scholarly journals Zika virus employs the host antiviral RNase L protein to support replication factory assembly

2021 ◽  
Vol 118 (22) ◽  
pp. e2101713118
Author(s):  
Jillian N. Whelan ◽  
Nicholas A. Parenti ◽  
Joshua Hatterschide ◽  
David M. Renner ◽  
Yize Li ◽  
...  
Keyword(s):  
Zika Virus ◽  
Virus Production ◽  
Rnase L ◽  
L Protein ◽  
Microtubule Stabilization ◽  
Replication Factory ◽  
Inactive Form ◽  
Route Of Transmission ◽  
Cytoskeleton Rearrangements ◽  
Ns3 Protease

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

scholarly journals The host antiviral ribonuclease L protein supports Zika virus replication factory formation to enhance infectious virus production

2019 ◽  
Author(s):  
Jillian N Whelan ◽  
Joshua Hatterschide ◽  
David M. Renner ◽  
Beihua Dong ◽  
Robert H Silverman ◽  
...  
Keyword(s):  
Zika Virus ◽  
Virus Production ◽  
Rnase L ◽  
Microtubule Stabilization ◽  
Replication Factory ◽  
Inactive Form ◽  
Antiviral Function ◽  
Cytoskeleton Rearrangements ◽  
Ribonuclease L

SummaryThe flavivirus Zika virus (ZIKV) activates ribonuclease L (RNase L) catalytic antiviral function during infection, yet deletion of RNase L decreases ZIKV production, suggesting a proviral role of RNase L. In this study, we reveal that latent RNase L supports ZIKV replication factory (RF) assembly. Deletion of RNase L induced broader cellular distribution of ZIKV dsRNA and NS3 compared with densely concentrated RFs detected in WT cells. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller area, which increased levels of viral RNA within RFs as well as infectious ZIKV released from the cell. We used a microtubule stabilization drug to demonstrate that RNase L deletion impaired the cytoskeleton rearrangements that are required for proper generation of RFs. During infection with dengue or West Nile Kunjin viruses, RNase L decreased virus production, suggesting that RNase L proviral function is specific to ZIKV.

scholarly journals Zika Virus Production Is Resistant to RNase L Antiviral Activity

2019 ◽  
Vol 93 (16) ◽  
Author(s):  
Jillian N. Whelan ◽  
Yize Li ◽  
Robert H. Silverman ◽  
Susan R. Weiss
Keyword(s):  
Antiviral Activity ◽  
Zika Virus ◽  
Viral Genome ◽  
Molecular Mechanisms ◽  
Virus Production ◽  
Type I ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Zikv Infection ◽  
Initial Report

SUMMARYThere is currently no knowledge of how the emerging human pathogen Zika virus (ZIKV) interacts with the antiviral endoribonuclease L (RNase L) pathway during infection. Since activation of RNase L during infection typically limits virus production dramatically, we used CRISPR-Cas9 gene editing technology to knockout (KO) targeted host genes involved in the RNase L pathway to evaluate the effects of RNase L on ZIKV infection in human A549 cells. RNase L was activated in response to ZIKV infection, which degraded ZIKV genomic RNA. Surprisingly, despite viral genome reduction, RNase L activity did not reduce ZIKV infectious titers. In contrast, both the flavivirus dengue virus and the alphavirus Sindbis virus replicated to significantly higher titers in RNase L KO cells compared to wild-type (WT) cells. Using MAVS/RNase L double KO cells, we demonstrated that the absence of increased ZIKV production in RNase L KO cells was not due to compensation by enhanced type I interferon transcripts to thus inhibit virus production. Finally, when synthetic double-stranded RNA was detected by OAS3 to induce RNase L antiviral activity prior to ZIKV infection, we observed reduced ZIKV replication factory formation, as well as a 42-fold reduction in virus yield in WT but not RNase L KO cells. This study proposes that ZIKV evades RNase L antiviral activity by generating a viral genome reservoir protected from RNase L cleavage during early infection, allowing for sufficient virus production before RNase L activation is detectable.IMPORTANCEWith the onset of the 2015 ZIKV outbreak, ZIKV pathogenesis has been of extreme global public health interest, and a better understanding of interactions with the host would provide insight into molecular mechanisms driving the severe neurological outcomes of ZIKV disease. Here is the initial report on the relationship between ZIKV and the host oligoadenylate synthetase-RNase L (OAS-RNase L) system, a potent antiviral pathway effective at restricting replication of diverse viruses. Our study elucidated a unique mechanism whereby ZIKV production is impervious to antiviral RNase L activity, through a mechanism of viral RNA protection that is not mimicked during infection with numerous other RNase L-activating viruses, thus identifying a distinct replication strategy potentially important for ZIKV pathogenesis.

scholarly journals Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3732
Author(s):  
Agnieszka Dabrowska ◽  
Aleksandra Milewska ◽  
Joanna Ner-Kluza ◽  
Piotr Suder ◽  
Krzysztof Pyrc
Keyword(s):  
Mass Spectrometry ◽  
Zika Virus ◽  
Viral Infections ◽  
Protein Detection ◽  
Extensive Discussion ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Infected Cells ◽  
Ns3 Protease ◽  
To Receive

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

Flavonoids from Pterogyne nitens as Zika virus NS2B-NS3 protease inhibitors

2021 ◽  
Vol 109 ◽  
pp. 104719
Author(s):  
Caroline Sprengel Lima ◽  
Melina Mottin ◽  
Leticia Ribeiro de Assis ◽  
Nathalya Cristina de Moraes Roso Mesquita ◽  
Bruna Katiele de Paula Sousa ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Zika Virus ◽  
Ns3 Protease Inhibitors ◽  
Ns3 Protease

NS2B‐NS3 protease inhibitors as promising compounds in the development of antivirals against Zika virus: A systematic review

2021 ◽  
Author(s):  
Damiana Antônia de Fátima Nunes ◽  
Felipe Rocha da Silva Santos ◽  
Sara Thamires Dias da Fonseca ◽  
William Gustavo de Lima ◽  
Waleska Stephanie da Cruz Nizer ◽  
...  
Keyword(s):  
Systematic Review ◽  
Protease Inhibitors ◽  
Zika Virus ◽  
Ns3 Protease Inhibitors ◽  
Ns3 Protease

scholarly journals Kinetics of Asian and African Zika Virus Lineages over Single-cycle and Multi-cycle Growth in Culture: gene expression, cell killing, virus production, and mathematical modeling

2021 ◽  
Author(s):  
Huicheng Shi ◽  
John Yin
Keyword(s):  
Cell Culture ◽  
Zika Virus ◽  
Virus Production ◽  
Congenital Defects ◽  
Scale Production ◽  
African Lineage ◽  
Intact Virus ◽  
One Step ◽  
Step Growth ◽  
Asian Lineage

Since 2014, an Asian lineage of Zika virus has caused outbreaks, and it has been associated with neurological disorders in adults and congenital defects in newborns. The resulting threat of the Zika virus to human health has prompted the development of new vaccines, which have yet to be approved for human use. Vaccines based on the attenuated or chemically inactivated virus will require large-scale production of the intact virus to meet potential global demands. Intact viruses are produced by infecting cultures of susceptible cells, a dynamic process that spans from hours to days and has yet to be optimized. Here, we infected Vero cells adhesively cultured in well-plates with two Zika virus strains: a recently isolated strain from the Asian lineage, and a cell-culture-adapted strain from the African lineage. At different time points post-infection, virus particles in the supernatant were quantified; further, microscopy images were used to quantify cell density and the proportion of cells expressing viral protein. These measurements were performed across multiple replicate samples of one-step infections every four hours over 60 hours and for multi-step infections every four to 24 hours over 144 hours, generating a rich dataset. For each set of data, mathematical models were developed to estimate parameters associated with cell infection and virus production. The African-lineage strain was found to produce a 14-fold higher yield than the Asian-lineage strain in one-step growth and a 7-fold higher titer in multi-step growth, suggesting a benefit of cell-culture adaptation for developing a vaccine strain. We found that image-based measurements were critical for discriminating among different models, and different parameters for the two strains could account for the experimentally observed differences. An exponential-distributed delay model performed best in accounting for multi-step infection of the Asian strain, and it highlighted the significant sensitivity of virus titer to the rate of viral degradation, with implications for optimization of vaccine production. More broadly, this work highlights how image-based measurements can contribute to discrimination of virus-culture models for the optimal production of inactivated and attenuated whole-virus vaccines.

scholarly journals Drug-Screening Strategies for Inhibition of Virus-Induced Neuronal Cell Death

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2317
Author(s):  
Durbadal Ojha ◽  
Tyson A. Woods ◽  
Karin E. Peterson
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Zika Virus ◽  
Treatment Options ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Virus Production ◽  
Neuronal Stem Cell ◽  
Stem Cell Lines ◽  
Simplex Virus

A number of viruses, including Herpes Simplex Virus (HSV), West Nile Virus (WNV), La Crosse Virus (LACV), Zika virus (ZIKV) and Tick-borne encephalitis virus (TBEV), have the ability to gain access to the central nervous system (CNS) and cause severe neurological disease or death. Although encephalitis cases caused by these viruses are generally rare, there are relatively few treatment options available for patients with viral encephalitis other than palliative care. Many of these viruses directly infect neurons and can cause neuronal death. Thus, there is the need for the identification of useful therapeutic compounds that can inhibit virus replication in neurons or inhibit virus-induced neuronal cell death. In this paper, we describe the methodology to test compounds for their ability to inhibit virus-induced neuronal cell death. These protocols include the isolation and culturing of primary neurons; the culturing of neuroblastoma and neuronal stem cell lines; infection of these cells with viruses; treatment of these cells with selected drugs; measuring virus-induced cell death using MTT or XTT reagents; analysis of virus production from these cells; as well as the basic understanding in mode of action. We further show direct evidence of the effectiveness of these protocols by utilizing them to test the effectiveness of the polyphenol drug, Rottlerin, at inhibiting Zika virus infection and death of neuronal cell lines.

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