Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly, Precursor Membrane (prM) Protein Cleavage, and Entry

Hsieh, Szu Chia; Wu, Yi; Zou, Gang; Nerurkar, Vivek R.; Shi, Pei Yong; Wang, Wei‐Kung

doi:10.1074/jbc.m114.610428

Cited by 42 publications

(38 citation statements)

References 65 publications

(22 reference statements)

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“…These investigations identified residues S112, E114, W117, A120, V123, E124, W126, I127, L128 and R129 as strongly influencing the assembly of virus particles, with substitutions at M111, E114, G115, W117, V123, E124, W126, I127, L128 and R129 also impairing particle infectivity. Interestingly, only E114 in DENV-1 was also demonstrated to be crucial for heterodimeric interactions between prM and E (Hsieh et al, 2014), indicating that the other residues may play a greater role in influencing the conformational rearrangement of structural proteins during virion maturation (Zhang et al, 2012). This hypothesis is particularly supported by recent evidence linking residue E125 in the JEV prM-H domain (corresponding to DENV E124) in electrostatic interactions with the K93 and H246 residues in E protein DII (Peng & Wu, 2014).…”

Section: Heterodimerization Of Prm and Esupporting

confidence: 64%

“…The role of the DENV prM-H domain in particle formation has also been revealed in two thorough proline/alanine mutation studies (Hsieh et al, 2011(Hsieh et al, , 2014. These investigations identified residues S112, E114, W117, A120, V123, E124, W126, I127, L128 and R129 as strongly influencing the assembly of virus particles, with substitutions at M111, E114, G115, W117, V123, E124, W126, I127, L128 and R129 also impairing particle infectivity.…”

Section: Heterodimerization Of Prm and Ementioning

confidence: 99%

“…This region (which is located adjacent to the fusion loop of E in heterotrimeric spikes) was deemed critical for the assembly and secretion of prM/E particles from both TBEV and JEV (Yoshii et al, 2012). The GXXXG motif within the prM protein of JEV (corresponding to residues 142-146 within the first transmembrane domain) was also deemed to be important for prM and E interaction, as alanine insertion mutants displayed reduced heterodimerization and prM/E particle secretion .The role of the DENV prM-H domain in particle formation has also been revealed in two thorough proline/alanine mutation studies (Hsieh et al, 2011(Hsieh et al, , 2014. These investigations identified residues S112, E114, W117, A120, V123, E124, W126, I127, L128 and R129 as strongly influencing the assembly of virus particles, with substitutions at M111, E114, G115, W117, V123, E124, W126, I127, L128 and R129 also impairing particle infectivity.…”

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confidence: 99%

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Post-translational regulation and modifications of flavivirus structural proteins

Roby

Setoh

Hall

et al. 2015

Journal of General Virology

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Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular -capsid (C), pre-membrane (prM) and envelope (E) -are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics. FlavivirusesThe genus Flavivirus in the family Flaviviridae comprises small, enveloped viruses with non-segmented genomes consisting of single-stranded, positive-sense RNA. These RNA genomes are flanked by 59 and 39 untranslated regions (UTRs) and encode a single polyprotein that is cleaved coand post-translationally to generate between 10 and 11 viral proteins. The 59 UTR contains a type 1 m 7 GpppNm cap structure and the 39 UTR lacks a polyadenylated tail. Structural elements within the UTRs regulate genome replication, translation and host immune responses. Viral proteins are divided between structural proteins, which form the virion, and non-structural proteins, which are involved in genomic replication and modulating host immunity (Fig. 1a) (Lindenbach et al., 2007;Roby et al., 2012).Flaviviruses are maintained predominantly in natural transmission cycles between vertebrate reservoir species (normally mammalian or avian) and haematophagous arthropod vectors (mosquitoes or ticks). Notable exceptions are the viruses that are maintained solely in mosquito hosts (insectspecific flaviviruses) and viruses with no known invertebrate vector (Cook et al., 2012;Mackenzie et al., 2004). As of 2013, the International Committee on Taxonomy of Viruses has classified 53 different viral species as belonging to ...

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Section: Heterodimerization Of Prm and Esupporting

confidence: 64%

Section: Heterodimerization Of Prm and Ementioning

confidence: 99%

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confidence: 99%

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Post-translational regulation and modifications of flavivirus structural proteins

Roby

Setoh

Hall

et al. 2015

Journal of General Virology

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“…This genetic complementation results in the production of pseudoinfectious virus-like particles capable of only a single round of infection, referred to herein as reporter virus particles (RVPs). RVPs have been used extensively to study flavivirus biology (43)(44)(45)(46)(47)(48) and virus-antibody interactions (35,36,(49)(50)(51) and as tools to screen antiviral compounds (19,52,53). The mechanism of flavivirus RNA packaging is not well understood and appears to be relatively nonselective.…”

Section: Resultsmentioning

confidence: 99%

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

VanBlargan

Davis

Dowd

et al. 2015

J Virol

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The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1= position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1= position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1= position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. West Nile virus (WNV) and the four serotypes of dengue virus (DENV1 to -4) are mosquito-borne viruses of the Flavivirus genus that significantly impact public health (1, 2). Despite a clear need, neither vaccines nor therapeutics for WNV or DENV have been licensed for use in humans. The flavivirus genome is an ϳ11-kb, single-stranded, positive-sense RNA that encodes a single open reading frame flanked by 5= and 3= untranslated regions. The viral genome is translated on endoplasmic reticulum (ER)-derived membranes into a single polyprotein that undergoes co-and posttranslational cleavage by the viral protease NS2B/3 and host proteases into 10 functionally distinct proteins, including the structural proteins capsid (C), premembrane (prM), and envelope (E) that form the virus particle. During assembly, membrane-anchored prM and E glycoproteins are incorporated into virions as they bud into the ER lumen. The C protein associates with the viral genome in the cytoplasm to form an unstructured nucleocapsid that is incorporated into the budding particle via unknown mechanisms (3). The carboxy terminus (C terminus) of the C protein includes a signal sequence, flanked by protease cleavage sites, that directs the translocation of prM into the ER lumen and tethers C to the cytosolic face of the ER membrane.Cleavage at both sites is essential for virion morphogenesis and occurs in a sequenti...

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“…The perimembrane and transmembrane helices serve to anchor the M protein to the membrane. In the case of DV and JEV, the perimembrane helix region was shown to be involved in virus assembly (30)(31)(32)(33) and entry (31,32). Subsequent mutagenesis analysis of the M protein proved the importance of an interactive network between E and M in those processes (16,34,35).…”

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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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Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly, Precursor Membrane (prM) Protein Cleavage, and Entry

Cited by 42 publications

References 65 publications

Post-translational regulation and modifications of flavivirus structural proteins

Post-translational regulation and modifications of flavivirus structural proteins

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

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

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