scholarly journals Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Benjamin O. Fulton ◽  
David Sachs ◽  
Megan C. Schwarz ◽  
Peter Palese ◽  
Matthew J. Evans
Keyword(s):  
Zika Virus ◽  
Viral Evolution ◽  
Rna Replication ◽  
Transposon Mutagenesis ◽  
Ns1 Protein ◽  
Genetic Constraints ◽  
E Protein ◽  
A Genome ◽  
Nucleotide Insertions ◽  
Fusion Loop

ABSTRACT The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus. IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses.

scholarly journals A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 603
Author(s):  
Beatrice Sarah Berneck ◽  
Alexandra Rockstroh ◽  
Jasmin Fertey ◽  
Thomas Grunwald ◽  
Sebastian Ulbert
Keyword(s):  
Zika Virus ◽  
Insect Cell ◽  
Neutralizing Antibodies ◽  
Structural Similarity ◽  
Cross Reactivity ◽  
Virus Envelope ◽  
E Protein ◽  
Neutralizing Capacity ◽  
Fusion Loop

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles.

scholarly journals Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Pralow ◽  
Alexander Nikolay ◽  
Arnaud Leon ◽  
Yvonne Genzel ◽  
Erdmann Rapp ◽  
...  
Keyword(s):  
Zika Virus ◽  
Animal Cell ◽  
Gradient Centrifugation ◽  
Viral Envelope ◽  
Protein Glycosylation ◽  
High Yield ◽  
Structural Protein ◽  
Site Specific ◽  
E Protein ◽  
The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

scholarly journals A human antibody against Zika virus crosslinks the E protein to prevent infection

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
S. Saif Hasan ◽  
Andrew Miller ◽  
Gopal Sapparapu ◽  
Estefania Fernandez ◽  
Thomas Klose ◽  
...  
Keyword(s):  
Zika Virus ◽  
Human Antibody ◽  
E Protein

scholarly journals Diterpenes/Diterpenoids and Their Derivatives as Potential Bioactive Leads against Dengue Virus: A Computational and Network Pharmacology Study

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6821
Author(s):  
Rasel Ahmed Khan ◽  
Rajib Hossain ◽  
Abolghasem Siyadatpanah ◽  
Khattab Al-Khafaji ◽  
Abul Bashar Ripon Khalipha ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Dengue Fever ◽  
Computational Study ◽  
Viral Proteins ◽  
Network Pharmacology ◽  
Binding Affinities ◽  
Ns1 Protein ◽  
Structural Protein ◽  
E Protein

Dengue fever is a dangerous infectious endemic disease that affects over 100 nations worldwide, from Africa to the Western Pacific, and is caused by the dengue virus, which is transmitted to humans by an insect bite of Aedes aegypti. Millions of citizens have died as a result of dengue fever and dengue hemorrhagic fever across the globe. Envelope (E), serine protease (NS3), RNA-directed RNA polymerase (NS5), and non-structural protein 1 (NS1) are mostly required for cell proliferation and survival. Some of the diterpenoids and their derivatives produced by nature possess anti-dengue viral properties. The goal of the computational study was to scrutinize the effectiveness of diterpenoids and their derivatives against dengue viral proteins through in silico study. Methods: molecular docking was performed to analyze the binding affinity of compounds against four viral proteins: the envelope (E) protein, the NS1 protein, the NS3 protein, and the NS5 protein. Results: among the selected drug candidates, triptolide, stevioside, alepterolic acid, sphaeropsidin A, methyl dodovisate A, andrographolide, caesalacetal, and pyrimethamine have demonstrated moderate to good binding affinities (−8.0 to −9.4 kcal/mol) toward the selected proteins: E protein, NS3, NS5, and NS1 whereas pyrimethamine exerts −7.5, −6.3, −7.8, and −6.6 kcal/mol with viral proteins, respectively. Interestingly, the binding affinities of these lead compounds were better than those of an FDA-approved anti-viral medication (pyrimethamine), which is underused in dengue fever. Conclusion: we can conclude that diterpenoids can be considered as a possible anti-dengue medication option. However, in vivo investigation is recommended to back up the conclusions of this study.

scholarly journals Adaptation to host cell environment during experimental evolution of Zika virus

2020 ◽  
Author(s):  
Vincent Grass ◽  
Emilie Hardy ◽  
Kassian Kobert ◽  
Soheil Rastgou Talemi ◽  
Elodie Décembre ◽  
...  
Keyword(s):  
Host Cell ◽  
Human Health ◽  
Zika Virus ◽  
Viral Evolution ◽  
Time Lag ◽  
Viral Escape ◽  
Host Responses ◽  
Type I ◽  
Zikv Infection ◽  
Antiviral Responses

AbstractZika virus (ZIKV) infection can cause developmental and neurological defects and represents a threat for human health. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment. We uncovered that serially passaged ZIKV acquired increased infectivity, defined as the probability for one virus to initiate infection, and simultaneously developed a resistance to TLR3-induced restriction. We built a mathematical model that suggests that the increased infectivity is due to a reduced time-lag between infection and viral replication. We found that this adaptation is cell-type specific, suggesting that different cell environments may drive viral evolution along different routes. Deep-sequencing of ZIKV quasi-species pinpointed mutations whose increased frequencies temporally coincide with the acquisition of the adapted phenotype. We functionally validated a point-mutation in ZIKV envelope (E) protein recapitulating the adapted phenotype. Its positioning on the E structure suggests a putative function in protein refolding/stability. Altogether, our results uncovered ZIKV adaptations to the cell environment leading to an accelerated replication onset coupled with resistance to TLR3-induced antiviral response. Our work provides insights into viral escape mechanisms and interactions with host cell and can serve as a framework to study other viruses.Significance StatementZika virus poses a major threat to Human health worldwide. To understand how Zika virus interacts with human cells, we studied its evolution in cell cultures. We found that the viruses adapted by initiating their replication sooner after cell entry. We sequenced the genomes of the viruses evolved over time and found mutations underlying the adaptation of the virus. One mutation in the envelope viral protein is sufficient to reproduce the faster initiation of replication. Our multidisciplinary approach based on analyzing viral evolution in a controlled environment and mathematical modeling revealed how Zika virus can escape antiviral responses, and can serve as framework to study other viruses.

scholarly journals Antibodies Elicited by an NS1-Based Vaccine Protect Mice against Zika Virus

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Mark J. Bailey ◽  
Felix Broecker ◽  
James Duehr ◽  
Fortuna Arumemi ◽  
Florian Krammer ◽  
...  
Keyword(s):  
Virus Vaccine ◽  
Zika Virus ◽  
Envelope Glycoprotein ◽  
Congenital Malformations ◽  
Ns1 Protein ◽  
Specific Antibodies ◽  
Antibody Dependent Enhancement ◽  
Effector Functions ◽  
Protective Antibodies ◽  
Infected Cells

ABSTRACTZika virus is a mosquito-borne flavivirus which can cause severe disease in humans, including microcephaly and other congenital malformations in newborns and Guillain-Barré syndrome in adults. There are currently no approved prophylactics or therapeutics for Zika virus; the development of a safe and effective vaccine is an urgent priority. Preclinical studies suggest that the envelope glycoprotein can elicit potently neutralizing antibodies. However, such antibodies are implicated in the phenomenon of antibody-dependent enhancement of disease. We have previously shown that monoclonal antibodies targeting the Zika virus nonstructural NS1 protein are protective without inducing antibody-dependent enhancement of disease. Here, we investigated whether the NS1 protein itself is a viable vaccine target. Wild-type mice were vaccinated with an NS1-expressing DNA plasmid followed by two adjuvanted protein boosters, which elicited high antibody titers. Passive transfer of the immune sera was able to significantly protect STAT2 knockout mice against lethal challenge by Zika virus. In addition, long-lasting NS1-specific IgG responses were detected in serum samples from patients in either the acute or the convalescent phase of Zika virus infection. These NS1-specific antibodies were able to functionally engage Fcγ receptors. In contrast, envelope-specific antibodies did not activate Fc-mediated effector functions on infected cells. Our data suggest that the Zika virus NS1 protein, which is expressed on infected cells, is critical for Fc-dependent cell-mediated immunity. The present study demonstrates that the Zika virus NS1 protein is highly immunogenic and can elicit protective antibodies, underscoring its potential for an effective Zika virus vaccine.IMPORTANCEZika virus is a global public health threat that causes microcephaly and congenital malformations in newborns and Guillain-Barré syndrome in adults. Currently, no vaccines or treatments are available. While antibodies targeting the envelope glycoprotein can neutralize virus, they carry the risk of antibody-dependent enhancement of disease (ADE). In contrast, antibodies generated against the NS1 protein can be protective without eliciting ADE. The present study demonstrates the effectiveness of an NS1-based vaccine in eliciting high titers of protective antibodies against Zika virus disease in a mouse model. Sera generated by this vaccine can elicit Fc-mediated effector functions against Zika virus-infected cells. Lastly, we provide human data suggesting that the antibody response against the Zika virus NS1 protein is long-lasting and functionally active. Overall, our work will inform the development of a safe and effective Zika virus vaccine.

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 Two Genetic Differences between Closely Related Zika Virus Strains Determine Pathogenic Outcome in Mice

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Derek L. Carbaugh ◽  
Shuntai Zhou ◽  
Wes Sanders ◽  
Nathaniel J. Moorman ◽  
Ronald Swanstrom ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mouse Models ◽  
Zika Virus ◽  
Clinical Manifestations ◽  
Amino Acid Sequences ◽  
Viral Envelope ◽  
Laboratory Research ◽  
Balanced Polymorphism ◽  
E Protein ◽  
Virus Strains

ABSTRACT Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here, we identify two strain-specific determinants of ZIKV virulence in mice. We found that strain H/PF/2013 caused 100% lethality in Ifnar1−/− mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in Ifnar1−/− Ifngr1−/− double-knockout (DKO) mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G-to-T change at nucleotide 1965 producing a Val-to-Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both virus strain backgrounds, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 noncoding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3′ quarter of the H/PF/2013 genome were sufficient to confer virulence in Ifnar1−/− mice. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis (Ifnar1−/− and Ifnar1−/− Ifngr1−/− DKO mice). IMPORTANCE Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here, we identify two strain-specific determinants of ZIKV virulence that are evident in only Ifnar1−/− mice but not Ifnar1−/− Ifngr1−/− DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence but not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.

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 Efficient Homologous RNA Recombination and Requirement for an Open Reading Frame during Replication of Equine Arteritis Virus Defective Interfering RNAs

2000 ◽  
Vol 74 (19) ◽  
pp. 9062-9070 ◽  
Author(s):  
Richard Molenkamp ◽  
Sophie Greve ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Virus ◽  
Rna Replication ◽  
Proximal Region ◽  
Full Length ◽  
Equine Arteritis Virus ◽  
Open Reading Frame ◽  
Replicase Gene ◽  
Rna Recombination ◽  
Reading Frame ◽  
A Genome

ABSTRACT Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156–3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3′- and 5′-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5′-proximal region was found to be approximately 100-fold lower than that in the 3′-proximal part of the genome.

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