scholarly journals A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production

2016 ◽  
Vol 90 (11) ◽  
pp. 5451-5461 ◽  
Author(s):  
Leopoldo G. Gebhard ◽  
Néstor G. Iglesias ◽  
Laura A. Byk ◽  
Claudia V. Filomatori ◽  
Federico A. De Maio ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Particle ◽  
Rna Replication ◽  
Particle Formation ◽  
Human Pathogen ◽  
Particle Assembly ◽  
Infectious Particle ◽  
Viral Particles ◽  
Reporter Systems ◽  
Viral Particle Assembly

ABSTRACTDengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation.IMPORTANCEDengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for dengue virus particle assembly. NS3 is a multifunctional enzyme that participates in different steps of the viral life cycle. Using reporter systems to dissect different viral processes, we identified a novel N-terminal unstructured region of the NS3 protein as crucial for production of viral particles. Based on our findings, we propose new ideas that include NS3 as a possible scaffold for the viral assembly process.

scholarly journals Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles

2015 ◽  
Vol 89 (24) ◽  
pp. 12441-12456 ◽  
Author(s):  
Juan Wan ◽  
Kaustuv Basu ◽  
Jeannie Mui ◽  
Hojatollah Vali ◽  
Huanquan Zheng ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Viral Particle ◽  
Rna Replication ◽  
Turnip Mosaic Virus ◽  
Infection Process ◽  
Viral Rna ◽  
Particle Assembly ◽  
Induced Membrane ◽  
Positive Strand Rna ◽  
Viral Particle Assembly

ABSTRACTPositive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.IMPORTANCEPositive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.

scholarly journals The GLN Family of Murine Endogenous Retroviruses Contains an Element Competent for Infectious Viral Particle Formation

2008 ◽  
Vol 82 (9) ◽  
pp. 4413-4419 ◽  
Author(s):  
David Ribet ◽  
Francis Harper ◽  
Cécile Esnault ◽  
Gérard Pierron ◽  
Thierry Heidmann
Keyword(s):  
Viral Particle ◽  
Leukemia Virus ◽  
Endogenous Retroviruses ◽  
Particle Assembly ◽  
New Class ◽  
Mouse Mammary Tumor ◽  
The Family ◽  
Tumor Virus ◽  
Mouse Cells ◽  
Viral Particle Assembly

ABSTRACT Several families of endogenous retroviruses (ERVs) have been identified in the mouse genome, in several instances by in silico searches, but for many of them it remains to be determined whether there are elements that can still encode functional retroviral particles. Here, we identify, within the GLN family of highly reiterated ERVs, one, and only one, copy that encodes retroviral particles prone to infection of mouse cells. We show that its envelope protein confers an ecotropic host range and recognizes a receptor different from mCAT1 and mSMIT1, the two previously identified receptors for other ecotropic mouse retroviruses. Electron microscopy disclosed viral particle assembly and budding at the cell membrane, as well as release of mature particles into the extracellular space. These particles are closely related to murine leukemia virus (MLV) particles, with which they have most probably been confused in the past. This study, therefore, identifies a new class of infectious mouse ERVs belonging to the family Gammaretroviridae, with one family member still functional today. This family is in addition to the two MLV and mouse mammary tumor virus families of active mouse ERVs with an extracellular life cycle.

scholarly journals Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

2009 ◽  
Vol 5 (10) ◽  
pp. e1000632 ◽  
Author(s):  
Marcelo M. Samsa ◽  
Juan A. Mondotte ◽  
Nestor G. Iglesias ◽  
Iranaia Assunção-Miranda ◽  
Giselle Barbosa-Lima ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Capsid Protein ◽  
Lipid Droplets ◽  
Viral Particle ◽  
Particle Formation ◽  
Virus Capsid ◽  
Virus Capsid Protein

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.

scholarly journals Rational pharmacological approach to clinical studies for the treatment of SARS-COV-2 infection

2020 ◽  
Vol 19 (4) ◽  
pp. 42
Author(s):  
Antônia Dailane Dos Santos Rabêlo ◽  
Gizelle Gomes De Souza ◽  
Rosilene Ribeiro De Sousa ◽  
Charllyton Luis Sena Da Costa
Keyword(s):  
Viral Particle ◽  
Scientific Information ◽  
Lessons Learned ◽  
Cell Replication ◽  
Particle Assembly ◽  
Rational Combination ◽  
Different Populations ◽  
Multiple Drugs ◽  
Molecular Components ◽  
Viral Particle Assembly

AbstractThe global emergency generated by the COVID-19 pandemic caused by the SARS-COV-2 virus has created serious impacts on the different populations of the planet and has triggered the generation of scientific information on an unprecedented scale until then for a single topic. One of the consequences of the global scientific effort lies in the large number of substances already tested, by different methods, the search for an effective treatment for the infection and the consequent disease, remaining without absolute success so far. Assimilating the lessons, learned from the successful adoption of therapies combining multiple drugs used in HIV infection, the evidence obtained from the large amount of published information regarding the action of many substances with different mechanisms, now allows the proposition, in this work, of tests clinical trials for the evaluation of regimens composed of at least three drugs in combination. Rational combination schemes can target different molecular components of the virus affecting different points in the SARS-COV-2 replication cycle, such as virus fusion to the host cell, replication and viral particle assembly generating a potentially more effective synergistic effect than attempts using a single substance.Keywords: antiviral, pandemic, combination therapy.

scholarly journals Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Kotaro Ishida ◽  
Simon Goto ◽  
Marina Ishimura ◽  
Misato Amanuma ◽  
Yumiko Hara ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Virus Particle ◽  
Capsid Protein ◽  
Viral Particle ◽  
Particle Formation ◽  
Molecular Surface ◽  
Functional Correlation ◽  
Viral Particles ◽  
Detergent Treatment ◽  
Virus Capsid Protein

ABSTRACT The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles. IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

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