Mechanisms Of Inclusion Bodies Formed By Measles Virus

Measles is a contagious acute infectious disease caused by measles virus(MeV)infection which incidence rate and mortality rate are very high.Measles virus,like rabies virus and respiratory syncytial virus,belongs to non-segmented single-stranded negative sense RNA virus.MeV is a member of the Paramyxoviridae family,are assumed to replicate in cytoplasmic inclusion bodies(IBs).These cytoplasmic viral factories are not membrane-bound and serve to concentrate the viral RNA replication machinery.It has been proven to increase the reaction rate and shields the viral components from innate immune surveillance responses.Although inclusion bodies are a prominent feature in MeV-infected cells,their biogenesis and regulation are not well understood.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.In our research,the formation mechanism of MeV inclusion bodies was studied by using Immunofluorescence technique.To test whether a relationship exists between the size and shape heterogeneity of IBs and the duration of infection,we carried out a time-course study at high multiplicity of infection.In the infected cells,IBs appeared as small,disperse spherical puncta at up to 12 h pi.Puncta of larger sizes and irregular shapes became more prevalent as the time of infection increased thereafter.The larger puncta were often irregularly shaped and positioned in a more central,perinuclear-like location.These observations suggest the possibility that nascent MeV IBs may exist initially as small spheres that subsequently increase in size and adopt different shapes.The WDR5 protein was recruited to and concentrated within IBs formed both in MeV virus-infected cells and N and P plasmid co-transfected cells,thereby providing us with a framework by which we could assess IB dynamics in live cells by monitoring EGFP or mCherry fluorescence.To examine these criteria in vivo,we employed live cells stably expressing an EGFP or mCherry fusion of the cellular WDR5 protein.To probe the exchange rate of IB-associated WDR5-EGFP,we performed Fluorescence Recovery After Photobleaching(FRAP)analyses using cells either infected with MeV or co-transfected with N and P plasmids.Under both conditions,infection and co-transfection,we observed a rapid and efficient recovery after photobleaching for the small IBs.By contrast,the fluorescence recovery was inefficient for the large IBs indicating that these structures had acquired a more gel-like property.Likewise,IBs induced both by MeV infection and by N and P co-transfection underwent fusion and relaxation.Thus,IBs triggered both by MeV infection and by N and P co-transfection not only shared similar time-dependent changes of size and morphology,but they also underwent fusion/relaxation and exhibited comparable photobleaching recovery efficiency of WDR5-EGFP fluorescence.Taken together,these data strongly suggest that the formation of IBs either by MeV infection or by N and P co-transfection is mediated via LLPS and lend further support to the notion that N and P proteins constitute core components of MeV IBs.Measles virus N and P proteins are sufficient to mediate formation of inclusion body-like puncta in transfected cells and this kind of inclusion bodies,like typical inclusion bodies induced by measles virus infection,also display increased size and altered morphology in a time-dependent manner.When three critical residues(K180,R194 and R354)shown to mediate the RNA binding activity of N were mutated to alanine,the formation of spherical puncta in transfected cells co-expressing N and P did not depend on the ability of N to bind RNA since co-expression of the N(KRR/AAA)mutant and P resulted in puncta of comparable size and morphology as co-expression of WT N with P.Upon identification of N and P viral proteins functioning together as minimal viral components sufficient to trigger MeV IB formation,we next attempted to gain insight into the regions of the proteins required for the IB assembly.We tested its role in the IB formation process using cells transfected with wild-type N and P or their mutants.Based on our observations,we conclude that the XD domain of P and the C-terminal MoRE-containing unstructured region of N are pivotal regions for cytoplasmic IB formation triggered by these two viral proteins.The measles virus P protein is phosphorylated at multiple sites.Among these,S86 and S151 are two major casein kinase 2(CK2)phosphorylation sites and their phosphorylation status is reported to change upon the binding of P to N.We therefore explored using a combination of approaches whether these CK2 sites played a role in IB formation.Treating MeV-infected cells with DMAT,a cell-permeable inhibitor of CK2,decreased the IB size without significantly affecting N protein expression measured by western analysis.To directly test the role of specific CK2 sites of P phosphorylation in IB assembly,we next examined whether the mutation of the serine residues at positions 86 and 151 to alanine also affected IB formation.The S86A/S151A phospho site double mutant of P was expressed at a similar level as wild-type P protein,yet the S86A/S151A mutant compared to WT P protein did not trigger the production of large IBs when co-expressed with N.The puncta size difference was verified by utilizing imaging software to quantify the fluorescent intensities of N and P proteins and the volumes of the largest puncta of individual cells.Further indicating a role for S86 and S151 phosphorylation in modulating the size of IBs formed by N and P co-expression.Whereas the small IBs were observed scattered throughout the cytoplasm of MeV-infected cells,the large IBs were often seen positioned in a more central location near the cell nucleus.We assessed the effect of HPI-4,a cell-permeable specific inhibitor of dynein motor function,on the size and spatial distribution of MeV IBs and on viral titer.Compared to the infected control cells not drug-treated,cells treated with 80 ?M HPI-4 exhibited a strikingly different IB pattern with the vast majority of IBs being smaller and uniformly distributed throughout the cytoplasm.The volume measurement of the largest punctum within a cell confirmed the inhibitory effect of HPI-4 on the formation of large IBs.Importantly,HPI-4 also led to lower levels of viral protein expression without significantly affecting the level of GAPDH or alpha tubulin.Moreover,viral titers were decreased 30-fold following treatment with 80 ?M HPI-4.Finally,similar HPI-4 effects on the IB size and distribution were also observed after treatment with dynarrestin,a drug that inhibits dynein by a mechanism different from that of HPI-4.Taken together,these data are consistent with the notion that dynein promotes viral replication by facilitating the formation of large IB s.Here,we show that measles virus triggers formation of IBs that are assembled by LLPS.In addition,we identified domain regions of the MeV N and P proteins that are essential for this LLPS process,and we showed that the phosphorylation of MeV P and the dynein-dependent transport system represent two mechanisms that affect the size and spatial distribution of IBs within MeV-infected cells.Finally,we provide evidence consistent with the notion that IB maturation facilitates MeV replication.Thus,our study not only further establishes LLPS as a common process to mediate IB formation across the Mononegavirales,but also sheds new insights into the biogenesis,regulation and function of viral inclusion bodies.