Respiratory Syncytial Virus M2-1 Protein Research Articles

In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts.

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants and the elderly. RSV is a non-segmented negative strand RNA virus. The viral M2-1 protein plays a key role in viral transcription, serving as an elongation factor to enable synthesis of full-length mRNAs. M2-1 contains an unusual CCCH zinc-finger motif that is conserved in the related human metapneumovirus M2-1 protein and filovirus VP30 proteins. Previous biochemical studies have suggested that RSV M2-1 might bind to specific virus RNA sequences, such as the transcription gene end signals or poly A tails, but there was no clear consensus on what RSV sequences it binds. To determine if M2-1 binds to specific RSV RNA sequences during infection, we mapped points of M2-1:RNA interactions in RSV-infected cells at 8 and 18 hours post infection using crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq). This analysis revealed that M2-1 interacts specifically with positive sense RSV RNA, but not negative sense genome RNA. It also showed that M2-1 makes contacts along the length of each viral mRNA, indicating that M2-1 functions as a component of the transcriptase complex, transiently associating with nascent mRNA being extruded from the polymerase. In addition, we found that M2-1 binds specific cellular mRNAs. In contrast to the situation with RSV mRNA, M2-1 binds discrete sites within cellular mRNAs, with a preference for A/U rich sequences. These results suggest that in addition to its previously described role in transcription elongation, M2-1 might have an additional role involving cellular RNA interactions.

RSV M2-1 Protein in Complex with RNA: Old Questions Are Answered and a New One Emerges

The respiratory syncytial virus (RSV) M2-1 protein is essential for virus transcription. In this issue of Structure, Gao etal. (2020) describe the crystal structure of M2-1 in complex with RNA. This structure provides new insight into the RSV transcription mechanism but also raises an intriguing question regarding M2-1's function.

Structure of the human respiratory syncytial virus M2-1 protein in complex with a short positive-sense gene-end RNA

Structure of the human respiratory syncytial virus M2-1 protein in complex with a short positive-sense gene-end RNA

Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA

The M2-1 protein of human respiratory syncytial virus (HRSV) is a transcription anti-terminator that regulates the processivity of the HRSV RNA-dependent RNA polymerase (RdRP). Here, we report a crystal structure of HRSV M2-1 bound to a short positive-sense gene-end RNA (SH7) at 2.7Å resolution. We identified multiple critical residues of M2-1 involved in RNA interaction and examined their roles using mutagenesis and MicroScale Thermophoresis (MST) assay. We found that hydrophobic residue Phe23 is indispensable for M2-1 to recognize the base of RNA. We also captured spontaneous binding of RNA (SH7) to M2-1 in all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method. Both experiments and simulations revealed that the interactions of RNA with two separate domains of M2-1, the zinc-binding domain (ZBD) and the core domain (CD), are independent of each other. Collectively, our results provided a structural basis for RNA recognition by HRSV M2-1.

Insights into Interactions of Flavanones with Target Human Respiratory Syncytial Virus M2-1 Protein from STD-NMR, Fluorescence Spectroscopy, and Computational Simulations.

The human Respiratory Syncytial Virus (hRSV) is the most frequent agent of respiratory infections in infants and children with no currently approved vaccine. The M2-1 protein is an important transcriptional antitermination factor and a potential target for viral replication inhibitor development. Hesperetin (HST) and hesperidin (HSD) are flavonoids from the flavanone group, naturally found in citrus and have, as one of their properties, antiviral activity. The present study reports on the interactions between hRSV M2-1 and these flavanones using experimental techniques in association with computational tools. STD-NMR results showed that HST and HSD bind to M2-1 by positioning their aromatic rings into the target protein binding site. Fluorescence quenching measurements revealed that HST had an interaction affinity greater than HSD towards M2-1. The thermodynamic analysis suggested that hydrogen bonds and van der Waals interactions are important for the molecular stabilization of the complexes. Computational simulations corroborated with the experimental results and indicated that the possible interaction region for the flavonoids is the AMP-binding site in M2-1. Therefore, these results point that HST and HSD bind stably to a critical region in M2-1, which is vital for its biological function, and thus might play a possible role antiviral against hRSV.

Reply to Rameix-Welti, "No Incongruity in Respiratory Syncytial Virus M2-1 Protein Remaining Bound to Viral mRNAs during Their Entire Life Time".

First, we thank Marie-Anne Rameix-Welti for the thoughtful comments on our recent paper (1). The M2-1 protein from respiratory syncytial virus (RSV) has for many years been rather enigmatic in allowing researchers to probe its structure and function, with details slow to emerge. In Selvaraj et al. (2), we suggested a model that posited that the requirement of M2-1 in RSV transcription involved binding of an M2-1 …

Recombinant live attenuated influenza vaccine viruses carrying CD8 T-cell epitopes of respiratory syncytial virus protect mice against both pathogens without inflammatory disease

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory disease in young children, elderly and immunocompromised adults. There is no licensed vaccine against RSV although development of an effective and safe RSV vaccine has been a high priority for several decades. Among the various vaccine platforms, the viral-vectored RSV vaccines based on licensed cold-adapted live attenuated influenza vaccine (LAIV) might offer an advantage of inducing adequate mucosal CD8 T cell immunity at the infection site of respiratory pathogens. We constructed two recombinant LAIV viruses expressing immunodominant T-cell epitopes of RSV M2-1 protein. The results in this study provide evidence that RSV CD8 T cell epitopes delivered by LAIV viral vector could confer protection against RSV infection without causing pulmonary eosinophilia and inflammatory RSV disease in mice. In addition, these chimeric LAIV-RSV vaccines retained their attenuated phenotype and ability to protect against virulent influenza virus, thus providing a unique approach to fight against two dangerous respiratory viral pathogens using a single vaccine preparation.

呼吸道合胞病毒M2-1蛋白Hep-2细胞稳定表达株的构建及其在呼吸道合胞病毒感染中的特点

呼吸道合胞病毒M2-1蛋白Hep-2细胞稳定表达株的构建及其在呼吸道合胞病毒感染中的特点

The Structure of the Human Respiratory Syncytial Virus M2-1 Protein Bound to the Interaction Domain of the Phosphoprotein P Defines the Orientation of the Complex.

Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause life-threatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90-110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences.IMPORTANCE Human respiratory syncytial virus (HRSV) is a leading cause of respiratory illness, particularly in the young, elderly, and immunocompromised, and has also been linked to the development of asthma. HRSV replication depends on P and L, whereas transcription also requires M2-1. M2-1 interacts with P and RNA at overlapping binding sites; while these interactions are necessary for transcriptional activity, the mechanism of M2-1 action is unclear. To better understand HRSV transcription, we solved the crystal structure of M2-1 in complex with the minimal P interaction domain, revealing molecular details of the M2-1/P interface and defining the orientation of M2-1 within the tripartite complex. The M2-1/P interaction is relatively weak, suggesting high-affinity RNAs may displace M2-1 from the complex, providing the basis for a new model describing the role of M2-1 in transcription. Recently, the small molecules quercetin and cyclopamine have been used to validate M2-1 as a drug target.

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.

Inactivation of respiratory syncytial virus by zinc finger reactive compounds

BackgroundInfectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (Pneumoviridae), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins.ResultsThree compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats S.hispidus and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease.ConclusionsThis work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.

The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner

The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and approximately 7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative alpha-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.

Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-κB). The activation of NF-κB has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-κB in A549 cells. Electromobility shift assays show increased NF-κB complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-κB depends on the C-terminus of the RSV M2-1 protein, as shown by NF-κB-induced gene expression of a reporter gene construct.

Identification of amino acids that are critical to the processivity function of respiratory syncytial virus M2-1 protein.

The M2-1 protein of respiratory syncytial virus (RSV) is a transcription processivity factor that is essential for virus replication. The function of RSV M2-1 protein can be examined by using an RSVlacZ minigenome assay in vitro since the expression of the lacZ gene is dependent on M2-1. The M2-1 protein of pneumonia virus of mice (PVM), also a member of the Pneumovirus genus, functions poorly in the RSVlacZ minigenome assay despite conservation of the Cys(3)-His(1) motif at its N terminus and an overall 40% amino acid identity with RSV M2-1. To identify the amino acids responsible for the differences between these two proteins, two chimeric proteins were constructed. The RSV/PVM (RP) M2-1 chimera that contains the N-terminal 30 amino acids from RSV and the remaining C-terminal 148 amino acids from PVM maintained a level of activity at an ca. 36% of RSV M2-1. However, the PVM/RSV (PR) M2-1 chimera with the N-terminal 29 amino acids from PVM and 164 amino acids from RSV had an activity of <5% of RSV M2-1, indicating that the functional determinants are mainly located in the N terminus of M2-1. Mutagenesis of the N terminus of PR M2-1 and RSV M2-1 identified that Leu-16 and Asn-17 of RSV M2-1 are critical to the M2-1 function. In addition, several charged residues in the N terminus of RSV M2-1 also contributed to the functional integrity of M2-1.

Respiratory syncytial virus M2-1 protein requires phosphorylation for efficient function and binds viral RNA during infection.

The M2-1 protein of respiratory syncytial (RS) virus is a transcriptional processivity and antitermination factor. The M2-1 protein has a Cys3His1 zinc binding motif which is essential for function, is phosphorylated, and has been shown to interact with the RS virus nucleocapsid (N) protein. In the work reported here, we determined the sites at which the M2-1 protein was phosphorylated and investigated the importance of these phosphorylated residues for M2-1 function in transcription. By combining protease digestion, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and site-directed mutagenesis, we identified the phosphorylated residues as serines 58 and 61, not threonine 56 and serine 58 as previously reported. Serines 58 and 61 and the surrounding amino acids are in a consensus sequence for phosphorylation by casein kinase I. Consistent with this, we showed that the unphosphorylated M2-1 protein synthesized in Escherichia coli could be phosphorylated in vitro by casein kinase I. The effect of eliminating phosphorylation by site-specific mutagenesis of serines 58 and 61 on the function of the M2-1 protein in transcription of RS virus subgenomic replicons was assayed. The activities of the M2-1 protein phosphorylation mutants in transcriptional antitermination were tested over a range of concentrations and were found to be substantially inhibited at all concentrations. The data show that phosphorylation is important for the M2-1 protein function in transcription. However, mutation of the M2-1 phosphorylation sites did not interfere with the ability of the M2-1 protein to interact with the N protein in transfected cells. The interaction of the M2-1 and N proteins in cotransfected cells was found to be sensitive to RNase A, indicating that the M2-1-N protein interaction was mediated via RNA. Furthermore, the M2-1 protein was shown to bind monocistronic and polycistronic RS virus mRNAs during infection.

Requirement of cysteines and length of the human respiratory syncytial virus M2-1 protein for protein function and virus viability.

The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.