Fig. 3

DENV and ZIKV genomes form extensive pair-wise interactions. a Arc plot showing the most abundant top 75 pair-wise interactions in the DENV1 genome inside virions. The known circularization signal is colored in dark blue. b Top: peak calling of the circularization interaction region shows that the peak of base pairing occurs at positions 95 and 10,641. Bottom: prediction of hybridization using RNAcofold program using sequences around the called peaks identified three of the best known circularization signals. c Bar charts showing the number of peaks that interact uniquely and with other regions along the genome. Greater than forty percent of DENV and ZIKV interactions are not unique. d Boxplot showing that regions with alternative base pairing (alternate) have higher Shannon entropies than all regions present in the genome (all) for both DENV1 and ZIKV French Polynesia strain. Box encompasses first and third quartiles, with the median indicated in the box. Whiskers are at minimum and maximum of points excluding deviations of 1.5× interquartile (Q3–Q1) range from Q1 to Q3 as outliers. Outliers are indicated as dots. p-Value calculated using T-test. e Arc plot showing one set of alternative interactions found along the ZIKV French Polynesia genome. f Structure models of the pair-wise interactions for the alternative structures in e, using the program RNAcofold. Mutations along each strand of the pair-wise interaction are in blue and green respectively. g qPCR analysis of the amount of mutant (from f) and WT virus that are produced in Huh7 cells and supernatant, 24 and 48 h post infection. The Y-axis shows that amount of mutant virus produced as compared to wild type. Statistical analysis was performed using Student’s T-test (one tail) to determine if the mutant viruses are attenuated compared to wild type. *p-value < 0.05. Error bars represent SEM. Two biological replicates of Gibson assembly were performed to generate WT and MT viruses and two biological replicates of Huh7 infection were performed