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 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 Deep mutational scanning comprehensively maps how Zika envelope protein mutations affect viral growth and antibody escape

2019 ◽  
Author(s):  
Marion Sourisseau ◽  
Daniel J.P. Lawrence ◽  
Megan C. Schwarz ◽  
Carina H. Storrs ◽  
Ethan C. Veit ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Natural Infection ◽  
Physical Structure ◽  
Functional Constraints ◽  
Viral Growth ◽  
Viral Neutralization ◽  
Future Evolution ◽  
E Protein ◽  
Amino Acid Mutations

AbstractFunctional constraints on viral proteins are often assessed by examining sequence conservation among natural strains, but this approach is relatively ineffective for Zika virus because all known sequences are highly similar. Here we take an alternative approach to map functional constraints on Zika virus’s envelope (E) protein by using deep mutational scanning to measure how all amino-acid mutations to the protein affect viral growth in cell culture. The resulting sequence-function map is consistent with existing knowledge about E protein structure and function, but also provides insight into mutation-level constraints in many regions of the protein that have not been well characterized in prior functional work. In addition, we extend our approach to completely map how mutations affect viral neutralization by two monoclonal antibodies, thereby precisely defining their functional epitopes. Overall, our study provides a valuable resource for understanding the effects of mutations to this important viral protein, and also offers a roadmap for future work to map functional and antigenic selection to Zika virus at high resolution.ImportanceZika virus has recently been shown to be associated with severe birth defects. The virus’s E protein mediates its ability to infect cells, and is also the primary target of the antibodies that are elicited by natural infection and vaccines that are being developed against the virus. Therefore, determining the effects of mutations to this protein is important for understanding its function, its susceptibility to vaccine-mediated immunity, and its potential for future evolution. We completely mapped how amino-acid mutations to E protein affected the virus’s ability to grow in cells in the lab and escape from several antibodies. The resulting maps relate changes in the E protein’s sequence to changes in viral function, and therefore provide a valuable complement to existing maps of the physical structure of the protein.

Co-evolution analysis to predict protein–protein interactions within influenza virus envelope

2014 ◽  
Vol 12 (02) ◽  
pp. 1441008 ◽  
Author(s):  
Ramil R. Mintaev ◽  
Andrei V. Alexeevski ◽  
Larisa V. Kordyukova
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Influenza A ◽  
Matrix Protein ◽  
Amino Acid Sequences ◽  
Viral Envelope ◽  
Research Database ◽  
Virus Envelope ◽  
Physical Constraints ◽  
Matrix Protein M1

Interactions between integral membrane proteins hemagglutinin (HA), neuraminidase (NA), M2 and membrane-associated matrix protein M1 of influenza A virus are thought to be crucial for assembly of functionally competent virions. We hypothesized that the amino acid residues located at the interface of two different proteins are under physical constraints and thus probably co-evolve. To predict co-evolving residue pairs, the EvFold ( http://evfold.org ) program searching the (nontransitive) Direct Information scores was applied for large samplings of amino acid sequences from Influenza Research Database ( http://www.fludb.org/ ). Having focused on the HA, NA, and M2 cytoplasmic tails as well as C-terminal domain of M1 (being the less conserved among the protein domains) we captured six pairs of correlated positions. Among them, there were one, two, and three position pairs for HA–M2, HA–M1, and M2–M1 protein pairs, respectively. As expected, no co-varying positions were found for NA–HA, NA–M1, and NA–M2 pairs obviously due to high conservation of the NA cytoplasmic tail. The sum of frequencies calculated for two major amino acid patterns observed in pairs of correlated positions was up to 0.99 meaning their high to extreme evolutionary sustainability. Based on the predictions a hypothetical model of pair-wise protein interactions within the viral envelope was proposed.

scholarly journals Deep Mutational Scanning Comprehensively Maps How Zika Envelope Protein Mutations Affect Viral Growth and Antibody Escape

2019 ◽  
Vol 93 (23) ◽  
Author(s):  
Marion Sourisseau ◽  
Daniel J. P. Lawrence ◽  
Megan C. Schwarz ◽  
Carina H. Storrs ◽  
Ethan C. Veit ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Natural Infection ◽  
Physical Structure ◽  
Functional Constraints ◽  
Viral Growth ◽  
Viral Neutralization ◽  
Future Evolution ◽  
E Protein ◽  
Amino Acid Mutations

ABSTRACT Functional constraints on viral proteins are often assessed by examining sequence conservation among natural strains, but this approach is relatively ineffective for Zika virus because all known sequences are highly similar. Here, we take an alternative approach to map functional constraints on Zika virus’s envelope (E) protein by using deep mutational scanning to measure how all amino acid mutations to the E protein affect viral growth in cell culture. The resulting sequence-function map is consistent with existing knowledge about E protein structure and function but also provides insight into mutation-level constraints in many regions of the protein that have not been well characterized in prior functional work. In addition, we extend our approach to completely map how mutations affect viral neutralization by two monoclonal antibodies, thereby precisely defining their functional epitopes. Overall, our study provides a valuable resource for understanding the effects of mutations to this important viral protein and also offers a roadmap for future work to map functional and antigenic selection to Zika virus at high resolution. IMPORTANCE Zika virus has recently been shown to be associated with severe birth defects. The virus’s E protein mediates its ability to infect cells and is also the primary target of the antibodies that are elicited by natural infection and vaccines that are being developed against the virus. Therefore, determining the effects of mutations to this protein is important for understanding its function, its susceptibility to vaccine-mediated immunity, and its potential for future evolution. We completely mapped how amino acid mutations to the E protein affected the virus’s ability to grow in cells in the laboratory and escape from several antibodies. The resulting maps relate changes in the E protein’s sequence to changes in viral function and therefore provide a valuable complement to existing maps of the physical structure of the protein.

scholarly journals Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins

2016 ◽  
Vol 90 (12) ◽  
pp. 5586-5600 ◽  
Author(s):  
Xiuyuan Ou ◽  
Wangliang Zheng ◽  
Yiwei Shan ◽  
Zhixia Mu ◽  
Samuel R. Dominguez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Fusion ◽  
Fusion Proteins ◽  
Virus Entry ◽  
Amino Acid Sequences ◽  
Viral Envelope ◽  
Class I ◽  
Viral Fusion ◽  
Viral Fusion Proteins

ABSTRACTThe fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide (955IAGVGWTAGL964) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other β-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of β-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution.IMPORTANCEWithin the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 β-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of β-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of β-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

scholarly journals Different pattern of haemagglutinin immunoreactivity of equine influenza virus strains isolated in Poland

2015 ◽  
Vol 59 (4) ◽  
pp. 463-466
Author(s):  
Małgorzata Kwaśnik ◽  
Ilona Marcelina Góra ◽  
Wojciech Rożek
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Amino Acid ◽  
Point Mutations ◽  
Amino Acid Sequences ◽  
Equine Influenza Virus ◽  
Equine Influenza ◽  
Antigenic Sites ◽  
Virus Strains

Abstract The immunoreactivity of haemagglutinin (HA) polypeptides of equine influenza virus was compared among the strains isolated in Poland, using H3 monoclonal antibody. A stronger signal in immunoblot reaction was observed for A/equi/Pulawy/2008 HA polypeptides compared to A/equi/Pulawy/2006, despite the fact that both strains are phylogenetically closely related and belong to Florida clade 2 of American lineage. The strongest signal, observed in the case of A/equi/Pulawy/2008, seemed to be connected with the presence of G135, I213, E379, and/or V530 instead of R135, M213, G379, and I530 present in A/equi/Pulawy/2006 HA sequence. This implies that point mutations within amino acid sequences of HA polypeptides of equine influenza virus may change their immunoreactivity even when they are not located within five basic antigenic sites.

Hydrophobic-at-Interface Regions in Viral Fusion Protein Ectodomains

2000 ◽  
Vol 20 (6) ◽  
pp. 519-533 ◽  
Author(s):  
José L. Nieva ◽  
Tatiana Suárez
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Viral Envelope ◽  
Fusion Pore ◽  
Viral Fusion ◽  
New Approach ◽  
Viral Fusion Protein ◽  
Membrane Partitioning ◽  
Viral Glycoproteins ◽  
New Procedures

In this chapter we shall describe how to apply the hydrophobicity-at-interface scale, as proposed by Wimley and White [Wimley, W. C. and White, S. H. (1996) Nature Struct. Biol. 3:842–848], to the detection of amino acid sequences of viral envelope glycoproteins putatively engaged in interactions with the target membranes. In addition, a new approach will be briefly introduced to infer the bilayer location at equilibrium of membrane-partitioning sequences. The use of these new procedures may be important in describing the molecular mechanism leading to the formation of a fusion pore by viral glycoproteins.

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