scholarly journals Replication is the key barrier during the dual-host adaption of mosquito-borne flaviviruses

2021 ◽  
Author(s):  
Aihua Zheng ◽  
Xiangxi Wang ◽  
Yanan Zhang ◽  
Dening Liang ◽  
Fei Yuan ◽  
...  
Keyword(s):  
Zika Virus ◽  
Reverse Genetics ◽  
Transmission Mechanism ◽  
Host Factors ◽  
Reporter Virus ◽  
Rna Structures ◽  
Ideal Model ◽  
Host Interaction ◽  
Post Entry ◽  
Key Barrier

Mosquito-borne flaviviruses (MBFs) adapt to a dual-host transmission circle between mosquitoes and vertebrates. Dual-host affiliated insect-specific flaviviruses (dISFs), discovered from mosquitoes, are phylogenetically similar to MBFs but do not infect vertebrates. Thus, dISF-MBF chimeras could be an ideal model to study the dual-host adaption of MBFs. Using the pseudo-infectious reporter virus particle and reverse genetics systems, we found dISFs entered vertebrate cells as efficiently as the MBFs, but failed to initiate replication. Exchange of the un-translational regions (UTRs) of Donggang virus (DONV), an dISF, with those from Zika virus (ZIKV) rescued DONV replication in vertebrate cells and critical secondary RNA structures were further mapped. Essential UTR-binding host factors were screened for ZIKV replication in vertebrate cells, displaying different binding patterns. Therefore, our data demonstrate a post-entry cross-species transmission mechanism of MBFs, while UTR-host interaction is critical for dual-host adaption.

scholarly journals A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 368 ◽  
Author(s):  
Maximilian Münster ◽  
Anna Płaszczyca ◽  
Mirko Cortese ◽  
Christopher Neufeldt ◽  
Sarah Goellner ◽  
...  
Keyword(s):  
Zika Virus ◽  
In Silico ◽  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Cdna Clones ◽  
In Silico Prediction ◽  
Fluorescent Reporter ◽  
Full Length Cdna ◽  
Spurious Transcription

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

scholarly journals TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 519
Author(s):  
Yuchen Wang ◽  
Ryuta Uraki ◽  
Jesse Hwang ◽  
Erol Fikrig
Keyword(s):  
Mass Spectrometry ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Host Factors ◽  
Ns1 Protein ◽  
Zikv Infection ◽  
Binding Partner ◽  
Promising Therapeutic Target ◽  
Critical Components ◽  
Congenital Microcephaly

Mosquito-borne Zika virus (ZIKV) can cause congenital microcephaly and Guillain–Barré syndrome, among other symptoms. Specific treatments and vaccines for ZIKV are not currently available. To further understand the host factors that support ZIKV replication, we used mass spectrometry to characterize mammalian proteins that associate with the ZIKV NS1 protein and identified the TRiC/CCT complex as an interacting partner. Furthermore, the suppression of CCT2, one of the critical components of the TRiC/CCT complex, inhibited ZIKV replication in both mammalian cells and mosquitoes. These results highlight an important role for the TRiC/CCT complex in ZIKV infection, suggesting that the TRiC/CCT complex may be a promising therapeutic target.

scholarly journals A Genome-Wide CRISPR Activation Screen Identifies Genes Involved in Protection from Zika Virus Infection

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 149
Author(s):  
Anna Dukhovny ◽  
Kevin Lamkiewicz ◽  
Qian Chen ◽  
Markus Fricke ◽  
Nabila Jabrane-Ferrat ◽  
...  
Keyword(s):  
Cell Death ◽  
Zika Virus ◽  
Early Stage ◽  
Specific Treatment ◽  
Febrile Illness ◽  
Neurological Complications ◽  
Host Factors ◽  
Zikv Infection ◽  
A Genome ◽  
Crispr Activation

Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated with the Guillain–Barré syndrome and other neurological complications. The vertical transmission of ZIKV can cause fetus demise, stillbirths or severe congenital abnormalities and neurological complications. There is still no vaccine or specific treatment for ZIKV infection. To identify the host factors that can rescue cells from ZIKV infection, we used a genome-scale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-lamda2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV infection. The activation of these genes had an effect at an early stage in the viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. These results highlighted a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death, thus substantiating CRISPR activation screens as a valid tool for identifying host factors impeding pathogen infection.

scholarly journals Sulfated Glycans and Related Digestive Enzymes in the Zika Virus Infectivity: Potential Mechanisms of Virus-Host Interaction and Perspectives in Drug Discovery

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Vitor H. Pomin
Keyword(s):  
Neural Development ◽  
Zika Virus ◽  
Molecular Mechanisms ◽  
Surface Proteins ◽  
Host Cells ◽  
Future Research ◽  
Virus Infectivity ◽  
Virus Attachment ◽  
Chemical Structures ◽  
Host Interaction

As broadly reported, there is an ongoing Zika virus (ZIKV) outbreak in countries of Latin America. Recent findings have demonstrated that ZIKV causes severe defects on the neural development in fetuses in utero and newborns. Very little is known about the molecular mechanisms involved in the ZIKV infectivity. Potential therapeutic agents are also under investigation. In this report, the possible mechanisms of action played by glycosaminoglycans (GAGs) displayed at the surface proteoglycans of host cells, and likely in charge of interactions with surface proteins of the ZIKV, are highlighted. As is common for the most viruses, these sulfated glycans serve as receptors for virus attachment onto the host cells and consequential entry during infection. The applications of (1) exogenous sulfated glycans of different origins and chemical structures capable of competing with the virus attachment receptors (supposedly GAGs) and (2) GAG-degrading enzymes able to digest the virus attachment receptors on the cells may be therapeutically beneficial as anti-ZIKV. This communication attempts, therefore, to offer some guidance for the future research programs aimed to unveil the molecular mechanisms underlying the ZIKV infectivity and to develop therapeutics capable of decreasing the devastating consequences caused by ZIKV outbreak in the Americas.

scholarly journals Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication

2010 ◽  
Vol 84 (13) ◽  
pp. 6720-6732 ◽  
Author(s):  
Ileana M. Cristea ◽  
Heather Rozjabek ◽  
Kelly R. Molloy ◽  
Sophiya Karki ◽  
Laura L. White ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Sindbis Virus ◽  
Specific Treatment ◽  
Host Factors ◽  
Host Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Replication Process ◽  
Flag Epitope ◽  
Factors Associated ◽  
Host Interaction

ABSTRACT Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3×Flag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.

scholarly journals Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

2018 ◽  
Author(s):  
Michael F. Wells ◽  
Max R. Salick ◽  
Federica Piccioni ◽  
Ellen J. Hill ◽  
Jana M. Mitchell ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Zika Virus ◽  
Large Scale ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Autism Spectrum ◽  
Host Factors ◽  
Protein Markers ◽  
Genome Wide

SUMMARYNeural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe humanStem cell-derivedNGN2-acceleratedProgenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum genePTEN.

scholarly journals Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1991
Author(s):  
Puxian Fang ◽  
Huichang Zhang ◽  
He Sun ◽  
Gang Wang ◽  
Sijin Xia ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput Screening ◽  
Reverse Genetics ◽  
Infectious Clone ◽  
Artificial Chromosome ◽  
Antiviral Drugs ◽  
Reporter Virus ◽  
Suckling Piglets ◽  
One Step

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

scholarly journals Secretory Carrier Membrane Protein 3 Interacts with 3A Viral Protein of Enterovirus and Participates in Viral Replication

2021 ◽  
Author(s):  
Jia-Ying Lu ◽  
Gary Brewer ◽  
Mei-Ling Li ◽  
Kai-Zhe Lin ◽  
Chien-Chih Huang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Viral Replication ◽  
Viral Protein ◽  
Host Factors ◽  
Replication Complex ◽  
Host Interaction ◽  
Enterovirus A71

Virus-host interaction plays an important role in viral replication. 3A protein of enterovirus A71 (EV-A71) recruits other viral and host factors to form a replication complex, which is important for viral replication. In this investigation, we utilized immunoprecipitation combined with proteomics approaches to identify 3A-interacting factors.

scholarly journals Leu-to-Phe substitution at prM146 decreases the growth ability of Zika virus and partially reduces its pathogenicity in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takuya Inagaki ◽  
Satoshi Taniguchi ◽  
Yasuhiro Kawai ◽  
Takahiro Maeki ◽  
Eri Nakayama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Reverse Genetics ◽  
Cultured Cells ◽  
Congenital Infection ◽  
Febrile Illness ◽  
Vero Cells ◽  
Lower Growth ◽  
Asian Strain ◽  
Growth Ability

AbstractZika virus (ZIKV) is a mosquito-borne flavivirus that causes febrile illness. The recent spread of ZIKV from Asia to the Americas via the Pacific region has revealed unprecedented features of ZIKV, including transplacental congenital infection causing microcephaly. Amino acid changes have been hypothesized to underlie the spread and novel features of American ZIKV strains; however, the relationship between genetic changes and the epidemic remains controversial. A comparison of the characteristics of a Southeast Asian strain (NIID123) and an American strain (PRVABC59) revealed that the latter had a higher replication ability in cultured cells and higher virulence in mice. In this study, we aimed to identify the genetic region of ZIKV responsible for these different characteristics using reverse genetics. A chimeric NIID123 strain in which the E protein was replaced with that of PRVABC59 showed a lower growth ability than the recombinant wild-type strain. Adaptation of the chimeric NIID123 to Vero cells induced a Phe-to-Leu amino acid substitution at position 146 of the prM protein; PRVABC59 also has Leu at this position. Leu at this position was found to be responsible for the viral replication ability and partially, for the pathogenicity in mouse testes.

scholarly journals A high throughput reporter virus particle microneutralization assay for quantitation of Zika virus neutralizing antibodies in multiple species

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250516
Author(s):  
Kelly Bohning ◽  
Stephanie Sonnberg ◽  
Hui-Ling Chen ◽  
Melissa Zahralban-Steele ◽  
Timothy Powell ◽  
...  
Keyword(s):  
Virus Particle ◽  
High Throughput ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Surface Proteins ◽  
Mouse Serum ◽  
Reporter Virus ◽  
Serum Samples ◽  
Economic Sectors ◽  
Virus Surface

Zika virus is a Flavivirus, transmitted via Aedes mosquitos, that causes a range of symptoms including Zika congenital syndrome. Zika has posed a challenging situation for health, public and economic sectors of affected countries. To quantitate Zika virus neutralizing antibody titers in serum samples, we developed a high throughput plate based Zika virus reporter virus particle (RVP) assay that uses an infective, non-replicating particle encoding Zika virus surface proteins and capsid (CprME) and a reporter gene (Renilla luciferase). This is the first characterization of a Zika virus RVP assay in 384-well format using a Dengue replicon Renilla reporter construct. Serially diluted test sera were incubated with RVPs, followed by incubation with Vero cells. RVPs that have not been neutralized by antibodies in the test sera entered the cells and expressed Renilla luciferase. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. The principle of limiting the infection to a single round increases the precision of the assay measurements. RVP log10EC50 titers correlated closely with titers determined using a plaque reduction neutralization test (PRNT) (R2>95%). The plate-based Zika virus RVP assay also demonstrated high levels of precision, reproducibility and throughput. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. Spiking studies indicated that the assay performs equally well in different species, producing comparable titers irrespective of the serum species. The assay is conducted in 384-well plates and can be automated to simultaneously achieve high throughput and high reproducibility.

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