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Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers

ABSTRACT: Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. To describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms, a full-length wild-type (WT) HTNV-L construct comprising an N-terminal his-tag was expressed in Hi5 insect cells and subsequently purified. Through a combination of size-exclusion chromatography and mass photometry analysis, the presence of distinct HTNV-L oligomers was observed. In buffers containing 250mM NaCl, several species were automatically detected including monomers (53%), dimers (17%), higher molecular weight oligomers up to hexamers (2%) and an unidentified 150kDa protein (18%). To characterize the proteins present in the 150kDa band, a bottom-up mass spectrometry (MS)-based proteomic analysis was performed. For this, proteins present in the 150 kDa band were in-gel digested with trypsin before analysis by nano liquid chromatography coupled to MS/MS. Obtained result revealed that the most abundant protein in this band was HTNV-L. The identified peptides were covering the entire HTNV-L sequence, suggesting that the 150kDa band may contain different cleaved forms of HTNV-L co-migrating in this particular SDS-PAGE gel band.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Hantaan Virus 76-118 Trichoplusia Ni

SUBMITTER: Yohann Couté

LAB HEAD: Yohann Couté

PROVIDER: PXD049232 | Pride | 2024-02-07

REPOSITORIES: Pride

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Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.

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Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers

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