Measles Virus Nucleoprotein Research Articles

Measles outbreak in 2019: a warning for the post-COVID-19 pandemic period.

Introduction. At the end of 2019 and the year before, there was a significant spread of measles in the World Health Organization (WHO) European Region.Gap statement. Among the countries that reported, a measles outbreak was Bosnia and Herzegovina (BiH).Aim. To describe the measles outbreak in BiH (an entity of the Federation of BiH, FBiH) in 2019.Methodology. Confirmatory IgM serology, measles nucleic acid detection by real-time RT-PCR and virus genotyping were done in the WHO-accredited laboratory for measles and rubella at the Clinical Center of the University of Sarajevo, Unit for Clinical Microbiology. Genotype was determined in all measles-RNA-positive cases by sequence analysis of the 450 nt fragment coding the C-terminal of measles virus nucleoprotein (N).Results. From 1 January to 31 December 2019, 1332 measles cases were reported, with the peak observed in April 2019 (413/1332, 31.01 %). Sarajevo Canton had the highest incidence, number of cases and percentage (206.4; 868/1332; 65.17 %) of measles cases. Around four-fifths of infected persons were unvaccinated (1086/1332, 81.53 %), while 4.58 % of the patients (61/1332) were immunized with one dose of measles-containing vaccine. The highest proportion of cases was found in children 0-6 years of age (738/1332, 55.41 %). Measles IgM positivity was determined in 75.88 % (346/456), while virus RNA was detected in 82.46 % (47/57) of the swab samples. All measles virus sequences belonged to genotype B3. SNP (position 216: C=>T) was detected in 1 of the 40 sequences obtained during this outbreak.Conclusion. Due to suboptimal immunization coverage, BiH belongs to countries at a high risk for measles outbreaks. Post-COVID-19 (coronavirus disease 2019) pandemic, targeted and tailored strategies are required to ensure routine vaccination demand and acceptance and broad partner and stakeholder group participation.

Folding-upon-binding pathways of an intrinsically disordered protein from a deep Markov state model

A central challenge in the study of intrinsically disordered proteins is the characterization of the mechanisms by which they bind their physiological interaction partners. Here, we utilize a deep learning-based Markov state modeling approach to characterize the folding-upon-binding pathways observed in a long timescale molecular dynamics simulation of a disordered region of the measles virus nucleoprotein NTAIL reversibly binding the X domain of the measles virus phosphoprotein complex. We find that folding-upon-binding predominantly occurs via two distinct encounter complexes that are differentiated by the binding orientation, helical content, and conformational heterogeneity of NTAIL. We observe that folding-upon-binding predominantly proceeds through a multi-step induced fit mechanism with several intermediates and do not find evidence for the existence of canonical conformational selection pathways. We observe four kinetically separated native-like bound states that interconvert on timescales of eighty to five hundred nanoseconds. These bound states share a core set of native intermolecular contacts and stable NTAIL helices and are differentiated by a sequential formation of native and non-native contacts and additional helical turns. Our analyses provide an atomic resolution structural description of intermediate states in a folding-upon-binding pathway and elucidate the nature of the kinetic barriers between metastable states in a dynamic and heterogenous, or "fuzzy", protein complex.

Distant electrostatic interactions dominate the dynamics of the disordered measles virus NTAIL

NTAIL is the intrinsically disordered C-terminal domain of the measles virus nucleoprotein N. NTAIL protrudes from the nucleocapsid where the viral RNA is packaged. It recruits the polymerase and enables viral replication upon binding to a folded domain of protein P (PXD). The binding occurs via a coupled folding and binding mechanism, in which a small NTAIL region acts as binding recognition motive and folds into an κ-helix (κ-MoRE). The remaining portion of NTAIL remains disordered upon binding.

Dynamical Heterogeneity in the Measles Virus IDP NTAIL in its Free and Bound States

The measles virus nucleoprotein N possesses an intrinsically disordered domain, NTAIL, that protrudes from the nucleocapsid and binds to the P protein, thereby recruiting the polymerase and enabling viral replication. A small NTAIL region (18 out of 125 aa) acts as a binding motive (α-MoRE), folding upon binding to the folded X domain of P (PXD). The rest of NTAIL remains disordered. Although the remaining disordered regions of NTAIL have been shown to dampen the binding affinity, the underlying mechanism and their role in NTAIL dynamical reconfiguration upon binding is not clear.

PIM 3 kinase, a proto-oncogene product, regulates phosphorylation of the measles virus nucleoprotein tail domain at Ser 479 and Ser 510

The tail domain of the measles virus (MeV) N protein is typically phosphorylated at S479 and S510. However, the protein kinase responsible for this phosphorylation has not been identified. To identify the protein kinase responsible, we conducted an in vitro kinase assay in the presence of various protein kinase inhibitors. Phosphorylation of S479 and S510 was suppressed in the presence of SP600125. We demonstrated that purified PIM 3 kinase, which is sensitive to SP600125, successfully phosphorylated both phosphorylation sites. Inhibitors of PIM kinase, CX6258 and LY294002, also suppressed phosphorylation of the N protein. These findings indicate that PIM 3 kinase is associated with the tail domain of the N protein and that PIM 3 kinase regulates N protein phosphorylation.

Mechanism of Coupled Folding-upon-Binding of an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs), which in isolation do not adopt a well-defined tertiary structure but instead populate a structurally heterogeneous ensemble of interconverting states, play important roles in many biological pathways. IDPs often fold into ordered states upon binding to their physiological interaction partners (a so-called "folding-upon-binding" process), but it has proven difficult to obtain an atomic-level description of the structural mechanisms by which they do so. Here, we describe in atomic detail the folding-upon-binding mechanism of an IDP segment to its binding partner, as observed in unbiased molecular dynamics simulations. In our simulations, we observed over 70 binding and unbinding events between the α-helical molecular recognition element (α-MoRE) of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the measles virus phosphoprotein complex. We found that folding-upon-binding primarily occurred through induced-folding pathways (in which intermolecular contacts form before or concurrently with the secondary structure of the disordered protein)-an observation supported by previous experiments-and that the transition state ensemble was characterized by formation of just a few key intermolecular contacts and was otherwise highly structurally heterogeneous. We found that when a large amount of helical content was present early in a transition path, NTAIL typically unfolded and then refolded after additional intermolecular contacts formed. We also found that, among conformations with similar numbers of intermolecular contacts, those with less helical content had a higher probability of ultimately forming the native complex than conformations with more helical content, which were more likely to unbind. These observations suggest that even after intermolecular contacts have formed, disordered regions can have a kinetic advantage over folded regions in the folding-upon-binding process.

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

Dissecting the Energetics of Intrinsically Disordered Proteins via a Hybrid Experimental and Computational Approach.

Intrinsically disordered proteins (IDPs) play important roles in biology, but little is known about the energetics of their inter-residue interactions. Methods that have been successfully applied to analyze the energetics of globular proteins are not applicable to the fluctuating partially ordered ensembles populated by IDPs. A combined computational experimental strategy is introduced for analyzing the energetic role of individual residues in the free state of IDPs. The approach combines experimental measurements of the binding of wild-type and mutant IDPs to their partners with alchemical free energy calculations of the structured complexes. These data allow quantitative information to be deduced about the free state via a thermodynamic cycle. The approach is validated by the analysis of the effects of mutations upon the binding free energy of the ovomucoid inhibitor third binding domain to its partners and is applied to the C-terminal domain of the measles virus nucleoprotein, a 125-residue IDP involved in the RNA transcription and replication of measles virus. The analysis reveals significant inter-residue interactions in the unbound IDP and suggests a biological role for them. The work demonstrates that advances in force fields and computational hardware have now led to the point where it is possible to develop methods, which integrate experimental and computational techniques to reveal insights that cannot be studied using either technique alone.

The Nucleoprotein and Phosphoprotein of Measles Virus.

Measles virus is a negative strand virus and the genomic and antigenomic RNA binds to the nucleoprotein (N), assembling into a helical nucleocapsid. The polymerase complex comprises two proteins, the Large protein (L), that both polymerizes RNA and caps the mRNA, and the phosphoprotein (P) that co-localizes with L on the nucleocapsid. This review presents recent results about N and P, in particular concerning their intrinsically disordered domains. N is a protein of 525 residues with a 120 amino acid disordered C-terminal domain, Ntail. The first 50 residues of Ntail extricate the disordered chain from the nucleocapsid, thereby loosening the otherwise rigid structure, and the C-terminus contains a linear motif that binds P. Recent results show how the 5′ end of the viral RNA binds to N within the nucleocapsid and also show that the bases at the 3′ end of the RNA are rather accessible to the viral polymerase. P is a tetramer and most of the protein is disordered; comprising 507 residues of which around 380 are disordered. The first 37 residues of P bind N, chaperoning against non-specific interaction with cellular RNA, while a second interaction site, around residue 200 also binds N. In addition, there is another interaction between C-terminal domain of P (XD) and Ntail. These results allow us to propose a new model of how the polymerase binds to the nucleocapsid and suggests a mechanism for initiation of transcription.

Molecular characterization and evolutionary dynamics of measles virus sequences isolated from children in Lagos and Ibadan, South Western, Nigeria

Measles infection is endemic in Nigeria, with outbreaks occurring yearly. Genotype B3 is the dominant genotype and the only genotype characterized from Nigeria. The current study investigated the phylogenetic and Bayesian evolutionary dynamics of Nigerian measles virus Nucleoprotein (N) sequences isolated from Lagos and Ibadan, Nigeria. A total of 120 throat swab samples were analysed by RT-PCR and Sanger sequencing. Phylogenetic analysis and Bayesian demographic reconstructions were done using MEGA and BEAST software. Measles RNA positivity was 14.2% (17/120), age range 0–1 recorded the highest rate with 40.83%. Study sequences clustered within clade B3.1. The evolutionary rate of analysed B3 sequences was 1.108×10−3, higher posterior density HPD interval (1.462×10−3 – 7.886×10−4)subs/site/year. The time to most recent common ancestor (TMRC), was 1991. The Bayesian skyride analysis(BSP) of West African MV cladeB3.1, showed a stable, steady state population demography. This study has reemphasised the dominance of clade B3.1 in Nigeria. We have shown that clade B3.1 was recently introduced into circulation and has a slow population expansion. We advocate for the institution of molecular surveillance country wide in order to help monitor strain diversity and genetic evolution of Measles in Nigeria.

Development of a high-throughput assay to measure measles neutralizing antibodies.

Measles virus is highly infectious and remains a leading cause of vaccine preventable deaths in children. Neutralizing antibody responses elicited by measles virus infection or immunization are a serological correlate of protection. We describe a high-throughput neutralization assay to improve surveillance for measles immunity. Measles virus-antibody mixtures were incubated on Vero cell monolayers and 24 hours later cell-lysates harvested and subjected to one-step SYBR green RT-qPCR to amplify a target sequence within the measles virus nucleoprotein gene. Neutralization endpoint titers were interpolated to determine the dilution that inhibited the relative amplicon copy number by at least 90% compared to the mean signal obtained in virus control wells in the absence of serum. Anti-measles virus and anti-measles hemagglutinin antisera specifically neutralized measles virus in the microneutralization RT-qPCR assay while pre-immune sera and sera raised against other viruses did not. The microneutralization RT-qPCR assay obeyed the Percentage Law for measles virus inputs ranging from 100–5000 TCID50/well. The linear range of the assay corresponds to measles antibody concentrations of 30 to 3000 mIU/mL. Bland-Altman analysis and two-way analysis of variance demonstrated that results obtained using the microneutralization RT-qPCR assay were comparable to those obtained using a plaque reduction neutralization test and correctly identified human serum samples that were seropositive (95% and 100%, sensitivity and specificity, respectively). Furthermore, these comparisons suggest that a concentration of 300 mIU/mL may be a conservative cut-point to use to identify individuals likely to be protected against severe measles disease when the endpoint is based on 90% inhibition of virus replication. Measles virus microneutralization RT-qPCR is a rapid, sensitive, specific, and robust assay for detecting measles neutralizing antibodies that may help to improve immunization strategies nationally and achieve measles elimination globally.

Modulation of Measles Virus NTAIL Interactions through Fuzziness and Sequence Features of Disordered Binding Sites

In this paper we review our recent findings on the different interaction mechanisms of the C-terminal domain of the nucleoprotein (N) of measles virus (MeV) NTAIL, a model viral intrinsically disordered protein (IDP), with two of its known binding partners, i.e., the C-terminal X domain of the phosphoprotein of MeV XD (a globular viral protein) and the heat-shock protein 70 hsp70 (a globular cellular protein). The NTAIL binds both XD and hsp70 via a molecular recognition element (MoRE) that is flanked by two fuzzy regions. The long (85 residues) N-terminal fuzzy region is a natural dampener of the interaction with both XD and hsp70. In the case of binding to XD, the N-terminal fuzzy appendage of NTAIL reduces the rate of α-helical folding of the MoRE. The dampening effect of the fuzzy appendage on XD and hsp70 binding depends on the length and fuzziness of the N-terminal region. Despite this similarity, NTAIL binding to XD and hsp70 appears to rely on completely different requirements. Almost any mutation within the MoRE decreases XD binding, whereas many of them increase the binding to hsp70. In addition, XD binding is very sensitive to the α-helical state of the MoRE, whereas hsp70 is not. Thus, contrary to hsp70, XD binding appears to be strictly dependent on the wild-type primary and secondary structure of the MoRE.

How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?

The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of NTAIL upon binding to XD by measuring the effect on both the folding and binding steps of NTAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of NTAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs.

Partner-Mediated Polymorphism of an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (NTAIL) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, NTAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the NTAIL/hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both NTAIL/XD and NTAIL/hsp70 interactions. Although the same NTAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased NTAIL/hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners.

Analyzing the Folding and Binding Steps of an Intrinsically Disordered Protein by Protein Engineering.

Intrinsically disordered proteins (IDPs) are functionally active despite lacking a well-defined three-dimensional structure. Such proteins often undergo a disorder-to-order transition, or induced folding, when binding to their specific physiological partner. Because of cooperativity, the folding and binding steps typically appear as a single event, and therefore, induced folding is extremely difficult to characterize experimentally. In this perspective, the interaction between the disordered C-terminal domain of the measles virus nucleoprotein NTAIL and the folded X domain of the viral phosphoprotein (XD) is particularly interesting because the inherent complexity of the observed kinetics allows characterization of the binding and folding steps individually. Here we present a detailed structural description of the folding and binding events occurring in the recognition between NTAIL and XD. This result was achieved by measuring the effect of single-amino acid substitutions in NTAIL on the reaction mechanism. Analysis of the experimental data allowed us (i) to identify the key residues involved in the initial recognition between the two molecules and (ii) to depict the general features of the folding pathway of NTAIL. Furthermore, an analysis of the changes in stability obtained for the whole set of variants highlights how the sequence of this IDP has not been selected during evolution to fold efficiently. This feature might be a consequence of the weakly funneled nature of the energy landscape of IDPs in their unbound state and represents a plausible explanation of their highly dynamic nature even in the bound state, typically defined as "fuzziness".

Simulation of coupled folding and binding of an intrinsically disordered protein in explicit solvent with metadynamics

The C-terminal domain of measles virus nucleoprotein is an intrinsically disordered protein that could bind to the X domain (XD) of phosphoprotein P to exert its physiological function. Experiments reveal that the minimal binding unit is a 21-residue α-helical molecular recognition element (α-MoRE-MeV), which adopts a fully helical conformation upon binding to XD. Due to currently limited computing power, direct simulation of this coupled folding and binding process with atomic force field in explicit solvent cannot be achieved. In this work, two advanced sampling methods, metadynamics and parallel tempering, are combined to characterize the free energy surface of this process and investigate the underlying mechanism. Starting from an unbound and partially folded state of α-MoRE-MeV, multiple folding and binding events are observed during the simulation and the energy landscape was well estimated. The results demonstrate that the isolated α-MoRE-MeV resembles a molten globule and rapidly interconverts between random coil and multiple partially helical states in solution. The coupled folding and binding process occurs through the induced fit mechanism, with the residual helical conformations providing the initial binding sites. Upon binding, α-MoRE-MeV can easily fold into helical conformation without obvious energy barriers. Two mechanisms, namely, the system tending to adopt the structure in which the free energy of isolated α-MoRE-MeV is the minimum, and the binding energy of α-MoRE-MeV to its partner protein XD tending to the minimum, jointly dominate the coupled folding and binding process. With the advanced sampling approach, more IDP systems could be simulated and common mechanisms concerning the coupled folding and binding process could be investigated in the future.

Crystal Structure of the Measles Virus Nucleoprotein Core in Complex with an N-Terminal Region of Phosphoprotein.

The enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N(0)) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N(0) assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N(0)₁₋₄₀₈ fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N(0)-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N(0) binding peptide, P₁₋₄₈, to the C terminus of an N(0)₂₁₋₄₀₈ fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N(0)-P complex. We solved the X-ray structure of the resulting N(0)-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N(0)-P interface and explains how P chaperones N(0), preventing both self-assembly of N(0) and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization. Measles virus is an important, highly contagious human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development to treat not only measles but also potentially other paramyxovirus diseases.

Demonstration of a folding after binding mechanism in the recognition between the measles virus NTAIL and X domains.

In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms.

The Importance of Intrinsic Order in a Disordered Protein Ligand

The Importance of Intrinsic Order in a Disordered Protein Ligand

Testing the validity of ensemble descriptions of intrinsically disordered proteins

Understanding biological function in the profusion of proteins containing significant levels of intrinsic disorder depends on how accurately we can describe their conformational behavior (1). Recently, Wang et al. used molecular dynamics (MD) techniques to study the molecular recognition element of the C-terminal domain of the measles virus nucleoprotein (MeV-NTAIL), an example of this enigmatic family of intrinsically disordered proteins (IDPs) (2). In justifying their approach, the authors state that “in general, it is not feasible to characterize IDPs by an ensemble averaged method due to the underlying structural heterogeneity.” Although the potential advantages of restraint-free MD in terms of dynamic time scales are evident, numerous developments in the field also exploit ensemble-averaged NMR data to derive molecular descriptions of IDPs.