Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Zhang, Wei; Chipman, Paul R.; Corver, Jeroen; Johnson, Patricia A.; Zhang, Ying; Mukhopadhyay, Suchetana; Baker, Timothy S.; Strauss, James H.; Rossmann, Michael G.; Kühn, Richard

doi:10.1038/nsb990

Cited by 395 publications

(211 citation statements)

References 35 publications

(17 reference statements)

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“…Other membrane topology evidence suggests that helix-4 could be transmembrane possibly in another state of the molecule (28). The lateral positioning of helix-4 along the surface of the cell membrane seen in this structure has been observed in several other membrane proteins, such as the E and M membrane proteins of the flavivirus (31).…”

Section: Resultsmentioning

confidence: 99%

Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli

Chang²

2004

Proc. Natl. Acad. Sci. U.S.A.

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Multidrug resistance efflux transporters threaten to reverse the progress treating infectious disease by extruding a wide range of drug and other cytotoxic compounds. One such drug transporter, EmrE, from the small multidrug resistance family, utilizes proton gradients as an energy source to drive substrate translocation. In an effort to understand the molecular structural basis of this transport mechanism, we have determined the structure of EmrE from Escherichia coli to 3.8 Å. EmrE is a tetramer comprised of two conformational heterodimers related by a pseudo two-fold symmetry axis perpendicular to the cell membrane. Based on the structure and biochemical evidence, we propose a mechanism by which EmrE accomplishes multidrug efflux by coupling conformational changes between two heterodimers with proton gradient. Because of its simplicity and compact size, the structure of EmrE can serve as an ideal model for understanding the general structural basis of proton:drug antiport for other drug efflux systems

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Section: Resultsmentioning

confidence: 99%

Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli

Chang²

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…8A). The symmetry of the tetramer was not considered an important element, as cryoelectron microscopy data revealed that the DV nucleocapsid is disordered (47,48,49).…”

Section: St-148 Alters Intracellular Distribution Of Capsid Proteinmentioning

confidence: 99%

Characterization of the Mode of Action of a Potent Dengue Virus Capsid Inhibitor

Scaturro

Trist

Paul

et al. 2014

J Virol

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Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts to develop vaccine candidates and antiviral drugs, there is a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small-molecule inhibitor exhibiting broad and potent antiviral activity against DV in vitro and in vivo (C. M. Byrd et al., Antimicrob. Agents Chemother. 57:15-25, 2013, doi:10.1128/AAC.01429-12). In the present study, we investigated the mode of action of this promising compound by using a combination of biochemical, virological, and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of bimodal antiviral activity affecting both assembly/ release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay, we observed an ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids, probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept that also is applicable to flaviviruses. Dengue virus (DV) belongs to the genus Flavivirus, a large group of emerging pathogens capable of causing severe human diseases. Among these, DV represents the most prevalent mosquito-borne human-pathogenic virus worldwide, comprising 4 serotypes that are transmitted mainly by infected Aedes mosquitoes during a blood meal. DV infections can lead to a wide range of clinical manifestations, ranging from asymptomatic infections to life-threatening dengue hemorrhagic fever and shock syndrome. A recent study estimated around 390 million DV infections each year, resulting in approximately 100 million symptomatic cases and around 25,000 deaths (1). Despite intense efforts and growing public interest, no licensed antiviral drug against DV infection is available, and the most advanced DV vaccine candidate did not meet expectations in a recent large clinical trial (2).DV has a single-stranded RNA genome of positive polarity that codes for a polyprotein, which is co-and posttranslationally processed into three structural proteins (capsid, prM, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3). The virus enters mammalian cells via receptor-mediated endocytosis. In the endosomal compartment, the low pH induces a conformational change in the envelope (E) protein, triggering membrane fusion and nucleocapsid release into the cytoplasm (4, 5). Disassembly of the nucleocapsid occur...

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“…The N-terminal coding sequence of CP, which overlaps with cis-acting RNA elements, is required for virus replication and translation (5, 6). The DENV virion is ϳ50 nm in diameter and contains an outer glycoprotein shell that is composed of M and E proteins on the surface, a host-derived lipid bilayer, and the nucleocapsid embedded within (7,8). The internal nucleocapsid has a relatively poorly ordered structure compared to its external icosahedral glycoprotein shell (7-9).…”

mentioning

confidence: 99%

“…The DENV virion is ϳ50 nm in diameter and contains an outer glycoprotein shell that is composed of M and E proteins on the surface, a host-derived lipid bilayer, and the nucleocapsid embedded within (7,8). The internal nucleocapsid has a relatively poorly ordered structure compared to its external icosahedral glycoprotein shell (7)(8)(9). CP is the building block of the nucleocapsid, and it has been postulated that multiple copies of CP and one copy of the RNA genome form one nucleocapsid.…”

mentioning

confidence: 99%

Maintenance of Dimer Conformation by the Dengue Virus Core Protein α4-α4′ Helix Pair Is Critical for Nucleocapsid Formation and Virus Production

Teoh

Huang

Pong

et al. 2014

J Virol

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The virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal ␣2 and ␣4 helices of each monomer. Pairing of ␣2-␣2= creates a continuous hydrophobic surface, while the ␣4-␣4= helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the ␣4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. The in vitro characterization assays further highlighted that the ␣4-␣4= helix pair conformation is critical in preserving the overall ␣-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improves in vitro nucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formation in vivo. This study provides the first direct link between the ␣4-␣4= helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production. IMPORTANCEStructure-based mutagenesis study of the dengue virus core protein (CP) reveals that the ␣4-␣4= helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus translation and replication were detected. In vitro inefficiency and size of nucleocapsid-like particle (NLP) formation offer a possible explanation for in vivo virus production inefficiency upon CP mutation. Further, the transition of NLP morphology from an incomplete state to an intact particle shown by ␣4-␣4= helix pair mutants in the presence of a nonionic detergent suggests the regulatory role of the intracellular lipid droplet (LD) in CP-LD interaction and in promoting nucleocapsid formation. This study provides the first direct link between the ␣4-␣4= helix pair interaction and CP dimer conformation that is the fundamental requirement of CP function, particularly in nucleocapsid formation during virion production. V iruses of the Flaviviridae family are enveloped and have a monopartite, linear, and positive-polarity single-stranded RNA genome. A member of this family in the Flavivirus genus, Dengue virus (DENV), is the most important human arthropodborne viral pathogen, putting 3 billion people at risk and resulting in 50 million to 100 million infections and around 22,000 deat...

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Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Cited by 395 publications

References 35 publications

Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli

Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli

Characterization of the Mode of Action of a Potent Dengue Virus Capsid Inhibitor

Maintenance of Dimer Conformation by the Dengue Virus Core Protein α4-α4′ Helix Pair Is Critical for Nucleocapsid Formation and Virus Production

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