Mechanism of Action for Respiratory Syncytial Virus Inhibitor RSV604

ABSTRACTRespiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children and other high-risk populations. RSV nucleoprotein (N) is essential for virus assembly and replication as part of the viral ribonucleoprotein (RNP) complex. RSV604 was a putative N inhibitor in phase 2 clinical trials whose molecular mechanism of action (MoA) was not well understood. This study investigated the cell line-dependent potency of RSV604 and demonstrated its direct binding to the N proteinin vitro, providing the first evidence of direct target engagement for this class of inhibitors reported to date. The affinity of RSV604 N binding was not affected by RSV604 resistance mutations in the N protein. RSV604 engaged in two different MoAs in HeLa cells, inhibiting both RSV RNA synthesis and the infectivity of released virus. The lack of inhibition of viral RNA synthesis in some cell lines explained the cell-type-dependent potency of the inhibitor. RSV604 did not inhibit viral RNA synthesis in the RSV subgenomic replicon cells or in the cell-free RNP assay, suggesting that it might act prior to viral replication complex formation. RSV604 did not alter N protein localization in the infected cells. Taken together, these results provide new insights leading to an understanding of the MoAs of RSV604 and other similar N inhibitors.

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Phosphorylation of the human respiratory syncytial virus N protein provokes a decrease in viral RNA synthesis

Virus Research ◽

10.1016/j.virusres.2011.10.004 ◽

2012 ◽

Vol 163 (1) ◽

pp. 396-400 ◽

Cited By ~ 1

Author(s):

A. Asenjo ◽

I. Cuesta ◽

A. Vivo ◽

N. Villanueva

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Synthesis ◽

Human Respiratory Syncytial Virus ◽

Viral Rna ◽

N Protein ◽

Syncytial Virus

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011602 ◽

2019 ◽

Vol 295 (3) ◽

pp. 883-895 ◽

Cited By ~ 1

Author(s):

Yunrong Gao ◽

Dongdong Cao ◽

Hyunjun Max Ahn ◽

Anshuman Swain ◽

Shaylan Hill ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Synthesis ◽

Genomic Rnas ◽

Viral Rnas ◽

N Protein ◽

Syncytial Virus ◽

Nucleoprotein N ◽

Negative Sense ◽

Transcription And Replication

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

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Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

Journal of Virology ◽

10.1128/jvi.03666-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3484-3496 ◽

Cited By ~ 31

Author(s):

Marie Galloux ◽

Gaëlle Gabiane ◽

Julien Sourimant ◽

Charles-Adrien Richard ◽

Patrick England ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Polymerase ◽

Rna Synthesis ◽

Polymerase Activity ◽

Viral Rna ◽

Free Form ◽

Mutant Form ◽

Content Type ◽

Amino Terminal ◽

Syncytial Virus

ABSTRACTThe RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy withParamyxoviridaeandRhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N0-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N0monomer. We used this N mutant, designated Nmono, as a substitute for N0in order to characterize the P regions involved in the N0-P complex formation. Using a series of P fragments, we determined by glutathioneS-transferase (GST) pulldown assays that the N and C termini of P are able to interact with Nmono. We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and Nmonoin vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N0-P interaction could constitute a potential antiviral strategy.IMPORTANCERespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N0) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.

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Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins

Virus Research ◽

10.1016/j.virusres.2015.10.011 ◽

2016 ◽

Vol 211 ◽

pp. 117-125 ◽

Cited By ~ 5

Author(s):

Ana Asenjo ◽

Nieves Villanueva

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Synthesis ◽

Human Respiratory Syncytial Virus ◽

Viral Rna ◽

P Protein ◽

Syncytial Virus ◽

P Proteins

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Viral RNA N6-methyladenosine modification modulates both innate and adaptive immune responses of human respiratory syncytial virus

PLoS Pathogens ◽

10.1371/journal.ppat.1010142 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1010142

Author(s):

Miaoge Xue ◽

Yuexiu Zhang ◽

Haitao Wang ◽

Elizabeth L. Kairis ◽

Mijia Lu ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Immune Responses ◽

Adaptive Immunity ◽

Human Respiratory Syncytial Virus ◽

Viral Rna ◽

Type I ◽

Adaptive Immune Responses ◽

Adaptive Immune ◽

Syncytial Virus ◽

Tract Infections

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N6-methyladenosine (m6A) and RSV grown in m6A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m6A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m6A methylation enhances innate immune responses which in turn promote adaptive immunity.

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Extracellular amoebal-vesicles: potential transmission vehicles for respiratory viruses

npj Biofilms and Microbiomes ◽

10.1038/s41522-021-00201-y ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Rafik Dey ◽

Melanie A. Folkins ◽

Nicholas J. Ashbolt

Keyword(s):

Respiratory Syncytial Virus ◽

Respiratory Tract ◽

Respiratory Tract Infections ◽

Respiratory Viruses ◽

Environmental Persistence ◽

Viral Dissemination ◽

Acute Respiratory Tract Infections ◽

High Concentrations ◽

Syncytial Virus ◽

Tract Infections

AbstractHuman respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and immunocompromised adults worldwide. Here we report that amoebae-release respirable-sized vesicles containing high concentrations of infectious RSV that persisted for the duration of the experiment. Given the ubiquity of amoebae in moist environments, our results suggest that extracellular amoebal-vesicles could contribute to the environmental persistence of respiratory viruses, including potential resistance to disinfection processes and thereby offering novel pathways for viral dissemination and transmission.

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Considerations for a Respiratory Syncytial Virus Vaccine Targeting an Elderly Population

Vaccines ◽

10.3390/vaccines9060624 ◽

2021 ◽

Vol 9 (6) ◽

pp. 624

Author(s):

Laura M. Stephens ◽

Steven M. Varga

Keyword(s):

Respiratory Syncytial Virus ◽

Disease Burden ◽

Elderly Population ◽

Vaccine Development ◽

The Elderly ◽

The United States ◽

Susceptible Population ◽

Age Related ◽

Syncytial Virus ◽

Tract Infections

Respiratory syncytial virus (RSV) is most commonly associated with acute lower respiratory tract infections in infants and children. However, RSV also causes a high disease burden in the elderly that is often under recognized. Adults >65 years of age account for an estimated 80,000 RSV-associated hospitalizations and 14,000 deaths in the United States annually. RSV infection in aged individuals can result in more severe disease symptoms including pneumonia and bronchiolitis. Given the large disease burden caused by RSV in the aged, this population remains an important target for vaccine development. Aging results in lowered immune responsiveness characterized by impairments in both innate and adaptive immunity. This immune senescence poses a challenge when developing a vaccine targeting elderly individuals. An RSV vaccine tailored towards an elderly population will need to maximize the immune response elicited in order to overcome age-related defects in the immune system. In this article, we review the hurdles that must be overcome to successfully develop an RSV vaccine for use in the elderly, and discuss the vaccine candidates currently being tested in this highly susceptible population.

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Bacterial and Viral Coinfections with the Human Respiratory Syncytial Virus

Microorganisms ◽

10.3390/microorganisms9061293 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1293

Author(s):

Gaspar A. Pacheco ◽

Nicolás M. S. Gálvez ◽

Jorge A. Soto ◽

Catalina A. Andrade ◽

Alexis M. Kalergis

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A ◽

Human Respiratory Syncytial Virus ◽

Respiratory Pathogens ◽

Parainfluenza Viruses ◽

Starting Point ◽

The Social ◽

Syncytial Virus ◽

Tract Infections ◽

Polymicrobial Infections

The human respiratory syncytial virus (hRSV) is one of the leading causes of acute lower respiratory tract infections in children under five years old. Notably, hRSV infections can give way to pneumonia and predispose to other respiratory complications later in life, such as asthma. Even though the social and economic burden associated with hRSV infections is tremendous, there are no approved vaccines to date to prevent the disease caused by this pathogen. Recently, coinfections and superinfections have turned into an active field of study, and interactions between many viral and bacterial pathogens have been studied. hRSV is not an exception since polymicrobial infections involving this virus are common, especially when illness has evolved into pneumonia. Here, we review the epidemiology and recent findings regarding the main polymicrobial infections involving hRSV and several prevalent bacterial and viral respiratory pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, human rhinoviruses, influenza A virus, human metapneumovirus, and human parainfluenza viruses. As reports of most polymicrobial infections involving hRSV lack a molecular basis explaining the interaction between hRSV and these pathogens, we believe this review article can serve as a starting point to interesting and very much needed research in this area.

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