Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases.
The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity.



The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.
Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USAInstituto de Biotecnologia/UNAM.
Lindy M
LM
Lutz
Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USAInstituto de Biotecnologia/UNAM.
Chandler R
CR
Pace
Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USAInstituto de Biotecnologia/UNAM marno2@lsuhsc.edu.
Michelle M
0000-0001-9219-3097
MM
Arnold
D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D007113:Immunity, Innate; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D034741:RNA, Small Interfering; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
C503645:CUL3 protein, human; C100318:Cullin 1; D044842:Cullin Proteins; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D034741:RNA, Small Interfering; D017361:Viral Nonstructural Proteins; C104282:nsp1 protein, Rotavirus; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex