Insect-Specific Flavivirus Replication in Mammalian Cells Is Inhibited by Physiological Temperature and the Zinc-Finger Antiviral Protein.

Agathe M.G Colmant,Jessica J Harrison,Jody Hobson-Peters,Gorben P Pijlman,Peter Simmonds,Teun A.P Slijkerman,Monique M Van Oers,Jelke J Fros,Roy A Hall

doi:10.3390/v13040573

Abstract

The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and insect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The zinc-finger antiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 °C. In ZAP-expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP’s antiviral function, did. Importantly, at 34 °C, both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection. This provides new insights into flavivirus evolution and the mechanisms associated with host switching.

Highlights

The genus Flavivirus contains many vertebrate-infecting flaviviruses (VIF), including severe human pathogens such as the dengue viruses, Zika virus, yellow fever virus, Japanese encephalitis virus and West Nile virus (WNV)
The obdinucleotide frequencies present in flavivirus genome sequences were analysed
Flaviviruses were grouped as lineage I or II insect-specific flaviviruses (ISF), and as mosquito-borne, tick-borne or no known vector flaviviruses (NKVF) VIFs (Figure 1)

Summary

Introduction

The genus Flavivirus (family Flaviviridae) contains many vertebrate-infecting flaviviruses (VIF), including severe human pathogens such as the dengue viruses, Zika virus, yellow fever virus, Japanese encephalitis virus and West Nile virus (WNV). In addition to the VIFs, the Flavivirus genus includes many insect-specific flaviviruses (ISF) These viruses have been isolated from mosquitoes and empirical evidence suggests these exclusively replicate in cells of invertebrate origin (reviewed in [1]). When temperatures were reduced from 37 to 34 ◦ C, mouse embryonic fibroblasts (MEF) with the interferon receptor (IFNAR) or endonuclease RNase L knocked out and RIG-I deficient BSR cells displayed low levels of BinJV protein expression [3,12] Together, this indicates that ISF replication in vertebrate cells is restricted at multiple stages of infection and suggests that vertebrate antiviral pathways may limit lineage II ISF RNA replication inside the vertebrate cell

Methods

Results

Conclusion