Lifetime of ground conformational state determines the activity of structured RNA.

Biomolecules continually sample alternative conformations. Consequently, even the most energetically favored ground conformational state has a finite lifetime. Here, we show that, in addition to the 3D structure, the lifetime of a ground conformational state determines its biological activity. Using hydrogen-deuterium exchange nuclear magnetic resonance spectroscopy, we found that Zika virus exoribonuclease-resistant RNA (xrRNA) encodes a ground conformational state with a lifetime that is ~105-107 longer than that of canonical base pairs. Mutations that shorten the apparent lifetime of the ground state without affecting its 3D structure decreased exoribonuclease resistance in vitro and impaired virus replication in cells. Additionally, we observed this exceptionally long-lived ground state in xrRNAs from diverse infectious mosquito-borne flaviviruses. These results demonstrate the biological significance of the lifetime of a preorganized ground state and further suggest that elucidating the lifetimes of dominant 3D structures of biomolecules may be crucial for understanding their behaviors and functions.

Structure and neutralization mechanism of a human antibody targeting a complex Epitope on Zika virus.

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

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

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.

Flavivirus Envelope Protein Glycosylation: Impacts on Viral Infection and Pathogenesis.

Flaviviruses encode one, two, or no N-linked glycosylation sites on their envelope proteins. Glycosylation can impact virus interactions with cell surface attachment factors and also may impact virion stability and virus replication. Envelope protein glycosylation has been identified as a virulence determinant for multiple flaviviruses, but the mechanisms by which glycosylation mediates pathogenesis remain unclear. In this Gem, we summarize current knowledge on flavivirus envelope protein glycosylation and its impact on viral infection and pathogenesis.

Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barré syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. All contemporary ZIKV isolates encode an N-linked glycosylation site in the envelope (E) protein (N154), but this glycosylation site is absent in many historical ZIKV isolates. Here, we investigated the role of E protein glycosylation in ZIKV pathogenesis using two contemporary Asian-lineage strains (H/PF/2013 and PRVABC59) and the historical African-lineage strain (MR766). We found that glycosylated viruses were highly pathogenic in Ifnar1-/- mice. In contrast, nonglycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously but remaining neurovirulent when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, we found that glycosylation facilitated infection of cells expressing the lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) or DC-SIGN-related (DC-SIGNR), suggesting that inefficient infection of lectin-expressing leukocytes could contribute to the attenuation of nonglycosylated ZIKV in mice.IMPORTANCE It is unclear why the ability of Zika virus (ZIKV) to cause serious disease, including Guillain-Barré syndrome and birth defects, was not recognized until recent outbreaks. One contributing factor could be genetic differences between contemporary ZIKV strains and historical ZIKV strains. All isolates from recent outbreaks encode a viral envelope protein that is glycosylated, whereas many historical ZIKV strains lack this glycosylation. We generated nonglycosylated ZIKV mutants from contemporary and historical strains and evaluated their virulence in mice. We found that nonglycosylated viruses were attenuated and produced lower viral loads in serum and brains. Our studies suggest that envelope protein glycosylation contributes to ZIKV pathogenesis, possibly by facilitating attachment to and infection of lectin-expressing leukocytes.

A Reverse Genetics Platform That Spans the Zika Virus Family Tree.

Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics.IMPORTANCE Viral emergence is a poorly understood process as evidenced by the sudden emergence of Zika virus in Latin America and the Caribbean. Malleable reagents that both predate and span an expanding epidemic are key to understanding the virologic determinants that regulate pathogenesis and transmission. We have generated representative cDNA molecular clones and recombinant viruses that span the known ZIKV family tree, including early Brazilian isolates. Recombinant viruses replicated efficiently in cell culture and were pathogenic in immunodeficient mice, providing a genetic platform for rational vaccine and therapeutic design.