Sub-nucleolar trafficking of Hendra virus matrix protein is regulated by ubiquitination and oligomerisation

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 in infected cells and in cells expressing HeV M protein alone, M protein localizes into Treacle-enriched FC-DFC at early time points, and that FC-DFC localization is subsequently lost due to relocalization into 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 compartments, important to the interplay between HeV M protein and host cell factors during infection. AUTHOR SUMMARYHenipaviruses, including Hendra (HeV) and Nipah viruses, cause deadly diseases in humans and livestock and are considered priority diseases by the World Health Organization due to their epidemic potential and lack of effective treatments. Understanding how these viruses interact with host cells is essential for developing new therapeutics. Our study examines the matrix (M) protein of henipaviruses and its interaction with the nucleolus, a cell structure that mediates ribosome production, and is a common target for various viruses, although their functions are largely unresolved. Previously, we showed that the HeV M protein targets a sub-nucleolar structure, called the FC-DFC, to modulate ribosome biogenesis. Here, we report that the M proteins movement between sub-nucleolar compartments is controlled by two processes: ubiquitination, which causes accumulation of the protein in the FC-DFC, and oligomerization, which is associated with exit. Similar mechanisms are also observed in other henipaviruses. Our findings reveal mechanisms regulating the hijacking of host cell functions by henipaviruses and suggest new potential targets for antiviral therapies. This study is the first to investigate how viral proteins move within the nucleolus, offering new insights into interactions that may be significant to multiple viruses.