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

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.

Zika Virus Subverts Stress Granules To Promote and Restrict Viral Gene Expression.

Flaviviruses limit the cell stress response by preventing the formation of stress granules (SGs) and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific short interfering RNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR, and YB1. Knockdown of TIA-1 and TIAR affected ZIKV protein and RNA levels but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia Luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection while HuR exhibits an antiviral effect. The results of ZIKV relocalizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis.IMPORTANCE Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.

Probing Zika Virus Neutralization Determinants with Glycoprotein Mutants Bearing Linear Epitope Insertions.

Zika virus (ZIKV) glycoproteins are the primary target of the humoral immune response. In this study, we explored the capacity of these glycoproteins to tolerate insertion of linear epitope sequences and the potential of antibodies that bind these epitopes to inhibit infection. We first created a panel of ZIKV mutants with the FLAG epitope inserted in the premembrane (prM) and envelope (E) glycoprotein regions. The insertion locations were based on the results of our recent transposon insertional mutagenesis screen. Although FLAG insertions in prM greatly impaired viral fitness, this sequence was tolerated in numerous surface-exposed E protein sites. We observed that mutants bearing FLAG epitopes in E domains I and II and the E domain I-II hinge region were all neutralized by FLAG antibody; however, the neutralization sensitivity varied highly. We measured the antibody binding efficiency and found that this closely matched the pattern of neutralization sensitivity. We determined that E glycosylation did not affect antibody binding to a nearby epitope or its capacity to serve as a neutralization target. Although we could not generate infectious viruses with FLAG epitope insertions in a buried region of E protein domain III, we found that the V5 epitope could be inserted at this site without greatly impacting fitness. Furthermore, this virus was efficiently neutralized by V5 antibodies, highlighting that even buried epitopes can function as neutralization targets. Finally, we analyzed the timing of antibody neutralization activity during cell entry and found that all antibodies blocked a step after cell attachment.IMPORTANCE Zika virus (ZIKV) infections are associated with severe birth defects and neurological disease. The structure of the mature ZIKV particle reveals a virion surface covered by the envelope glycoprotein, which is the dominant target of the humoral immune response. It is unclear if all regions of the envelope protein surface or even buried epitopes can function as neutralization targets. To test this, we created a panel of ZIKV mutants with epitope insertions in different regions of the envelope protein. In characterizing these viruses, we found that the strength of antibody binding to an epitope is the major determinant of the neutralization potential of an antibody, that even a buried region of the envelope protein can be efficiently targeted, and that the sole potential envelope glycan does not impact nearby epitope antibody binding and neutralization. Furthermore, this work provides important insights into our understanding of how antibodies neutralize ZIKV.

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

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.

A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses.

Zika virus (ZIKV) is a mosquito borne flavivirus, which was a neglected tropical pathogen until it emerged and spread across the Pacific Area and the Americas, causing large human outbreaks associated with fetal abnormalities and neurological disease in adults. The factors that contributed to the emergence, spread and change in pathogenesis of ZIKV are not understood. We previously reported that ZIKV evades cellular antiviral responses by targeting STAT2 for degradation in human cells. In this study, we demonstrate that Stat2-/- mice are highly susceptible to ZIKV infection, recapitulate virus spread to the central nervous system (CNS), gonads and other visceral organs, and display neurological symptoms. Further, we exploit this model to compare ZIKV pathogenesis caused by a panel of ZIKV strains of a range of spatiotemporal history of isolation and representing African and Asian lineages. We observed that African ZIKV strains induce short episodes of severe neurological symptoms followed by lethality. In comparison, Asian strains manifest prolonged signs of neuronal malfunctions, occasionally causing death of the Stat2-/- mice. African ZIKV strains induced higher levels of inflammatory cytokines and markers associated with cellular infiltration in the infected brain in mice, which may explain exacerbated pathogenesis in comparison to those of the Asian lineage. Interestingly, viral RNA levels in different organs did not correlate with the pathogenicity of the different strains. Taken together, we have established a new murine model that supports ZIKV infection and demonstrate its utility in highlighting intrinsic differences in the inflammatory response induced by different ZIKV strains leading to severity of disease. This study paves the way for the future interrogation of strain-specific changes in the ZIKV genome and their contribution to viral pathogenesis.

Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone.

The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. This ZIKV infectious clone will be useful for investigating the genetic determinants of ZIKV infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of ZIKV isolates. IMPORTANCE The study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and pathogenesis.

A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I.

While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties. IMPORTANCE: Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.

Viral Determinants of miR-122-Independent Hepatitis C Virus Replication.

Hepatitis C virus (HCV) replication requires binding of the liver-specific microRNA (miRNA) miR-122 to two sites in the HCV 5' untranslated region (UTR). Although we and others have shown that viral genetics impact the amount of active miR-122 required for replication, it is unclear if HCV can replicate in the complete absence of this miRNA. To probe the absolute requirements for miR-122 and the genetic basis for those requirements, we used clustered regularly interspaced short palindromic repeat (CRISPR) technology to knock out miR-122 in Huh-7.5 cells and reconstituted these knockout (KO) cells with either wild-type miR-122 or a mutated version of this miRNA. We then characterized the replication of the wild-type virus, as well as a mutated HCV bearing 5' UTR substitutions to restore binding to the mutated miR-122, in miR-122 KO Huh-7.5 cells expressing no, wild-type, or mutated miR-122. We found that while replication was most efficient when wild-type or mutated HCV was provided with the matched miR-122, inefficient replication could be observed in cells expressing the mismatched miR-122 or no miR-122. We then selected viruses capable of replicating in cells expressing noncognate miR-122 RNAs. Unexpectedly, these viruses contained multiple mutations throughout their first 42 nucleotides that would not be predicted to enhance binding of the provided miR-122. These mutations increased HCV RNA replication in cells expressing either the mismatched miR-122 or no miR-122. These data provide new evidence that HCV replication can occur independently of miR-122 and provide unexpected insights into how HCV genetics influence miR-122 requirements. IMPORTANCE Hepatitis C virus (HCV) is the leading cause of liver cancer in the Western Hemisphere. HCV infection requires miR-122, which is expressed only in liver cells, and thus is one reason that replication of this virus occurs efficiently only in cells of hepatic origin. To understand how HCV genetics impact miR-122 usage, we knocked out miR-122 using clustered regularly interspaced short palindromic repeat (CRISPR) technology and adapted virus to replicate in the presence of noncognate miR-122 RNAs. In doing so, we identified viral mutations that allow replication in the complete absence of miR-122. This work provides new insights into how HCV genetics influence miR-122 requirements and proves that replication can occur without this miRNA, which has broad implications for how HCV tropism is maintained.

Zika Virus Targets Human STAT2 to Inhibit Type I Interferon Signaling.

The ongoing epidemic of Zika virus (ZIKV) illustrates the importance of flaviviruses as emerging human pathogens. All vector-borne flaviviruses studied thus far have to overcome type I interferon (IFN) to replicate and cause disease in vertebrates. The mechanism(s) by which ZIKV antagonizes IFN signaling is unknown. Here, we report that the nonstructural protein NS5 of ZIKV and other flaviviruses examined could suppress IFN signaling, but through different mechanisms. ZIKV NS5 expression resulted in proteasomal degradation of the IFN-regulated transcriptional activator STAT2 from humans, but not mice, which may explain the requirement for IFN deficiency to observe ZIKV-induced disease in mice. The mechanism of ZIKV NS5 resembles dengue virus (DENV) NS5 and not its closer relative, Spondweni virus (SPOV). However, unlike DENV, ZIKV did not require the E3 ubiquitin ligase UBR4 to induce STAT2 degradation. Hence, flavivirus NS5 proteins exhibit a remarkable functional convergence in IFN antagonism, albeit by virus-specific mechanisms.