Dengue Virus Genome Uncoating Requires Ubiquitination

Abstract

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process.

Biochemistry and Molecular Biology of Flaviviruses

“…380 Similarly, Byk et al reported that inhibition of E1 enzyme activity blocked DENV uncoating, possibly due to retention of internalized virions in endosomes. 221 This observation supports an earlier study that observed reduced DENV infection due to inhibition of the E1-activating enzyme, UBE1. 381 Another study reported that the E3 ubiquitin ligase, Nedd4, stimulated JEV replication without affecting virus entry, 382 suggesting that the ubiquitin system is important at multiple phases of the flavivirus life cycle.…”

Zika Virus Dependence on Host Hsp70 Provides a Protective Strategy against Infection and Disease

“…However, entry and capsid maturation in flaviviruses appear to share a strict requirement for Hsp70 function. The role in entry has not been explored in detail but could in principle be linked to capsid uncoating (Byk et al, 2016; Goodwin et al, 2011). Hsp70 inhibition strongly affects capsid function, reducing its stability (Figure 4B), preventing its association with LDs (Figure 4C), and altering the formation of capsid complexes in the cell (Figure 4F).…”

Vero Cell Proteomic Changes Induced by Zika Virus Infection

“…The UPS system involves tagging of proteins for enzymatic degradation. Some flaviviruses like Dengue‐2 and Japaneses encephalitis, as well as ZIKV, have been reported to adapt the UPS in their replicative cycles . ISG15 colocalizes with histone deacetylase 6 (HDAC6) and p62 in autophagic degradation of ubiquitinated cellular components .…”