Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

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Immunogenicity of Recombinant DNA Vaccine Encoding Non-Structural Protein-1 Dengue Virus Serotype-2 in Balb/c Mice

Microbiology Indonesia ◽

10.5454/mi.15.2.4 ◽

2021 ◽

Vol 15 (2) ◽

pp. 4

Author(s):

Elitha Pulungan

Keyword(s):

Immune Response ◽

Dengue Virus ◽

Dna Vaccine ◽

Humoral Immune Response ◽

Structural Proteins ◽

Ns1 Protein ◽

Structural Protein ◽

Humoral Immune ◽

Virus Serotype ◽

Positive Strand Rna

Background: Dengue Hemorrhagic Fever (DHF) is an infectious disease caused by the dengue virus (DENV) which spread widely in tropical and subtropical regions of the world. DENV is a single-positive strand RNA virus with a genome size of ± 11kb which encodes three structural proteins, seven non-structural proteins, and two untranslated regions (UTR). The non-structural protein-1 (NS1) of DENV is known to have important role in dengue pathogenesis also promising to be developed as dengue vaccine. Lately, novel vaccine approach by DNA immunization have given new perspective for a safe, stable, and immunogenic vaccine platform. Previously, we have successfully construct DNA vaccine encoding NS1 protein of DENV2 (pUNS1) which express recombinant NS1 protein in-vitro. Thus, in this current study the ability of pUNS1 to induce humoral immune response will be further analyzed by in mice immunization. Methods: Sixteen BALB/c mice aged of 4 weeks were immunized 3 times with 100 µg of pUNS1 or pUMVC4a on 2 week time interval. Blood sampling was carried out just before immunization and termination was done 2 week after last immunization. Titer from individual mice sera against DENV-2 were measure with in-house ELISA. Results: IgG against NS1 protein of DENV2 titer from mice group immunized with recombinant pUNS1 shown high ELISA absorbancies, 5 times higher than pUMVC4a group. This result suggest the ability of pUNS1 to induce humoral immune response against NS1 DENV-2 in-vivo. Conclusion: Recombinant pUNS1 can induce humoral immune response in mice.

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Dengue Virus Non-Structural Protein 5 as a Versatile, Multi-Functional Effector in Host–Pathogen Interactions

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.574067 ◽

2021 ◽

Vol 11 ◽

Author(s):

Priya Bhatnagar ◽

Gopinathan Pillai Sreekanth ◽

Kaja Murali-Krishna ◽

Anmol Chandele ◽

Ramakrishnan Sitaraman

Keyword(s):

Immune Responses ◽

Dengue Virus ◽

Structural Proteins ◽

Structural Protein ◽

Human Virus ◽

Human Host ◽

Adaptive Immune ◽

Interferon Signalling ◽

Rna Capping ◽

Stat Pathway

Dengue is emerging as one of the most prevalent mosquito-borne viral diseases of humans. The 11kb RNA genome of the dengue virus encodes three structural proteins (envelope, pre-membrane, capsid) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), all of which are translated as a single polyprotein that is subsequently cleaved by viral and host cellular proteases at specific sites. Non-structural protein 5 (NS5) is the largest of the non-structural proteins, functioning as both an RNA-dependent RNA polymerase (RdRp) that replicates the viral RNA and an RNA methyltransferase enzyme (MTase) that protects the viral genome by RNA capping, facilitating polyprotein translation. Within the human host, NS5 interacts with several proteins such as those in the JAK-STAT pathway, thereby interfering with anti-viral interferon signalling. This mini-review presents annotated, consolidated lists of known and potential NS5 interactors in the human host as determined by experimental and computational approaches respectively. The most significant protein interactors and the biological pathways they participate in are also highlighted and their implications discussed, along with the specific serotype of dengue virus as appropriate. This information can potentially stimulate and inform further research efforts towards providing an integrative understanding of the mechanisms by which NS5 manipulates the human-virus interface in general and the innate and adaptive immune responses in particular.

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Structural proteins of West Nile virus are a major determinant of infectious particle production and fitness in astrocytes

Journal of General Virology ◽

10.1099/vir.0.065474-0 ◽

2014 ◽

Vol 95 (9) ◽

pp. 1991-2003 ◽

Cited By ~ 9

Author(s):

Katherine L. Hussmann ◽

Rianna Vandergaast ◽

Kang Zheng ◽

Lisa I. Hoover ◽

Brenda L. Fredericksen

Keyword(s):

West Nile Virus ◽

Protease Activity ◽

Particle Production ◽

Viral Fitness ◽

Major Determinant ◽

Structural Proteins ◽

West Nile ◽

Infectious Particle ◽

Recombinant Viruses ◽

Viral Determinants

The molecular basis for the increased resistance of astrocytes to a non-neuropathogenic strain of West Nile virus (WNV), WNV-MAD78, compared with the neuropathogenic strain WNV-NY remains unclear. Here, we demonstrated that the reduced susceptibility of astrocytes to WNV-MAD78 is due to a combination of both cellular activities as well as viral determinants. Analyses of the viral particle indicated that astrocyte-derived WNV-MAD78 particles were less infectious than those of WNV-NY. Additionally, inhibition of cellular furin-like proteases increased WNV-MAD78 infectious particle production in astrocytes, suggesting that high levels of furin-like protease activity within these cells acted in a cell- and strain-specific manner to inhibit WNV-MAD78 replication. Moreover, analysis of recombinant viruses indicated that the structural proteins of WNV-MAD78 were responsible for decreased particle infectivity and the corresponding reduction in infectious particle production compared with WNV-NY. Thus, the composition of the WNV virion was also a major determinant for viral fitness within astrocytes and may contribute to WNV propagation within the central nervous system. Whether the WNV-MAD78 structural genes reduce virus replication and particle infectivity through the same mechanism as the cellular furin-like protease activity or whether these two determinants function through distinct pathways remains to be determined.

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The dengue virus non-structural protein 1 (NS1) is secreted from mosquito cells in association with the intracellular cholesterol transporter chaperone caveolin complex

10.1101/370932 ◽

2018 ◽

Cited By ~ 1

Author(s):

Romel Rosales Ramirez ◽

Juan E. Ludert

Keyword(s):

Dengue Virus ◽

Brefeldin A ◽

Structural Proteins ◽

Infected Mosquito ◽

Dependent Manner ◽

Structural Protein ◽

Caveolin 1 ◽

Virion Release ◽

Mosquito Cells ◽

Infected Cells

ABSTRACTDengue virus (DENV) is a mosquito-borne virus of the familyFlaviviridae.The RNA viral genome encodes for a polyprotein that is co-translationally processed into three structural proteins and seven non-structural proteins. The non-structural protein 1 (NS1) is a multifunctional viral protein actively secreted in vertebrate and mosquito cells during DENV infection. In mosquito cells, NS1 is secreted in a caveolin-1 (CAV-1) dependent manner by an unconventional pathway. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40 and CyA which is responsible for cholesterol traffic inside the cell. In this work, we demonstrate that in infected mosquito cells, DENV NS1 is secreted by an early and unconventional route that bypasses the Golgi apparatus in close association with the CCC. Treatment of mosquito cells with classic secretion inhibitors such as brefeldin A, golgicide A and Fli-06 showed no effect on NS1 secretion, but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV1, FKBP52 with siRNAs or the inhibition of CyA by cyclosporine A resulted in significant decrease in NS1 secretion without affecting virion release. Using co-localization, co-inmunoprecipitation and proximity ligation assays, NS1 was found to co-localize and interact with all the protein components of the CCC in mosquito infected cells. In addition, CAV-1 and FKBP52 expression was found augmented in DENV infected cells. Finally, the treatment of ZIKV infected mosquito cells with brefeldin A and golgicide A showed no effect on NS1 secretion, while affecting virion release. ZIKV NS1 was also found to co-localize with CAV-1 in infected mosquito cells. These results suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway observed for DENV NS1. The association of NS1 with the cholesterol transporter CCC agrees with the lipoprotein nature of secreted hexameric NS1.AUTHOR SUMMARYDengue protein NS1 is secreted in infected mosquito and vertebrate cells. In humans, secreted NS1 have been associated with pathogenesis. In mosquito cells, NS1 follows an unconventional secretion pathway that is dependent on Caveolin-1. This work shows that in mosquito cells, NS1 secretion is associated to the chaperone caveolin complex, a complex formed by caveolin-1 and several chaperones, in charge of cholesterol transport within the cells. Reduction of the expression or the activity of chaperone caveolin complex in mosquito infected cells, diminished the secretion of NS1 without affecting virion release. Direct interaction between NS1 and the chaperone caveolin complex proteins was demonstrated by several assays. Moreover, increased expression of the caveolin-1 and co-chaperone FKBP52 during dengue infection was found, presumably in response to the higher requirements of these proteins during dengue virus infection. Results obtained with ZIKV infected mosquito cells suggest that also ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. The functions of secreted NS1 within mosquito are unclear. However, giving the importance of the soluble NS1 in the vertebrate host, manipulation of the NS1 secretory route may prove a valuable strategy for dengue mosquito control and patient treatment.

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Structure and Function of the Non-Structural Protein of Dengue Virus and its Applications in Antiviral Therapy

Current Topics in Medicinal Chemistry ◽

10.2174/1568026616666160829155327 ◽

2016 ◽

Vol 17 (3) ◽

pp. 371-380 ◽

Cited By ~ 4

Author(s):

Qian Xie ◽

Bao Zhang ◽

JianHai Yu ◽

Qinghua Wu ◽

Fangji Yang ◽

...

Keyword(s):

Dengue Virus ◽

Antiviral Therapy ◽

Structure And Function ◽

Structural Protein ◽

And Function

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An experimental and theoretical approach to understand Fever, DENF & its cure

Infectious Disorders - Drug Targets ◽

10.2174/1871526520999200905122052 ◽

2020 ◽

Vol 20 ◽

Author(s):

Vijay Kumar Vishvakarma ◽

Ramesh Chandra ◽

Prashant Singh

Keyword(s):

Catalytic Activity ◽

Dengue Virus ◽

Theoretical Approach ◽

Major Part ◽

Genomic Structure ◽

Denv Infection ◽

Structural Proteins ◽

Experimental Approaches ◽

Ns3 Protease ◽

Prime Target

: Fever is a response of human body due to an increase the temperature against the certain stimuli. It may be associated with several reasons and one of the major causes of fever is mosquito bite. Fever due to dengue virus (DENV) infection is being paid most attention out of several other fevers because of a large number of deaths reported worldwide. Dengue virus is transmitted by biting of the mosquitoes, Aedes aegypti and Aedes albopictus. DENV1, DENV2, DENV3 and DENV4 are the four serotypes of dengue virus and these serotypes have 65% similarities in their genomic structure. Genome of DENV is composed of single stranded RNA and it encodes for the polyprotein. Structural and non-structural proteins (nsP) are the two major part of protese. Researchers have paid high attention on the non-structural protease (nsP) of DENV like nsP1, nsP2A, nsP2B, nsP3, nsP4A, nsP4B and nsP5. The NS2B-NS3 protease of DENV is the prime target of the researchers as it is responsible for the catalytic activity. In the present time, Dengvaxia (vaccine) is being recommended to the patients suffering severely due to DENV infection in few countries only. Till date, neither a vaccine nor an effective medicine is available to combat with all four serotypes. This review describes the fever, its causes and studies to cure the infection due to DENV using theoretical and experimental approaches.

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Nuclear localization of non-structural protein 3 (NS3) during dengue virus infection

Archives of Virology ◽

10.1007/s00705-021-05026-w ◽

2021 ◽

Vol 166 (5) ◽

pp. 1439-1446 ◽

Cited By ~ 1

Author(s):

Selvin Noé Palacios-Rápalo ◽

Luis Adrián De Jesús-González ◽

José Manuel Reyes-Ruiz ◽

Juan Fidel Osuna-Ramos ◽

Carlos Noe Farfan-Morales ◽

...

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

Nuclear Localization ◽

Structural Protein ◽

Dengue Virus Infection

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Hepatitis E Virus RNA‐Dependent RNA Polymerase is Involved in RNA Replication and Infectious Particle Production

Hepatology ◽

10.1002/hep.32100 ◽

2021 ◽

Author(s):

Noémie Oechslin ◽

Nathalie Da Silva ◽

Dagmara Szkolnicka ◽

François‐Xavier Cantrelle ◽

Xavier Hanoulle ◽

...

Keyword(s):

Rna Polymerase ◽

Hepatitis E Virus ◽

Particle Production ◽

Rna Replication ◽

Hepatitis E ◽

Rna Dependent Rna Polymerase ◽

Infectious Particle ◽

Virus Rna

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Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

Vaccines ◽

10.3390/vaccines9070726 ◽

2021 ◽

Vol 9 (7) ◽

pp. 726

Author(s):

Nikole L. Warner ◽

Kathryn M. Frietze

Keyword(s):

Dengue Virus ◽

Population Based ◽

Ns1 Protein ◽

Structural Protein ◽

Denv Serotypes ◽

Conserved Regions ◽

Virus Like Particle ◽

Dependent Cell ◽

Infected Cells ◽

Plasma Leakage

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

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