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

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.

Host-Driven Phosphorylation Appears to Regulate the Budding Activity of the Lassa Virus Matrix Protein.

Lassa mammarenavirus (LASV) is an enveloped RNA virus that can cause Lassa fever, an acute hemorrhagic fever syndrome associated with significant morbidity and high rates of fatality in endemic regions of western Africa. The arenavirus matrix protein Z has several functions during the virus life cycle, including coordinating viral assembly, driving the release of new virus particles, regulating viral polymerase activity, and antagonizing the host antiviral response. There is limited knowledge regarding how the various functions of Z are regulated. To investigate possible means of regulation, mass spectrometry was used to identify potential sites of phosphorylation in the LASV Z protein. This analysis revealed that two serines (S18, S98) and one tyrosine (Y97) are phosphorylated in the flexible N- and C-terminal regions of the protein. Notably, two of these sites, Y97 and S98, are located in (Y97) or directly adjacent to (S98) the PPXY late domain, an important motif for virus release. Studies with non-phosphorylatable and phosphomimetic Z proteins revealed that these sites are important regulators of the release of LASV particles and that host-driven, reversible phosphorylation may play an important role in the regulation of LASV Z protein function.