Viral evolution identifies a regulatory interface between paramyxovirus polymerase complex and nucleocapsid that controls replication dynamics.

Paramyxoviruses are negative-polarity RNA viruses of major clinical importance. The dynamic interaction of the RNA-dependent RNA polymerase (RdRP) complex with the encapsidated RNA genome is mechanistically and structurally poorly understood. Having generated recombinant measles (MeV) and canine distemper (CDV) viruses with truncated nucleocapsid (N) protein showing defects in replication kinetics, we have applied a viral evolution approach to the problem. Passaging of recombinants resulted in long-range compensatory mutations that restored RdRP bioactivity in minigenome assays and efficient replication of engineered viruses. Compensatory mutations clustered at an electronically compatible acidic loop in N-core and a basic face of the phosphoprotein X domain (P-XD). Co-affinity precipitations, biolayer interferometry, and molecular docking revealed an electrostatic-driven transiently forming interface between these domains. The compensatory mutations reduced electrostatic compatibility of these microdomains and lowered coprecipitation efficiency, consistent with a molecular checkpoint function that regulates paramyxovirus polymerase mobility through modulation of conformational stability of the P-XD assembly.

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design.IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family.

The structurally disordered paramyxovirus nucleocapsid protein tail domain is a regulator of the mRNA transcription gradient.

The paramyxovirus RNA-dependent RNA-polymerase (RdRp) complex loads onto the nucleocapsid protein (N)-encapsidated viral N:RNA genome for RNA synthesis. Binding of the RdRp of measles virus (MeV), a paramyxovirus archetype, is mediated through interaction with a molecular recognition element (MoRE) located near the end of the carboxyl-terminal Ntail domain. The structurally disordered central Ntail section is thought to add positional flexibility to MoRE, but the functional importance of this Ntail region for RNA polymerization is unclear. To address this question, we dissected functional elements of Ntail by relocating MoRE into the RNA-encapsidating Ncore domain. Linker-scanning mutagenesis identified a microdomain in Ncore that tolerates insertions. MoRE relocated to Ncore supported efficient interaction with N, MoRE-deficient Ntails had a dominant-negative effect on bioactivity that was alleviated by insertion of MoRE into Ncore, and recombinant MeV encoding N with relocated MoRE grew efficiently and remained capable of mRNA editing. MoRE in Ncore also restored viability of a recombinant lacking the disordered central Ntail section, but this recombinant was temperature-sensitive, with reduced RdRp loading efficiency and a flattened transcription gradient. These results demonstrate that virus replication requires high-affinity RdRp binding sites in N:RNA, but productive RdRp binding is independent of positional flexibility of MoRE and cis-acting elements in Ntail. Rather, the disordered central Ntail section independent of the presence of MoRE in Ntail steepens the paramyxovirus transcription gradient by promoting RdRp loading and preventing the formation of nonproductive polycistronic viral mRNAs. Disordered Ntails may have evolved as a regulatory element to adjust paramyxovirus gene expression.

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors.

Respiratory syncytial virus (RSV) is a leading pediatric pathogen that is responsible for a majority of infant hospitalizations due to viral disease. Despite its clinical importance, no vaccine prophylaxis against RSV disease or effective antiviral therapeutic is available. In this study, we established a robust high-throughput drug screening protocol by using a recombinant RSV reporter virus to expand the pool of RSV inhibitor candidates. Mechanistic characterization revealed that a potent newly identified inhibitor class blocks viral entry through specific targeting of the RSV fusion (F) protein. Resistance against this class was induced and revealed overlapping hotspots with diverse, previously identified RSV entry blockers at different stages of preclinical and clinical development. A structural and biochemical assessment of the mechanism of unique, broad RSV cross-resistance against structurally distinct entry inhibitors demonstrated that individual escape hotspots are located in immediate physical proximity in the metastable conformation of RSV F and that the resistance mutations lower the barrier for prefusion F triggering, resulting in an accelerated RSV entry kinetics. One resistant RSV recombinant remained fully pathogenic in a mouse model of RSV infection. By identifying molecular determinants governing the RSV entry machinery, this study spotlights a molecular mechanism of broad RSV resistance against entry inhibition that may affect the impact of diverse viral entry inhibitors presently considered for clinical use and outlines a proactive design for future RSV drug discovery campaigns.