Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex

Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals. IMPORTANCERespiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals. H uman respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in infants worldwide (1).No RSV vaccine is presently available, and the development of pediatric vaccines is particularly challenging. Currently, antiviral therapy is limited to palivizumab, a humanized mouse monoclonal antibody that targets the RSV fusion protein and is licensed for prophylactic use, and ribavirin, which has been used to treat severe infections despite its toxicity, its teratogenicity, and the limited evidence...

Section: Specificity Of Rnp:p Recognition In Rsvmentioning

confidence: 99%

A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus

Ouizougun-Oubari

Pereira

Tarus

et al. 2015

“…The structure of the nucleocapsid or a nucleocapsid-like particle has been solved for several members of Rhabdoviridae and Paramyxoviridae by X-ray crystallography or cryo-electron microscopy (cryo-EM) three-dimensional (3D) reconstruction (8)(9)(10)(11)(12). The common features among various structures are that the N protein has an N-terminal domain and C-terminal domain in its core, composed mostly of ␣-helices.…”

mentioning

confidence: 99%

“…The P N-terminal domain alone can enhance viral RNA synthesis, which has not been reported for other NSVs. Recently, the crystal structure of a truncated PIV5 N-RNA ring was reported (12). The N protein of PIV5 has the same typical two-domain fold as the N protein of other NSVs and is most homologous to that of Nipah virus (NiV) (19).…”

mentioning

confidence: 99%

Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

Severin

Terrell

Zengel

et al. 2016

In a negative-strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (N) to make the RNA accessible to the viral polymerase during this process. The structure of an empty mumps virus (MuV) nucleocapsid-like particle was determined to 10.4-Å resolution by cryo-electron microscopy (cryo-EM) image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it was shown that the ␣-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of N is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus N and its polymerase, instead of large N domain rotations, lead to exposure of the sequestered genomic RNA. Some pathogens appear to reemerge in spite of available vaccines, such as mumps virus and measles virus (3-5). Effective controls are needed to combat these pathogens. In order to develop more effective countermeasures, the mechanism of NSV replication should be better understood. One of the unique features in NSVs is that the genomic RNA is sequestered in the nucleocapsid (6). During transcription and replication, the viral RNA-dependent RNA polymerase (vRdRp) must be able to gain access to the sequestered genomic RNA in order to use it as the template. For Rhabdoviridae and Paramyxoviridae, the virus encodes a single nucleocapsid protein (N) that polymerizes as a linear capsid to encapsidate the genomic RNA (7). The viral polymerase complex consists of the large protein (L) and the phosphoprotein (P). IMPORTANCE Mumps virus (MuVThe structure of the nucleocapsid or a nucleocapsid-like particle has been solved for several members of Rhabdoviridae and Paramyxoviridae by X-ray crystallography or cryo-electron microscopy (cryo-EM) three-dimensional (3D) reconstruction (8-12). The common features among various structures are that the N protein has an N-terminal domain and C-terminal domain in its core, composed mostly of ␣-helices. When the N subunits assemble into a polymeric capsid, they are aligned in parallel in a linear fashion (13). There are extensive side-by-side interactions between the neighboring domains and domain swaps of extended loops and long termini. The genomic RNA is encapsidated in a cavity formed between the two core domains. Most of the RNA bases are stacked, some of which face the exterior and some the interior of the N protein core. The tight assembly of the nucleocapsid clearly suggests that vRdRp must open the N protein core in order to unveil the genomic RNA. How this action is carried out remains to be discovered. The interaction of the polymerase cofactor P with the nucleocapsid may provide some insights on this subject. The C-terminal domain of vesicular stomatitis virus (VSV) P protein binds between the extended loops in the C-terminal domains ...

“…Ten amino acids of PIV5 NP directly contact the RNA, namely, K194, R195, Y260, Q202, M266, G267, Y350, A351, R354, and S355 (10), and are strictly conserved between hPIV2 and PIV5 NP (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, the high-resolution crystal structure of the PIV5 NP-RNA ring complex assembled in Escherichia coli was determined (10). PIV5 NP encapsidates RNA in its positively charged groove between the NTD and CTD, similar to the case for other nsNSV.…”

mentioning

confidence: 84%

A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity

Matsumoto

Ohta

Kolakofsky

et al. 2017

The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA.IMPORTANCE We examined the importance of amino acids in the putative RNAbinding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression. KEYWORDS human parainfluenza virus type 2, encapsidation, nucleoprotein, polymerase H uman parainfluenza virus type 2 (hPIV2) is a major respiratory pathogen and a member of the Rubulavirus genus of the family Paramyxoviridae, which includes simian virus 41, hPIV4, mumps virus, and PIV5. The hPIV2 genome RNA contains six tandemly linked genes (3=-NP-P/V-M-F-HN-L-5=) and is tightly coated by the nucleoprotein (NP) to form a helical nucleocapsid that serves as the template for viral RNA synthesis. The phosphoprotein (P) and large protein (L) together form the viral RNAdependent RNA polymerase (RdRp), which is responsible for both transcription to produce mRNAs and replication to produce genomes and antigenomes (1). hPIV2 NP contains 543 amino acids, whose functional domains are partially identified. The N-terminal 294 amino acids are required for NP self-assembly, and two C-terminal