The Henipavirus genus includes the highly lethal zoonotic viruses Hendra (HeV) and Nipah (NiV), which have caused severe outbreaks in humans, horses and pigs. Henipaviruses are non-segmented negative-sense RNA viruses (NNSVs) that mediate genome replication exclusively in the host cell cytoplasm, yet their matrix protein (M) traffics between the cytoplasm and nucleus/nucleolus, with the nucleolar trafficking required to occur prior to roles in virus particle assembly and budding. Recently we identified the first nucleolar function for M protein, whereby M regulates ribosomal RNA (rRNA) production through targeting of a subset of host proteins within specific subnucleolar compartments; this identified a novel mechanism of pathogen modulation of ribosome biogenesis.
In order to elucidate the precise molecular mechanisms and functional scope of henipavirus M nucleolar targeting, we are comparing M proteins from a panel of diverse henipaviruses/henipa-like viruses that differ significantly in their genomic sequences, geographical distribution and pathogenesis. Using a combination of immunoprecipitation/mass-spectrometry (IP/MS) analysis, live-cell confocal microscopy and nucleolar functional assays, this work is revealing that, despite a broadly conserved capacity to target the nucleolus, henipavirus M proteins differ significantly in the quantitative levels of nucleolar targeting, and the nature of the subnucleolar sites targeted, as well as in the identity of nucleolar interactors that they bind. A number of novel nucleolar interactions are identified, including proteins known to form several distinct interaction networks within nucleoli. Intriguingly, despite differences in the subnucleolar interactions formed, the capacity of M proteins to inhibit rRNA production appears to be conserved, suggesting that different proteins might target distinct interactors to achieve the same ends. These data support an emerging picture of the nucleolus as an extensive and critical interface for henipaviruses and, potentially, many other NNSVs.