Class II ADP-ribosylation Factors Are Required for Efficient Secretion of Dengue Viruses

Kudelko, Mateusz; Brault, Jean‐Baptiste; Kwok, Kevin; Li, Ming Yuan; Pardigon, Nathalie; Peiris, Malik; Bruzzone, Roberto; Desprès, Philippe; Nal, Béatrice; Wang, Peigang

doi:10.1074/jbc.m111.270579

Cited by 55 publications

(71 citation statements)

References 51 publications

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“…As expected, in both cases BFA treatment drastically reduced titers of both viral RNA and infectivity ( Fig. 3D and E; black columns), consistent with an earlier report (22). In conclusion, these results suggest that ST-148 reduces DV particle production.…”

Section: St-148 Is a Potent Inhibitor Of DV St-148supporting

confidence: 81%

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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Section: St-148 Is a Potent Inhibitor Of DV St-148supporting

confidence: 81%

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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“…To further examine the specificity of the Arf4 interaction with Scyl1, we used affinity-selection assays with western blots. Owing to the lack of availability of antibodies against specific Arf isoforms, we used constructs encoding C-terminally tagged fluorescent Arf proteins, which are widely used for the study of Arfs (Chun et al, 2008;Duijsings et al, 2009;Jain et al, 2012;Kudelko et al, 2012). Scyl1 bound specifically to class II Arfs, with a preference for Arf4, but did not bind to class I Arfs (Fig.…”

Section: Scyl1 Interacts Specifically With Class II Arfsmentioning

confidence: 99%

Scyl1 scaffolds class II Arfs to selective subcomplexes of coatomer via the γ-COP appendage domain

Hamlin

Schroeder

Fotouhi

et al. 2014

Journal of Cell Science

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Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. We now demonstrate that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO 2 motif to interact directly with the appendage domain of coatomer subunit c-2 (also known as COPG2 or c2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to c2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cisGolgi, similar to that observed following the loss of Arf and Arf-guaninenucleotide-exchange factor (GEF) function. Our data reveal that Scyl1 is a key organizer of a subset of the COPI machinery.

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“…While there are limited data on insect host proteins interacting with flavivirus prM/M (38), several interacting proteins were identified in mammalian cells, such as members of the ADP-ribosylation factor (39), vacuolar ATPases (40), a light chain of dynein (36), a DEAD box helicase, FUNDC1/2 (37), and claudin-1 (41). In some cases, this interaction was even involved in viral particle secretion (39,40). Even though those mammalian prM/M interacting proteins have orthologs in insects, the strength of such an interaction could differ among hosts and be more or less sensitive to the introduction of mutations in the M protein.…”

Section: Discussionmentioning

confidence: 99%

“…Subsequent mutagenesis analysis of the M protein proved the importance of an interactive network between E and M in those processes (16,34,35). The flavivirus prM/M protein was also shown to interact with mammalian and mosquito host factors (36)(37)(38)(39)(40)(41), thus indicating that this small protein may be involved in additional nonstructural aspects of the flavivirus infectious cycle. Notably, a peptide identified in flavivirus M proteins was shown to potently trigger apoptosis in mammalian cells (42,43).…”

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confidence: 99%

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

Wispelaere

Khou

Frenkiel

et al. 2016

J Virol

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Japanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuation in vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virus in vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts. IMPORTANCEJEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines. F laviviruses such as Japanese encephalitis virus (JEV) are arthropod-borne pathogens (arboviruses) that are transmitted through the bite of an infected mosquito and cause serious human diseases worldwide (1). JEV is the causative agent for Japanese encephalitis, one of the most important viral encephalitis diseases of medical interest in Asia, with an incidence of approximately 67,900 human cases per year (2). Up to 30% of the symptomatic cases are fatal, while 30 to 50% of survivors can develop long-term neurologic sequelae (3).JEV has a positive-sense RNA genome encoding a single polyprotein. This polyprotein is processed by host-and JEV-encoded proteases into 10 proteins: three structural proteins (core [C], premembrane [prM], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Like other flaviviruses, JEV enters cells via receptor-mediated endocytosis (4, 5). The acidic environment in the host endosome serves as the physiological trigger for a major conformational change in the E protein...

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Class II ADP-ribosylation Factors Are Required for Efficient Secretion of Dengue Viruses

Cited by 55 publications

References 51 publications

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

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

Scyl1 scaffolds class II Arfs to selective subcomplexes of coatomer via the γ-COP appendage domain

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

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