Post-translational regulation of rotavirus protein NSP1 expression in mammalian cells

Abstract

The nonstructural rotavirus protein NSP1 binds specifically to viral mRNAs and to interferon regulatory factor 3 (IRF3), inducing IRF3 degradation through a proteasome-dependent pathway. By using a vaccinia virus expression system in mammalian cells, we found that the yield of NSP1 was 8- and 13-fold lower than the viral proteins VP2 or NSP3, respectively; while in the presence of proteasome inhibitors such difference could be reduced to 2- to 2.5-fold, respectively. The susceptibility of NSP1 to proteasome degradation was fully reversed in a dose-dependent manner by transfection with the full complement of 11 molecules of translation-competent rotavirus mRNAs, but this effect was abrogated by the protein synthesis inhibitor cycloheximide. These results demonstrate that NSP1 is degraded through a proteasome-dependent pathway, and viral proteins, alone or in combination with viral mRNAs, interfere with such degradation.

Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation

“…In order to examine a possible role for NSP1 in STAT1 inhibition, we expressed the NSP1 proteins from three rotavirus strains (simian RRV, bovine UK, and murine EW) that share the common property of IRF3 degradation in HT29 cells (22) as GFP-tagged fusion proteins (23). Previous studies reported that NSP1 is a poorly expressed protein due to inefficient recruitment of ribosomes on NSP1 transcripts (42) and due to posttranslational NSP1 proteasomal degradation (23,37,43). Therefore, we chose a single-cell analytical approach to directly compare the pY701-STAT1 status within NSP1-expressing cells in the presence or absence of exogenous stimulation (Fig.…”

Rotavirus NSP1 Mediates Degradation of Interferon Regulatory Factors through Targeting of the Dimerization Domain

“…NSP1 has several characteristics that suggest it functions as a viral E3 ubiquitin ligase. Primarily, NSP1 contains a RING domain similar to that of some E3 ligases (41,53,54), and the protein induces the proteasomal degradation of target proteins, such as IRF3 and IRF7 (37,53). Although the features of NSP1 indicate it functions as an E3 ubiquitin ligase, direct evidence in support of this has not yet been obtained.…”

Rotavirus Infections in Galapagos Sea Lions

“…The antigen was rhesus rotavirus (strain RRV obtained from H. Greenberg, Stanford University, Palo Alto, California). Virus was cultivated in MA104 African green monkey (Cercopithecus aethiops) kidney cells as described previously (Pina-Vazquez et al, 2007). Virions from cell lysates were semipurified by ultracentrifugation and were then treated with EDTA to remove their outer layer, thereby exposing the group antigen, as described (Pina-Vazquez et al, 2007).…”

“…Virus was cultivated in MA104 African green monkey (Cercopithecus aethiops) kidney cells as described previously (Pina-Vazquez et al, 2007). Virions from cell lysates were semipurified by ultracentrifugation and were then treated with EDTA to remove their outer layer, thereby exposing the group antigen, as described (Pina-Vazquez et al, 2007). Double-shelled viral particles were concentrated by ultracentrifugation at 150,000 3 G in a SW50 rotor (Beckman, Palo Alto, California) and resuspended in 1/600th volume of Tris 50 mM, pH 8.0.…”

“…Antigen for competition enzyme immunoassay was RRV purified by rate zonal centrifugation as described (Pina-Vazquez et al, 2007), with minor modifications. Briefly, virus lysates were treated with GenetronH 11 (trichlorofluoromethane, Cydsa, S. A. de C.V., San Pedro Garza García, Mexico), and the supernatant was concentrated by ultracentrifugation, resuspending the pellet in TNC (10 mM Tris, pH 8.0, 150 mM NaCl, 2 mM CaCl 2 ).…”