Dengue Virus Genome Uncoating Requires Ubiquitination.

Laura A. Byk,Andrea V. Gamarnik,Mario Rossi,Leopoldo G. Gebhard,Federico A. De Maio,Néstor G. Iglesias,Vincent R. Racaniello

doi:10.1128/mbio.00804-16

Abstract

ABSTRACTThe 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.

Highlights

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle
After Dengue virus (DENV) entry, the viral particle remains inside endosomes, and after endosome maturation and membrane fusion, the nucleocapsid is released into the cytoplasm (Fig. 1A)
Under these conditions, silencing ubiquitin-like modifier activating enzyme 1 (UBA1) resulted in a 4-fold inhibition of viral translation similar to that observed for the Renilla luciferase small interfering RNAs (siRNAs) positive control (Fig. 3F)

Summary

RESULTS

Fate of the incoming DENV capsid protein during viral infection. After DENV entry, the viral particle remains inside endosomes, and after endosome maturation and membrane fusion, the nucleocapsid is released into the cytoplasm (Fig. 1A). The results confirm that the ubiquitin E1 enzyme activity is necessary during an early step in DENV infection, after internalization but previous to viral translation This requirement was found to be independent of the proteasome activity (Fig. 2C). After confirming a reduction of about 85% of the levels of UBA1 mRNA at 48 h posttransfection, cells were infected with DV-R, and translation was measured at early time points to evaluate the first rounds of translation Under these conditions, silencing UBA1 resulted in a 4-fold inhibition of viral translation similar to that observed for the Renilla luciferase siRNA positive control (Fig. 3F).

C FMDV Renilla FMDV prM-E-NS1-2A-2B-3-4A-4B-5

DISCUSSION

MATERIALS AND METHODS