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

Abstract

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 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 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. 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.

Nipah shell disorder, modes of infection, and virulence

“…Interestingly, past experiments using mutations of N proteins of NiV cousins, canine distemper virus (CDV) and MeV, have shown that greater disorder at the N tail cause the viruses to be more virulent to ferrets in a laboratory [64]. While this was done experimentally using MeV and CDV in a laboratory setting, our results show for the first time that this also can be the case for NiV, but using an empirical analysis of NiV in the wild.…”

“…The N, P, and L proteins play important roles in the replication of the viral RNA [64,65]. The N protein when bound to the P protein, encapsidates the viral RNA during the replication of the latter.…”

“…When P binds to N, the complex stabilizes in order to have greater affinity for the viral RNA. Also, when P protein binds to N protein, it induces the binding of the L protein close to the binding region of the N protein, and together the N-P-L complex functions as an RNA polymerase during the viral replication [52,64].…”

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

“…These findings demonstrated that paramyxovirus polymerase bioactivity does not depend on the physical presence of the unstructured N-tail section. However, genetic evaluation of the N-tail truncated CDV revealed that robust virus growth coincided with the appearance of a mutation at residue 156 in N-core (15), implying that the deleted N-tail section may function as a long-range regulator of RdRP activity.…”

CryoEM structure of the Nipah virus nucleocapsid assembly

“…Another regulatory role may be contributed by the flexible Ntail region located at the Cterminus of the N protein. The N protein Ntail region has been shown both to be the site for P protein interaction 21,22 and to play a role in regulating transcriptional activity 23,24 . It is likely that this region is orientated toward the outside of the spiral 25 , allowing contact with the P protein.…”