Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts

Abstract

The high abundance of most viruses in infected host cells benefits their structural characterization; endogenous viruses are present in low copy numbers, however, and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity while stacked dsRNA bundles and the sub-stoichiometric polymerase localize underneath the capsid surface. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

Enabling cryo‐EM density interpretation from yeast native cell extracts by proteomics data and AlphaFold structures

“…Two high‐molecular weight fractions of yeast native cell extracts were previously analyzed by Schmidt et al. [25] to retrieve the endogenous L‐A helper virus and various states of translating ribosomes [25]. Here, we re‐analyzed the reported MS results as well as 12,795 cryo‐EM micrographs that were acquired at a pixel size of 3.177 Å/pix to capture the cell extract content beyond these abundant biomolecules (Figure S1A).…”

“…All MS and cryo‐EM data have been previously reported in Schmidt et al. [25] MS data have been deposited in the PRIDE repository under the accession PXD034431. Cryo‐EM data are available from the EMPIAR database under the accession EMPIAR‐11069.…”

“…Two high-molecular weight fractions of yeast native cell extracts were previously analyzed by Schmidt et al [25] to retrieve the endogenous L-A helper virus and various states of translating ribosomes [25].…”

“…To address this issue, the 150 most abundant proteins were selected, ribosomal proteins were excluded, and modeling was focused on less abundant protein signatures. Ribosomes display distinct structural signatures in the raw micrograph data, 2D classes, and 3D reconstruction and can be easily distinguished from other, less abundant, protein signatures in native cell extract [25,37]. The remaining 61 proteins were systematically fitted in the derived reconstruction (Figure 2F).…”