Crystal Structure and Thermostability Characterization of Enterovirus D68 3Dpol

ABSTRACT Enterovirus D68 (EV-D68) is one of the many nonpolio enteroviruses that cause mild to severe respiratory illness. The nonstructural protein 3Dpol is an RNA-dependent RNA polymerase (RdRP) of EV-D68 which plays a critical role in the replication of the viral genome and represents a promising drug target. Here, we report the first three-dimensional crystal structure of the RdRP from EV-D68 in complex with the substrate GTP to 2.3-Å resolution. The RdRP structure is similar to structures of other viral RdRPs, where the three domains, termed the palm, fingers, and thumb, form a structure resembling a cupped right hand. Particularly, an N-terminal fragment (Gly1 to Phe30) bridges the fingers and the thumb domains, which accounts for the enhanced stability of the full-length enzyme over the truncation mutant, as assessed by our thermal shift assays and the dynamic light scattering studies. Additionally, the GTP molecule bound proximal to the active site interacts with both the palm and fingers domains to stabilize the core structure of 3Dpol. Interestingly, using limited proteolysis assays, we found that different nucleoside triphosphates (NTPs) stabilize the polymerase structure by various degrees, with GTP and CTP being the most and least stabilizing nucleosides, respectively. Lastly, we derived a model of the core structure of 3Dpol stabilized by GTP, according to our proteolytic studies. The biochemical and biophysical characterizations conducted in this study help us to understand the stability of EV-D68-3Dpol, which may extend to other RdRPs as well. IMPORTANCE Enterovirus D68 (EV-D68) is an emerging viral pathogen, which caused sporadic infections around the world. In recent years, epidemiology studies have reported an increasing number of patients with respiratory diseases globally due to the EV-D68 infection. Moreover, the infection has been associated with acute flaccid paralysis and cranial nerve dysfunction in children. However, there are no vaccines and antiviral treatments specifically targeting the virus to date. In this study, we solved the crystal structure of the RNA-dependent RNA polymerase of EV-D68 and carried out systematic biophysical and biochemical characterizations on the overall and local structural stability of the wild-type (WT) enzyme and several variants, which yields a clear view on the structure-activity relationship of the EV-D68 RNA polymerase.

Download Full-text

Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain

Journal of Virology ◽

10.1128/jvi.00324-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 5

Author(s):

Weiwei Li ◽

Baixing Wu ◽

Wibowo Adian Soca ◽

Lei An

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Structural Basis ◽

Economic Losses ◽

Rna Dependent Rna Polymerase ◽

Content Type ◽

Terminal Domain

ABSTRACTClassical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by ade novomechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCEPigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genusPestivirus, familyFlaviviridae. Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

Download Full-text

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

Structure ◽

10.1016/s0969-2126(00)80031-3 ◽

1999 ◽

Vol 7 (11) ◽

pp. 1417-1426 ◽

Cited By ~ 305

Author(s):

Hideo Ago ◽

Tsuyoshi Adachi ◽

Atsuhito Yoshida ◽

Masaki Yamamoto ◽

Noriyuki Habuka ◽

...

Keyword(s):

Crystal Structure ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Dependent Rna Polymerase

Download Full-text

Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation

Journal of Virology ◽

10.1128/jvi.02085-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 249-261 ◽

Cited By ~ 16

Author(s):

Jiqin Wu ◽

Guoliang Lu ◽

Bo Zhang ◽

Peng Gong

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Full Length ◽

Intramolecular Interactions ◽

Rna Dependent Rna Polymerase ◽

Hydrophobic Residues ◽

Functional Relevance

ABSTRACTThe flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through ade novomechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.IMPORTANCEThe flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried outin vitropolymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

Download Full-text

A crystal structure of alphavirus nonstructural protein 4 (nsP4) reveals an intrinsically dynamic RNA-dependent RNA polymerase

10.1101/2021.05.27.445971 ◽

2021 ◽

Author(s):

Yaw Bia Tan ◽

Laura Sandra Lello ◽

Xin Liu ◽

Yee-Song Law ◽

Congbao Kang ◽

...

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Nonstructural Protein ◽

3D Structure ◽

Rna Replication ◽

Viral Rna ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Intrinsically Disordered ◽

Rdrp Domain

Alphaviruses such as Ross River virus (RRV), chikungunya virus and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report a crystal structure of the RdRp domain of nsP4 from RRV determined at a resolution of 2.6 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses and picornaviruses. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, we showed that in-solution nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were able to catalyze in vitro RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Download Full-text