NEDD4 family ubiquitin ligases associate with LCMV Z's PPXY domain and are required for virus budding, but not via direct ubiquitination of Z

• Published in: PLoS pathogens Vol. 15; no. 11; p. e1008100
• Main Authors: Ziegler, Christopher M, Dang, Loan, Eisenhauer, Philip, Kelly, Jamie A, King, Benjamin R, Klaus, Joseph P, Manuelyan, Inessa, Mattice, Ethan B, Shirley, David J, Weir, Marion E, Bruce, Emily A, Ballif, Bryan A, Botten, Jason
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11-11-2019; Public Library of Science (PLoS)
• Subjects: Biology; Biology and Life Sciences; Biosynthesis; Budding; Cleavage; Domains; Humans; Intracellular Signaling Peptides and Proteins - metabolism; Leukemia; Ligases; Lymphocytic Choriomeningitis - metabolism; Lymphocytic Choriomeningitis - virology; Lymphocytic choriomeningitis virus - physiology; Lysine; Mass spectrometry; Mass spectroscopy; Matrix protein; Medicine; Nedd4 Ubiquitin Protein Ligases - metabolism; Physical Sciences; Protein Domains; Protein Interaction Domains and Motifs; Protein purification; Proteins; Recruitment; Scientific equipment industry; Spectroscopy; Ubiquitin; Ubiquitin-protein ligase; Ubiquitination; Viral meningitis; Virus Assembly; Virus Replication; Viruses; United States; Vermont; New York; United States > US
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1008100

Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1-3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment.