Structure of the Respiratory Syncytial Virus Polymerase Complex.

Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.

The methyltransferase domain of the Respiratory Syncytial Virus L protein catalyzes cap N7 and 2'-O-methylation.

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at µM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion.

Structure-Activity Relationship of Oxacyclo- and Triazolo-Containing Respiratory Syncytial Virus Polymerase Inhibitors.

Despite the availability of medicines preventing respiratory syncytial virus (RSV) infection, post-exposure treatment options are needed for addressing patient's needs. RSV non-nucleoside polymerase inhibitors (NNI) have emerged as a promising asset for which our group previously disclosed JNJ-8003 with potent in vitro antiviral activity and pronounced in vivo efficacy. In this work, a structural-guided design to modify the linker vector of JNJ-8003 resulted in the identification of 2-oxacyclo pyridine-containing derivatives whose various ring closing strategies are described. In addition, bioisosteric replacement of an amide bond with triazole retained potency, and cryo-electron microscopy (cryo-EM) confirmed binding in the capping domain. Subsequent NMR conformational analysis suggested a correlation between the potency and conformations. Our efforts have fulfilled the aim of identifying linker modifications with maintained biological activity while enriching structural diversity and allowing modulations of other parameters.

Discovery of gem-Dimethyl-hydroxymethylpyridine Derivatives as Potent Non-nucleoside RSV Polymerase Inhibitors.

Respiratory syncytial virus (RSV) is an RNA virus infecting the upper and lower respiratory tract and is recognized as a major respiratory health threat, particularly to older adults, immunocompromised individuals, and young children. Around 64 million children and adults are infected every year worldwide. Despite two vaccines and a new generation monoclonal antibody recently approved, no effective antiviral treatment is available. In this manuscript, we present the medicinal chemistry efforts resulting in the identification of compound 28 (JNJ-8003), a novel RSV non-nucleoside inhibitor displaying subnanomolar activity in vitro as well as prominent efficacy in mice and a neonatal lamb models.

Spiro-Azetidine Oxindoles as Long-Acting Injectables for Pre-Exposure Prophylaxis against Respiratory Syncytial Virus Infections.

Respiratory syncytial virus (RSV) is a major cause of hospitalization in infants, the elderly, and immune-compromised patients. While a half-life extended monoclonal antibody and 2 vaccines have recently been approved for infants and the elderly, respectively, options to prevent disease in immune-compromised patients are still needed. Here, we describe spiro-azetidine oxindoles as small molecule RSV entry inhibitors displaying favorable potency, developability attributes, and long-acting PK when injected as an aqueous suspension, suggesting their potential to prevent complications following RSV infection over a period of 3 to 6 months with 1 or 2 long-acting intramuscular (IM) or subcutaneous (SC) injections in these immune-compromised patients.

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase.

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor.

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.

Therapeutic efficacy of a respiratory syncytial virus fusion inhibitor.

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.

Lactobacillus priming of the respiratory tract: Heterologous immunity and protection against lethal pneumovirus infection.

We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We show here that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for 5months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity. We undertook the present study in order to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (CXCL1, CCL3, CCL2, CXCL10, TNF-alpha and IL-17A) but not Th1 cytokines. IFNγ does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities.

Depletion of alveolar macrophages prolongs survival in response to acute pneumovirus infection.

Alveolar macrophages are immunoregulatory effector cells that interact directly with respiratory virus pathogens in vivo. We examined the role of alveolar macrophages in acute infection with pneumonia virus of mice (PVM), a rodent pneumovirus that replicates the clinical sequelae of severe human respiratory syncytial virus disease. We show that PVM replicates in primary mouse macrophage culture, releasing infectious virions and proinflammatory cytokines. Alveolar macrophages isolated from PVM-infected mice express activation markers Clec43 and CD86, cytokines TNFα, IL-1, IL-6, and numerous CC and CXC chemokines. Alveolar macrophage depletion prior to PVM infection results in small but statistically significant increases in virus recovery but paradoxically prolonged survival. In parallel, macrophage depleted PVM-infected mice exhibit enhanced NK cell recruitment and increased production of IFNγ by NK, CD4(+) and CD8(+) T cells. These results suggest a protective, immunomodulatory role for IFNγ, as overproduction secondary to macrophage depletion may promote survival despite increased virus recovery.