scholarly journals Membrane Anchors of the Structural Flavivirus Proteins and Their Role in Virus Assembly

2016 ◽  
Vol 90 (14) ◽  
pp. 6365-6378 ◽  
Author(s):  
Janja Blazevic ◽  
Harald Rouha ◽  
Victoria Bradt ◽  
Franz X. Heinz ◽  
Karin Stiasny
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Envelope Protein ◽  
Transmembrane Domains ◽  
Encephalitis Virus ◽  
Acidic Ph ◽  
Particle Assembly ◽  
Virus Maturation ◽  
Polyprotein Processing ◽  
Viral Polyprotein

ABSTRACTThe structural proteins of flaviviruses carry a unique set of transmembrane domains (TMDs) at their C termini that are derived from the mode of viral polyprotein processing. They function as internal signal and stop-transfer sequences during protein translation, but possible additional roles in protein interactions required during assembly and maturation of viral particles are ill defined. To shed light on the role of TMDs in these processes, we engineered a set of tick-borne encephalitis virus mutants in which these structural elements were replaced in different combinations by the homologous sequences of a distantly related flavivirus (Japanese encephalitis virus). The effects of these modifications were analyzed with respect to protein synthesis, viral particle secretion, specific infectivity, and acidic-pH-induced maturation processes. We provide evidence that interactions involving the double-membrane anchor of the envelope protein E (a unique feature compared to other viral fusion proteins) contribute substantially to particle assembly, stability, and maturation. Disturbances of the inter- and intra-TMD interactions of E resulted in the secretion of a larger proportion of capsidless subviral particles at the expense of whole virions, suggesting a possible role in the still incompletely understood mechanism of capsid integration during virus budding. In contrast, the TMD initially anchoring the C protein to the endoplasmic reticulum membrane does not appear to take part in envelope protein interactions. We also show that E TMDs are involved in the envelope protein rearrangements that are triggered by acidic pH in thetrans-Golgi network and represent a hallmark of virus maturation.IMPORTANCEThe assembly of flaviviruses occurs in the endoplasmic reticulum and leads to the formation of immature, noninfectious particles composed of an RNA-containing capsid surrounded by a lipid membrane, with the two integrated envelope proteins, prM and E, arranged in an icosahedral lattice. The mechanism by which the capsid is formed and integrated into the budding viral envelope is currently unknown. We provide evidence that the transmembrane domains (TMDs) of E are essential for the formation of capsid-containing particles and that disturbances of these interactions lead to the preferential formation of capsidless subviral particles at the expense of whole virions. E TMD interactions also appear to be essential for the envelope protein rearrangements required for virus maturation and for the generation of infectious virions. Our data thus provide new insights into the biological functions of E TMDs and extend their role during viral polyprotein processing to additional functions in particle assembly and maturation.

scholarly journals Dengue virus capsid anchor modulates the efficiency of polyprotein processing and assembly of viral particles

2019 ◽  
Vol 100 (12) ◽  
pp. 1663-1673 ◽  
Author(s):  
Jyoti Rana ◽  
José Luis Slon Campos ◽  
Monica Poggianella ◽  
Oscar R. Burrone
Keyword(s):  
Dengue Virus ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Sequence Length ◽  
Efficient Production ◽  
Processing Efficiency ◽  
Particle Assembly ◽  
Polyprotein Processing ◽  
Viral Polyprotein ◽  
Viral Particle Assembly

The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavages mediated by both viral and host proteases. Among these, sequential cleavage at the N and C termini of the hydrophobic capsid anchor (Ca) is crucial in deciding the fate of viral infection. Here, using a refined dengue pseudovirus production system, along with cleavage and furin inhibition assays, immunoblotting and secondary structure prediction analysis, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and viral particle assembly. Replacement of the DENV2 Ca with the homologous regions from West nile or Zika viruses or, alternatively, increasing its length, improved cleavage and hence particle assembly. Further, we showed that substitution of the Ca conserved proline residue (P110) to alanine abolishes pseudovirus production, regardless of the Ca sequence length. Besides providing the results of a biochemical analysis of DENV2 structural polyprotein processing, this study also presents a system for efficient production of dengue pseudoviruses.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

scholarly journals Proteomic Discovery of VEEV E2-Host Partner Interactions Identifies GRP78 Inhibitor HA15 as a Potential Therapeutic for Alphavirus Infections

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 283
Author(s):  
Michael D. Barrera ◽  
Victoria Callahan ◽  
Ivan Akhrymuk ◽  
Nishank Bhalla ◽  
Weidong Zhou ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Trafficking ◽  
Viral Protein ◽  
Sindbis Virus ◽  
Encephalitis Virus ◽  
Host Proteins ◽  
Atp Production ◽  
Equine Encephalitis Virus ◽  
E2 Glycoprotein ◽  
Late Stages

Alphaviruses are a genus of the Togaviridae family and are widely distributed across the globe. Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), cause encephalitis and neurological sequelae while chikungunya virus (CHIKV) and Sindbis virus (SINV) cause arthralgia. There are currently no approved therapeutics or vaccines available for alphaviruses. In order to identify novel therapeutics, a V5 epitope tag was inserted into the N-terminus of the VEEV E2 glycoprotein and used to identify host-viral protein interactions. Host proteins involved in protein folding, metabolism/ATP production, translation, cytoskeleton, complement, vesicle transport and ubiquitination were identified as VEEV E2 interactors. Multiple inhibitors targeting these host proteins were tested to determine their effect on VEEV replication. The compound HA15, a GRP78 inhibitor, was found to be an effective inhibitor of VEEV, EEEV, CHIKV, and SINV. VEEV E2 interaction with GRP78 was confirmed through coimmunoprecipitation and colocalization experiments. Mechanism of action studies found that HA15 does not affect viral RNA replication but instead affects late stages of the viral life cycle, which is consistent with GRP78 promoting viral assembly or viral protein trafficking.

scholarly journals Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

2003 ◽  
Vol 77 (9) ◽  
pp. 5401-5414 ◽  
Author(s):  
Maria Dimitrova ◽  
Isabelle Imbert ◽  
Marie Paule Kieny ◽  
Catherine Schuster
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

scholarly journals Engineered pH-Sensitive Protein G / IgG Interaction

2020 ◽  
Author(s):  
Ramesh K. Jha ◽  
Allison Yankey ◽  
Kalifa Shabazz ◽  
Leslie Naranjo ◽  
Nileena Velappan ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Protein Design ◽  
Protein Interactions ◽  
Rational Design ◽  
Protein G ◽  
Acidic Ph ◽  
Protein Protein Interactions ◽  
Natural Interface ◽  
Complex Stimuli ◽  
Igg Purification

ABSTRACTWhile natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G-IgG Fc domain, when incorporated with histidine and glutamic acid on Protein G (PrG-EHHE), showed a switch in binding affinity by 50-fold when pH was altered from mild acidic to mild basic. The wild type (WT) interface only showed negligible switch. The overall binding affinity at mild acidic pH for PrG-EHHE outperformed the WT PrG interaction. The new reagent PrG-EHHE will be revolutionary in IgG purification since the traditional method of using an extreme acidic pH for elution can be circumvented.Abstract Figure

The pre membrane and envelope protein is the crucial virulence determinant of Japanese encephalitis virus

2020 ◽  
Vol 148 ◽  
pp. 104492
Author(s):  
Sheng-ling Leng ◽  
Rong Huang ◽  
Ya-nan Feng ◽  
Li-juan Peng ◽  
Jian Yang ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Envelope Protein ◽  
Encephalitis Virus ◽  
Virulence Determinant

scholarly journals Endoplasmic Reticulum Detergent-Resistant Membranes Accommodate Hepatitis C Virus Proteins for Viral Assembly

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 487 ◽  
Author(s):  
Audrey Boyer ◽  
Julie Dreneau ◽  
Amélie Dumans ◽  
Julien Burlaud-Gaillard ◽  
Anne Bull-Maurer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Gradient Centrifugation ◽  
Cellular Membrane ◽  
Structural Protein ◽  
Membrane Structures ◽  
Detergent Resistant Membranes ◽  
Lysis Buffer

During Hepatitis C virus (HCV) morphogenesis, the non-structural protein 2 (NS2) brings the envelope proteins 1 and 2 (E1, E2), NS3, and NS5A together to form a complex at the endoplasmic reticulum (ER) membrane, initiating HCV assembly. The nature of the interactions in this complex is unclear, but replication complex and structural proteins have been shown to be associated with cellular membrane structures called detergent-resistant membranes (DRMs). We investigated the role of DRMs in NS2 complex formation, using a lysis buffer combining Triton and n-octyl glucoside, which solubilized both cell membranes and DRMs. When this lysis buffer was used on HCV-infected cells and the resulting lysates were subjected to flotation gradient centrifugation, all viral proteins and DRM-resident proteins were found in soluble protein fractions. Immunoprecipitation assays demonstrated direct protein–protein interactions between NS2 and E2 and E1 proteins, and an association of NS2 with NS3 through DRMs. The well-folded E1E2 complex and NS5A were not associated, instead interacting separately with the NS2-E1-E2-NS3 complex through less stable DRMs. Core was also associated with NS2 and the E1E2 complex through these unstable DRMs. We suggest that DRMs carrying this NS2-E1-E2-NS3-4A-NS5A-core complex may play a central role in HCV assembly initiation, potentially as an assembly platform.

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