a non-canonical arrangement between two type-1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor.

microbiology↗

bioRxiv2019

Structure of D. melanogaster ARC1 reveals a repurposed molecule with characteristics of retroviral Gag

The tetrapod neuronal protein ARC and its D. melanogaster homologue, dARC1, have important but differing roles in neuronal development. Both are thought to originate through exaptation of ancient Ty3/Gypsy retrotransposon Gag genes, with their novel function relying on an original capacity for self-assembly and encapsidation of nucleic acids. Here, we present the crystal structure of dARC1 CA and examine the relationship between dARC1, mammalian ARC and the CA protein of circulating retroviruses. We show that whilst the overall architecture is highly related to that of orthoretroviral and spumaretroviral CA, there are significant deviations in both N- and C-terminal domains, potentially affecting recruitment of partner proteins and particle assembly. The degree of sequence and structural divergence suggests that Ty3/Gypsy Gag has been exapted on two separate occasions and that, although mammalian ARC and dARC1 share functional similarity, the structures have undergone different adaptations after appropriation into the tetrapod and insect genomes.

evolutionary biology↗