Sub-nucleolar Trafficking of Hendra Virus Matrix Protein is Regulated by Ubiquitination and Oligomerisation

ABSTRACT Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (order Mononegavirales ), the replication cycle of which occurs primarily in the cytoplasm. The HeV matrix protein (HeV M) plays critical roles in viral assembly and budding at the plasma membrane, but also undergoes nuclear/nucleolar trafficking, to accumulate in nucleoli early in infection and, later, localise predominantly at the plasma membrane. Previously we found that HeV M protein targets specific sub-nucleolar compartments (corresponding to the FC-DFC (fibrillar centre (FC)/dense fibrillar component (DFC)) where it interacts with the nucleolar protein Treacle and modulates rRNA biogenesis by subverting the host nucleolar DNA damage response, indicating the importance of specific sub-nucleolar trafficking to infection. However, the mechanisms underlying targeting and movement between sub-nucleolar compartments by viral or cellular proteins remain poorly defined. Here, we assessed the molecular regulation of HeV M protein nucleolar/sub-nucleolar trafficking, finding that HeV M localizes into Treacle-enriched FC-DFC early after expression, but this localization is progressively depleted through relocalization to the surrounding granular component (GC) of the nucleolus. Analysis using mutated M proteins and pharmacological modulation of ubiquitination indicate that this dynamic localization is regulated by ubiquitination and oligomerisation, with ubiquitination required for retention of HeV M in Treacle-enriched sub-nucleolar compartments, and oligomerisation required for egress. To our knowledge, this study provides the first direct insights into the dynamics and mechanisms of viral protein trafficking between sub-nucleolar compartment, shedding light on the intricate interplay between HeV M and host cell factors during infection.