Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ∼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L–P complex from parainfluenza virus 5 (PIV5) at 4.3-Å resolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Å resolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5′ end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.

Preparing antiviral defenses Antiviral drugs are an important tool in the battle against COVID-19. Both remdesivir and molnupiravir, which target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase, were first developed against other RNA viruses. This highlights the importance of broad-spectrum antivirals that can be rapidly deployed against related emerging pathogens. Sourimant et al . used respiratory syncytial virus (RSV) as a primary indication in identifying further drugs that target the polymerase enzyme of RNA viruses. The authors explored derivatives of molnupiravir and identified 4′ fluorouridine (EIDD-2749) as an inhibitor of the polymerase of RSV and SARS-CoV-2. This drug can be delivered orally and was effective against RSV in mice and SARS-CoV-2 in ferrets. —VV

Parainfluenza virus 5 (PIV5) belongs to the family , which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (NP) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 NP consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The NP structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication. Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.