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. 2022 Jan 26;96(2):e0177421.
doi: 10.1128/JVI.01774-21. Epub 2021 Nov 10.

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production

Margarita V Rangel  1 , Nicholas Catanzaro  2 , Sara A Thannickal  1 , Kelly A Crotty  1 , Maria G Noval  1 , Katherine E E Johnson  3 , Elodie Ghedin  3   4 , Helen M Lazear  2 , Kenneth A Stapleford  1
Affiliations

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production

Margarita V Rangel et al. J Virol. .

Abstract

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in β-strand c (V114M) of domain II. We have previously shown that the analogous β-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E β-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Keywords: alphavirus; assembly; flavivirus; fusion protein; glycoprotein.

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Figures

FIG 1
FIG 1
Identification of emerging ZIKV minority variants after vector-borne transmission. ZIKV MR766 vector-transmitted synonymous (A) and nonsynonymous (B) minor variants (<50%) present in individual mice (n = 3). Boxes indicate variants present in multiple organs of individual mice. (C) Structure of the tip of ZIKV envelope protein (PDB ID 5JHM) with the V114M variant in red. (D) Structure of the ZIKV NS3 protease (PDB ID 5GJ4) with the R59K mutation in red and the 14-3-3 RLDP binding motif in black. (E) Schematic representation of the ZIKV NS2A protein. The I33V mutation is depicted in red.
FIG 2
FIG 2
ZIKV variants impact weight loss and virus replication in Ifnar1−/− mice. (A) Survival of 5- to 6-week-old Ifnar1−/− mice infected with 103 FFU of wild-type (WT) (n = 4), EV114M (n = 5), NS2AI33V (n = 6), or NS3R59K (n = 6) ZIKV. No significant difference between groups. (B) Weight loss of mice from panel A. Data are censored after the first mouse in each group died. ***, P < 0.001; *, P < 0.05 (two-way analysis of variance [ANOVA]). Viral loads in serum (C) and spleen (D) from Ifnar1−/− mice infected with WT ZIKV or variants. n = 8 for each virus. *, P < 0.05 (one-way ANOVA, Kruskal-Wallis test).
FIG 3
FIG 3
Replication of ZIKV envelope and NS2A variants is attenuated in A549 cells. Multistep growth curves of WT ZIKV and variants in A549 (A), Vero (B), and C6/36 cells (C). Each cell line was infected with each virus at an MOI of 0.1, and virus titers in the supernatant were quantified by focus-forming assay. Dotted lines indicate the limit of detection. Data are from 3 independent experiments. **, P < 0.01 (two-way ANOVA).
FIG 4
FIG 4
ZIKV V114M is structurally analogous to chikungunya virus E1 V80 and sensitive to ammonium chloride inhibition. (A) ZIKV MR766 envelope (PDB ID 5JHM) and CHIKV E1 (PDB ID 3N42). The fusion loop, ij loop, and bc loop are labeled, and the V114 and V80 variants are in red. (B) Flavivirus envelope protein sequence alignment around residue V114. (C) 293T cells were transfected with a ZIKV WT or ZIKV EV114M infectious clone. Cells were harvested at 72 h postinfection (hpi) and lysed in Laemmli buffer, and ZIKV proteins were analyzed by SDS-PAGE and immunoblotting. The blot is representative of at least three independent transfections. (D). Vero cells were pretreated for 1 h with increasing concentrations of NH4Cl and infected at an MOI of 0.1. Supernatant was collected 36 hpi, and viral titers were quantified by plaque assay. Data are means and standard errors of the means (SEM). There were 3 independent experiments with internal technical triplicates. Student's t test was used.
FIG 5
FIG 5
Replication of WT and E V114M ZIKV in neonatal mice and A. aegypti mosquitoes. Four-day-old (A) and 7-day-old (B) C57BL/6J mice were infected subcutaneously with 104 PFU of each virus. Virus titers were quantified in each organ at 7 days postinfection. Data are means and SEM from two independent infections. For four-day-old mice, n = 11 (WT) and 7 (EV114M). For seven-day-old mice, n = 7 (WT) and 8 (EV114M). The Mann-Whitney test was used. (C to F) A. aegypti mosquitoes were infected with 106 PFU of each virus, and viral titers were determined in the bodies (C and E) and legs and wings (D and F) at 14 days postinfection. Data are means and SEM from two independent infections. WT, n = 39; EV114M, n = 36. There was no significant difference between groups (Mann-Whitney compare-ranks test).
FIG 6
FIG 6
A single point mutation in the flavivirus E ij loop inhibits ZIKV infectious particle production and impacts envelope protein accumulation. (A) ZIKV MR766 envelope (PDB ID 5JHM) and CHIKV E1 (PDB ID 3N42). The ij loop and ZIKV A250 and CHIKV A226 residues are in red. (B) 293T cells were transfected with WT ZIKV or ZIKV EA250V infectious clone plasmids, and virus supernatants were collected at 48 hpi. Virus titers were quantified by plaque assay. n = 2 with internal technical duplicates. Extracellular (C) and intracellular (D) ZIKV RNA was quantified by RT-qPCR at 72 h posttransfection. RNA is normalized to WT ZIKV. There were three independent experiments with internal technical duplicates. (E) 293T cells were transfected with WT ZIKV or ZIKV EA250V plasmids. Cells were harvested at 72 h posttransfection and lysed in Laemmli buffer, and ZIKV proteins were analyzed by SDS-PAGE and immunoblotting. The immunoblot is representative of at least 3 independent transfections.
FIG 7
FIG 7
The alanine at the tip of flavivirus envelope inhibits infectious particle production of yellow fever virus but not West Nile virus. (A) Flavivirus envelope ij loop protein sequence alignment. The orange box indicates the conserved alanine at the tip of the ij loop. (B) Domain II of the WNV (PDB ID 2HG0) and YFV (PDB ID 6IW4) envelope protein. The ij loop and YFV A239 and WNV A247 residues are in red. (C and D) Vero cells were transfected with WT or variant WNV and YFV in vitro-transcribed RNA, and virus-containing supernatant was collected at 48 hpi. Virus titers were quantified by plaque assay. There were 3 independent experiments. (E) C6/36 cells were transfected with WT or variant YFV RNA, and virus-containing supernatant was collected at 72 and 96 hpi. Virus titers were quantified by plaque assay. There were 3 independent experiments.
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