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Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.

ABSTRACT: Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.

SUBMITTER: Arragain B

PROVIDER: S-EPMC7368059 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.

Arragain Benoît B Effantin Grégory G Gerlach Piotr P Reguera Juan J Schoehn Guy G Cusack Stephen S Malet Hélène H

Nature communications 20200717 1

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the ca ...[more]

PMID: 32681014

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Project description:Mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) causes up to 100 annual cases of severe meningoencephalitis in children and young adults in the United States. A major virulence factor of LACV is the nonstructural protein NSs, which inhibits host cell mRNA synthesis to prevent the induction of antiviral type I interferons (IFN-α/β). To achieve this host transcriptional shutoff, LACV NSs drives the proteasomal degradation of RPB1, the large subunit of mammalian RNA polymerase II. Here, we show that NSs acts in a surprisingly rapid manner, as RPB1 degradation was commencing already at 1 h postinfection. The RPB1 degradation was partially dependent on the cellular E3 ubiquitin ligase subunit Elongin C. Consequently, removal of Elongin C, but also of the subunits Elongin A or B by siRNA transfection partially rescued general RNAP II transcription and IFN-beta mRNA synthesis from the blockade by NSs. In line with these results, LACV NSs was found to trigger the redistribution of Elongin C out of nucleolar speckles, which, however, is an epiphenomenon rather than part of the NSs mechanism. Our study also shows that the molecular phenotype of LACV NSs is different from RNA polymerase II inhibitors like α-amanitin or Rift Valley fever virus NSs, indicating that LACV is unique in involving the Elongin complex to shut off host transcription and IFN response.IMPORTANCE The mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) is prevalent in the United States and can cause severe childhood meningoencephalitis. Its main virulence factor, the nonstructural protein NSs, is a strong inhibitor of the antiviral type I interferon (IFN) system. NSs acts by imposing a global host mRNA synthesis shutoff, mediated by NSs-driven proteasomal degradation of the RPB1 subunit of RNA polymerase II. Here, we show that RPB1 degradation commences as early as 1 h postinfection, and identify the E3 ubiquitin ligase subunit Elongin C (and its binding partners Elongins A and B) as an NSs cofactor involved in RPB1 degradation and in suppression of global as well as IFN-related mRNA synthesis.

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Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.

Publications

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.

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