Measles Virus Forms Inclusion Bodies with Properties of Liquid Organelles

Abstract

Nonsegmented negative-strand RNA viruses, including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane bound, and they serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here, we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying that they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggests that Mononegavirales have broadly evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells. IMPORTANCE Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur, and globally ∼100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WD repeat-containing protein 5 (WDR5) enhances MeV growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here, we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies, that these structures display properties characteristic of phase-separated liquid organelles, and that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.

Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein

“…With these lines of evidence in mind, our work points to the possibility that a liquid-like matrix of phosphorylated N protein and loosely bound RNA, linked to RTC membranes by Nsp3, provides a compartment to concentrate and protect the viral replication and transcription machinery. Similar mechanisms are likely to exist in negative-sense RNA viruses, where replication is focused in dynamic biomolecular condensates [64][65][66] . In Measles virus, these condensates have also been implicated in nucleocapsid assembly 67 .…”

Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly

“…Inclusion bodies from MeV have been shown to colocalize host proteins such as WDR5 (WD repeat-containing protein 5) (28) and other viral proteins [the polymerase L as well as C that is involved in controlling host immune response (30)]. Recent confirmation of the liquid-like nature of Negri bodies formed in rabies (31) and colocalization of N and P in infection-induced cytoplasmic inclusion bodies in MeV (32) suggests that LLPS represents a widespread mechanism exploited by mononegaviruses during infection.…”

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

“…Accordingly, mutations affecting the phase separation propensity of proteins are often linked to neurodegeneration and other pathological conditions (Alberti and Dormann, 2019). Whereas LLPS seems to be a heavily exploited process in eukaryotes and viruses (Heinrich et al, 2018;Nikolic et al, 2017Nikolic et al, , 2018Zhou et al, 2019), up until today only a few bacterial proteins were reported to undergo LLPS. These include Caulobacter crescentus Ribonuclease E (RNase E) that orchestrates RNA degradosomes (Al-Husini et al, 2018), Escherichia coli (E. coli) FtsZ that forms the division ring during cell divisions (Monterroso et al, 2019) and Mycobacterium tuberculosis Rv1747 that is a membrane transporter of cell wall biosynthesis intermediates (Heinkel et al, 2019).…”