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 Impact of Capsid Anchor Length and Sequential Processing on the Assembly and Infectivity of Dengue Virus

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 32 ◽  
Author(s):  
Oscar R. Burrone ◽  
José L. Slon Campos ◽  
Monica Poggianella ◽  
Jyoti Rana
Keyword(s):  
Dengue Virus ◽  
Signal Peptidase ◽  
Sequence Length ◽  
Processing Efficiency ◽  
Particle Assembly ◽  
Luminal Side ◽  
Cytosolic Side ◽  
Experimental Approaches ◽  
Viral Polyprotein ◽  
Ns3 Protease

: The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavage events mediated by both viral and host proteases. Among these, sequential cleavage at the N- and C-termini of the hydrophobic capsid anchor (Ca) at the junction of C-PrM has been considered essential for the production of flaviviruses. Here, using a refined dengue pseudovirus production system, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and the assembly of viral particles. The replacement of the relatively short DENV2 Ca with the homologous regions from West Nile or Zika viruses or, alternatively, the increase in its length, improved cleavage, and hence particle assembly. Furthermore, we show that the substitution of the Ca conserved proline residue (Pro-110), as alanine abolishes pseudovirus production, regardless of the Ca sequence length. Using two experimental approaches, we investigated the need for sequential cleavage (first on the cytosolic side, then on the luminal side) and found that, while cleavage at the Ca-Pr boundary is essential for the assembly of infective particles, the same is not true for cleavage at the C-Ca boundary. We show that both the mature (C) and unprocessed capsids (C-Ca) of DENV2 were equally efficient in packaging the viral RNA and in assembling the infective particles. This was further confirmed with mutants, in which cleavage at the luminal side, by the signal peptidase, occurred independently of cleavage at the cytosolic side, by the viral NS2B/NS3 protease. We thus demonstrate that, unlike other flaviviruses, DENV2 capsid does not require a cleavable Ca sequence and that sequential cleavage is not an obligatory requirement for the morphogenesis of infective particles.

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 Predicting RNA secondary structure via adaptive deep recurrent neural networks with energy-based filter

2019 ◽  
Vol 20 (S25) ◽  
Author(s):  
Weizhong Lu ◽  
Ye Tang ◽  
Hongjie Wu ◽  
Hongmei Huang ◽  
Qiming Fu ◽  
...  
Keyword(s):  
Neural Networks ◽  
Secondary Structure ◽  
Structure Prediction ◽  
Rna Secondary Structure ◽  
Predictive Accuracy ◽  
Secondary Structure Prediction ◽  
Learning Model ◽  
Fixed Number ◽  
Sequence Length ◽  
Biological Sequence

Abstract Background RNA secondary structure prediction is an important issue in structural bioinformatics, and RNA pseudoknotted secondary structure prediction represents an NP-hard problem. Recently, many different machine-learning methods, Markov models, and neural networks have been employed for this problem, with encouraging results regarding their predictive accuracy; however, their performances are usually limited by the requirements of the learning model and over-fitting, which requires use of a fixed number of training features. Because most natural biological sequences have variable lengths, the sequences have to be truncated before the features are employed by the learning model, which not only leads to the loss of information but also destroys biological-sequence integrity. Results To address this problem, we propose an adaptive sequence length based on deep-learning model and integrate an energy-based filter to remove the over-fitting base pairs. Conclusions Comparative experiments conducted on an authoritative dataset RNA STRAND (RNA secondary STRucture and statistical Analysis Database) revealed a 12% higher accuracy relative to three currently used methods.

scholarly journals Conserved Secondary Structures in Viral mRNAs

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 401 ◽  
Author(s):  
Kiening ◽  
Ochsenreiter ◽  
Hellinger ◽  
Rattei ◽  
Hofacker ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Sequence Length ◽  
Viral Mrnas ◽  
Protein Coding ◽  
Web Resource ◽  
Coding Regions ◽  
Regulatory Processes

RNA secondary structure in untranslated and protein coding regions has been shown to play an important role in regulatory processes and the viral replication cycle. While structures in non-coding regions have been investigated extensively, a thorough overview of the structural repertoire of protein coding mRNAs, especially for viruses, is lacking. Secondary structure prediction of large molecules, such as long mRNAs remains a challenging task, as the contingent of structures a sequence can theoretically fold into grows exponentially with sequence length. We applied a structure prediction pipeline to Viral Orthologous Groups that first identifies the local boundaries of potentially structured regions and subsequently predicts their functional importance. Using this procedure, the orthologous groups were split into structurally homogenous subgroups, which we call subVOGs. This is the first compilation of potentially functional conserved RNA structures in viral coding regions, covering the complete RefSeq viral database. We were able to recover structural elements from previous studies and discovered a variety of novel structured regions. The subVOGs are available through our web resource RNASIV (RNA structure in viruses; http://rnasiv.bio.wzw.tum.de).

scholarly journals Caveats to deep learning approaches to RNA secondary structure prediction

2021 ◽  
Author(s):  
Christoph Flamm ◽  
Julia Wielach ◽  
Michael T. Wolfinger ◽  
Stefan Badelt ◽  
Ronny Lorenz ◽  
...  
Keyword(s):  
Machine Learning ◽  
Deep Learning ◽  
Secondary Structure ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Secondary Structures ◽  
Training Data ◽  
Sequence Length ◽  
Learning Approaches ◽  
Rna Secondary Structures

Machine learning (ML) and in particular deep learning techniques have gained popularity for predicting structures from biopolymer sequences. An interesting case is the prediction of RNA secondary structures, where well established biophysics based methods exist. These methods even yield exact solutions under certain simplifying assumptions. Nevertheless, the accuracy of these classical methods is limited and has seen little improvement over the last decade. This makes it an attractive target for machine learning and consequently several deep learning models have been proposed in recent years. In this contribution we discuss limitations of current approaches, in particular due to biases in the training data. Furthermore, we propose to study capabilities and limitations of ML models by first applying them on synthetic data that can not only be generated in arbitrary amounts, but are also guaranteed to be free of biases. We apply this idea by testing several ML models of varying complexity. Finally, we show that the best models are capable of capturing many, but not all, properties of RNA secondary structures. Most severely, the number of predicted base pairs scales quadratically with sequence length, even though a secondary structure can only accommodate a linear number of pairs.

RNALL: AN EFFICIENT ALGORITHM FOR PREDICTING RNA LOCAL SECONDARY STRUCTURAL LANDSCAPE IN GENOMES

2006 ◽  
Vol 04 (05) ◽  
pp. 1015-1031 ◽  
Author(s):  
XIU-FENG WAN ◽  
GUOHUI LIN ◽  
DONG XU
Keyword(s):  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Energy Landscape ◽  
Query Sequence ◽  
Window Size ◽  
Structural Motif ◽  
Sequence Length ◽  
Worst Case ◽  
Running Time ◽  
Prediction Tools

Background: The information of RNA local secondary structures (LSSs) can help retrieve biologically important motifs and study functions of RNA molecules. Most of the current RNA secondary structure prediction tools are not suitable for RNA LSS prediction on the genome scale due to high computational complexity. Methods: We developed a new computer package Rnall based on a dynamic programming technique, which scans an RNA sequence with a sliding window and extracts all RNA LSSs with sizes no larger than the window size using the nearest neighbor thermodynamic parameters. The worst case running time of Rnall is O(W3L), where W is the window size and L is the query sequence length. In practice we observed a running time of O(W2L). We further introduced the concept of energy landscape for illustrating RNA LSS, which may facilitate RNA motif mining on the genomic scale. Results: Rnall shows better prediction accuracy than two other prediction tools Lfold and Quickfold. Rnall is also applied to scan for RNA LSSs in three genomes, and the prediction maps well with known RNA motifs. Conclusions: Rnall is designed for RNA LSS prediction and together with the energy landscape, it has unique features that could be used for RNA structural motif mining. Rnall is freely available for download at or .

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.

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