Analysis of the Zika and Japanese Encephalitis Virus NS5 Interactomes

Abstract

Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein–protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology.

Chasing Intracellular Zika Virus Using Proteomics

“…In addition to capsid and NS4B, several novel ZIKV NS5 binders were recently identified in these proteomics surveys. Among these, multiple members of the PAF1C complex (Leo1, CDC73, CTR9 and PAF1), confirming similar observations previously made on DENV-NS5 by the Gamarnik group [53] and recently confirmed by others [54]. Importantly, while these interactions appear highly conserved among DENV, ZIKV, JEV and WNV-NS5s, a moderate and reciprocal effect on viral replication was observed upon gene silencing (2-fold increase and 2-fold decrease for ZIKV and JEV infectivity, respectively) [41,54].…”

“…Among these, multiple members of the PAF1C complex (Leo1, CDC73, CTR9 and PAF1), confirming similar observations previously made on DENV-NS5 by the Gamarnik group [53] and recently confirmed by others [54]. Importantly, while these interactions appear highly conserved among DENV, ZIKV, JEV and WNV-NS5s, a moderate and reciprocal effect on viral replication was observed upon gene silencing (2-fold increase and 2-fold decrease for ZIKV and JEV infectivity, respectively) [41,54]. Further mechanistic studies suggest that PAF1 might be recruited by NS5 to reduce expression of interferon-stimulated genes and therefore dampen immune responses [41].…”

Flavivirus Nonstructural Protein NS5 Dysregulates HSP90 to Broadly Inhibit JAK/STAT Signaling

“…Thus, HSP90 inhibitors phenocopy JAK/STAT antagonism by flaviviruses, a similarity of function that we confirmed with side-by-side analyses. Our data revealed for the first time that flavivirus NS5 binds HSP90 to mediate broad STAT inhibition, which extends upon descriptive reports from other groups that identified JEV, DENV, and ZIKV NS5 interaction with HSP90, but did not define the relevance of this interaction [39][40][41]. Several DENV proteins have also been shown to bind HSP90 [37].…”

Importance of Zika Virus NS5 Protein for Viral Replication

“…Furthermore, flavivirus NS5 was shown to interact with some of the proteins that make up the Paf1 complex. It was found that inhibition of this complex increased infectivity of ZIKV and DENV but inhibited JEV thus showing that some host proteins interact differently with different flaviviruses [62]. Finally, NS5 is highly conserved among different flaviviruses [26,50,53,63], so identifying possible host protein interactions might prove an effective way to develop antivirals.…”