Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment

Nicholas S Eyre, Amanda L Aloia, Michael A Joyce, Monrat Chulanetra, D Lorne Tyrrell, Michael R Beard

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis.

Original languageEnglish
Pages (from-to)20-31
Number of pages12
JournalVirology
Volume507
DOIs
Publication statusPublished - Jul 2017
Externally publishedYes

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