Abstract
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.
Highlights
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
We have previously shown that the NSs of the orthobunyaviruses Bunyamwera virus (BUNV) and LACV directly interfere with mRNA synthesis by the cellular RNA polymerase II (RNAP II) [28, 44]
260-kDa large subunit RPB1 of RNAP II gets hyperphosphorylated at the 52 heptad repeat sequences that are situated at the CTD [46, 47]
Summary
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. The L segment encodes the RNA-dependent RNA polymerase (RdRP), the M segments encodes a polyprotein that is processed to the envelope glycoproteins Gn, NSm (nonstructural, M segment), and Gc, and the S segments encodes the nucleocapsid protein N and the nonstructural protein NSs. All genomic segments are encapsidated by N protein and contain noncoding regions at their 5= and 3= ends that have the potential to anneal to a so-called “panhandle structure” due to partial sequence complementarities. After entering the host cell via clathrin-mediated endocytosis [18, 19] and subsequent low pH-driven membrane fusion, mRNAs are transcribed from the incoming genome RNA nucleocapsids by L RdRP (“primary transcription”). The newly generated vRNA nucleocapsids can give rise to more mRNAs produced by secondary transcription or become packaged by peptidase-processed Gn/Gc on Golgi membranes and leave the cell via the exocytosis pathway [19, 21, 22]
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