Cryo-EM structure of the respiratory syncytial virus RNA polymerase (original) (raw)
Related papers
Structure of the Respiratory Syncytial Virus Polymerase Complex
Cell, 2019
Highlights d Cryo-EM structure of RSV L bound by tetrameric RSV P solved to 3.2 Å d P tetramer adopts an asymmetric tentacular arrangement when bound to L d L priming loop adopts elongation-compatible state without PRNTase-RdRp separation d Structure rationalizes escape from small-molecule antivirals
Journal of Virology, 2015
ABSTRACTThe minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluatedin cellulausing an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region.IMPORTANCERespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the vira...
Towards a structural understanding of RNA synthesis by negative strand RNA viral polymerases
Current opinion in structural biology, 2016
Negative strand RNA viruses (NSVs), which may have segmented (sNSV) or non-segmented genomes (nsNSV) are responsible for numerous serious human infections such as Influenza, Measles, Rabies, Ebola, Crimean Congo Haemorrhagic Fever and Lassa Fever. Their RNA-dependent RNA polymerases transcribe and replicate the nucleoprotein coated viral genome within the context of a ribonucleoprotein particle. We review the first high resolution crystal and cryo-EM structures of representative NSV polymerases. The heterotrimeric Influenza and single-chain La Crosse orthobunyavirus polymerase structures (sNSV) show how specific recognition of both genome ends is achieved and is required for polymerase activation and how the sNSV specific 'cap-snatching' mechanism of transcription priming works. Vesicular Stomatitis Virus (nsNSV) polymerase shows a similar core architecture but has different flexibly linked C-terminal domains which perform mRNA cap synthesis. These structures pave the way fo...
PLOS Pathogens, 2020
The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.
Virology, 2019
Respiratory syncytial virus (RSV) is significant for public health, capable of causing respiratory tract disease in infants, the elderly and the immunocompromised. The RSV polymerase is an attractive target for antiviral drug development, but as yet, there is no high throughput assay for analyzing RSV polymerase activity, specifically. In this study, using a primer elongation assay as a basis, we analyzed the tolerance of the RSV polymerase for modifications at the 5′ end of the primer, and nucleotide analogs. The RSV polymerase was found to accept primers containing 5′ biotin or digoxygenin modifications, and nucleotide analogs that are reactive or fluorescent, including 5-ethynyl UTP, 8-azido ATP, 2-amino PTP, and thieno-GTP. These findings provide a menu of options for developing non-isotopic high throughput assays for RSV polymerase RNA synthesis activity, and yield insight regarding the molecular biology of the polymerase complex.
Structural architecture of a dimeric paramyxovirus polymerase complex
2021
Human parainfluenza virus type 3 (hPIV3), a member of non-segmented, negative-strand RNA viruses (nsNSVs), is the second most common cause of severe respiratory disease in pediatrics. The transcription and replication processes of nsNSVs are catalyzed by a multi-functional RNA-dependent RNA polymerase (RdRp) complex composed of the large protein (L) and the phosphoprotein (P). Previous studies have shown that the polymerase can adopt a dimeric form, however, the structure of the dimer and how it functions are not understood. Here we determined the cryo-EM structure of hPIV3 L-P complex at 2.7 Å with substantial structural details. A putative catalytic magnesium ion could be built in our structure, and structural comparison revealed atomic features conserved with other RNA viruses. Interactions identified between the two priming and intrusion loops and the connector domain potentially trigger the spatial movement of three C-terminal L domains for different steps of transcription and ...
World Journal Of Advanced Research and Reviews, 2023
Human respiratory syncytial virus (hRSV) is one of the triple epidemic viruses that causes infections of the respiratory tract and lungs. For multiplication of the virus in human cells, its RNA polymerase is the crucial enzyme and it forms a part of a large protein (LP). The LP is a multicomponent and multifunctional protein harboring at least 3 different enzymes. The RNA polymerase belongs to RNA-dependent RNA polymerase (RdRp) (EC: 2.7.7.48) type and performs the synthesis of both mRNAs (transcription) and genomic RNA (gRNA) (replication). In addition to the RNA polymerase, the LP also harbours two more enzymes, viz. enzymes for cap addition and cap methylation of mRNAs. The polymerase domain of the LP is analyzed for its active site amino acids and its proofreading (PR) domain. Two polymerase active site regions and a DEDD-superfamily of 3'→5' PR exonuclease active site domain are identified in the polymerase region. The signature metal-binding motifs, viz.-GDNQ-and-SDD-which are commonly found in the RdRps of all the (-) strand RNA viral pathogens are also found in the hRSV RNA polymerase. The two highly conserved polymerase catalytic core regions identified by sequence similarity are in close agreement with other DNA/RNA polymerases already reported and hence, proposed to function in the nucleotidyl transfer reactions. Presence of the two catalytic regions also suggest that the polymerase may function in a dual mode, one for transcription and the other one for replication, using the same invariant catalytic Mg 2+-binding-GDNQ-and-SDD-motifs.
2020
ABSTRACTBunyavirales is an order of segmented negative stranded RNA viruses comprising several life-threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales genome constitute essential reactions of their viral cycle that are catalysed by the virally encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of key C-terminal domains, notably the cap-binding domain that undergoes large movements related to its role in transcription initiation and a zinc-binding domain that displays a fold not previously observed. We capture the structure of LACV-L FL in two functionally relevant states, pre-initiation and elongation, that reveal large conformational change...
Crystal Structure of Complete Rhinovirus RNA Polymerase Suggests Front Loading of Protein Primer
Journal of Virology, 2005
Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3D pol ) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3D pol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3D po1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.
Biomolecules
The phosphoprotein P of Mononegavirales (MNV) is an essential co-factor of the viral RNA polymerase L. Its prime function is to recruit L to the ribonucleocapsid composed of the viral genome encapsidated by the nucleoprotein N. MNV phosphoproteins often contain a high degree of disorder. In Pneumoviridae phosphoproteins, the only domain with well-defined structure is a small oligomerization domain (POD). We previously characterized the differential disorder in respiratory syncytial virus (RSV) phosphoprotein by NMR. We showed that outside of RSV POD, the intrinsically disordered N-and C-terminal regions displayed a structural and dynamic diversity ranging from random coil to high helical propensity. Here we provide additional insight into the dynamic behavior of PCα, a domain that is C-terminal to POD and constitutes the RSV L-binding region together with POD. By using small phosphoprotein fragments centered on or adjacent to POD, we obtained a structural picture of the POD–PCα regi...