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Integrative structural approaches identify the dynamic interactions between NS2B C-terminus and NS3 protease from DENV4 at post cleavage stage

ABSTRACT: Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. The NS2B-NS3 viral protease cleaves the viral polyprotein. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. Moreover, while presence of NS2B/NS3 cleavage inhibits the activity of unlinked ZIKV NS2B-NS3 protease, no inhibition of protease activity was observed for unlinked DENV NS2B-NS3 full length with NS2B/NS3 cleavage site. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of flexible NS2B and NS3 N- and C-termini. We identified a second cis-cleavage site prior to NS2B C terminus and captured the crystal structure of self-cleaved NS2B47NS3 protease in post cleavage state. We report that the cleaved NS2B C-terminus occupy the neighbouring NS2B-NS3 molecule identifying a different mechanism where DENV NS2B C-terminus modulates NS2B-NS3 protease activity via trans-interaction in contrast to the cis-interacting NS2B C-terminus from the unlinked ZIKV protease. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the solvent-exposed beta-barrel of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.

ORGANISM(S): Cellular Organisms

SUBMITTER: Wint Wint Phoo

PROVIDER: PXD033312 | JPOST Repository | Thu Apr 20 00:00:00 BST 2023

REPOSITORIES: jPOST

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			Action	DRS
	20210814_ZS_WP_beD4Pro_DSSO_R1-calib.mzML	Mzml
	20210814_ZS_WP_beD4Pro_DSSO_R1.raw	Raw
	20210814_ZS_WP_beD4Pro_DSSO_R2-calib.mzML	Mzml
	20210814_ZS_WP_beD4Pro_DSSO_R2.raw	Raw
	20210814_ZS_WP_beD4Pro_DSSO_R3-calib.mzML	Mzml

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Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.

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Integrative structural approaches identify the dynamic interactions between NS2B C-terminus and NS3 protease from DENV4 at post cleavage stage

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