Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Abstract

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

Flavivirus Infection Uncouples Translation Suppression from Cellular Stress Responses

“…S1C). Similar translation repression of host mRNAs upon DENV infection was observed in human lung epithelial A549 cells, which are immunocompetent, in contrast to Huh7 cells (41, 42) (Fig. S1D), arguing that the observed phenotype does not depend on interferon.…”

Biochemistry and Molecular Biology of Flaviviruses

“…146,147 The resulting slowdown of viral protein synthesis has been linked to a heightened innate immune response that ultimately prevents productive infection. 148,149 Taken together, these reports indicate that cap 2′-O-methylation allows cells to detect and restrict nonself RNAs using IFIT proteins.…”

Antiviral interferon response at single‐cell resolution

“…ISGs are then typically activated by STAT1‐STAT2 heterodimers in complex with IRF9, which binds to DNA sequences known as IFN‐stimulated response elements (ISRE). Live‐imaging studies with fluorescent reporters driven by ISG gene loci (using IRF‐7, Rand et al, and IFIT1 and Mx1, Schmid et al) have shown a binary pattern of gene regulation: cells either expressed or did not express the ISG reporter, where the expressing fraction increased with the interferon concentration applied. At very high interferon concentrations, more than 90% of the cells could be made to express the ISG reporters, but such IFN levels were not reached after RNA virus infection of fibroblasts or epithelial cells in culture.…”

“…This kind of apparently stochastic expression has been observed more broadly also for other cytokines in different contexts, including immune responses in vivo; it is thus a typical feature of cytokine expression. Moreover, the induction of ISGs after acute viral infections has been found to be highly heterogeneous at the single‐cell level, with only a fraction of cells upregulating an antiviral gene program at physiological concentrations of IFNs …”