All Four Sendai Virus C Proteins Bind Stat1, but only the Larger Forms also Induce Its Mono-ubiquitination and Degradation

Abstract

Sendai virus infection strongly induces interferon (IFN) production and has recently been shown to interdict the subsequent IFN signaling through the Jak/Stat pathway. This anti-IFN activity of SeV is due to its "C" proteins, a nested set of four proteins (C', C, Y1, Y2) that carry out a nested set of functions in countering the innate immune response. We previously reported that all four C proteins interact with Stat1 to prevent IFN signaling through the Jak/Stat pathway. Nevertheless, only the longer C proteins reduced Stat1 levels and prevented IFN from inducing an antiviral (VSV) state, or apoptosis, in IFN-competent murine cells. Here, we investigate the mechanism by which the various C proteins differentially affect the host antiviral defenses. All four C proteins were found to physically associate with Stat1 during cell culture infections, and in vitro in the absence of other viral gene products (as evidenced by co-immunoprecipitation). In addition, the inability of a null mutant (C(F170S)) to bind Stat1 suggests that this interaction is physiologically relevant. We have also shown that the proteasomal inhibitor MG132 can prevent the C protein-induced dismantling of the antiviral (VSV) state in murine cells; thus, the turnover of Stat1 correlates with the C protein-mediated counteraction of the antiviral (VSV) state. The C protein-induced instability of Stat1 was accompanied by a clear increase in the level of mono-ubiquinated Stat1, an unexpected hallmark of protein degradation. Finally, we show that a rSeV with mutant C proteins but wild-type Y proteins (CDelta10-15, that does not counteract the endogenous antiviral (VSV) state of MEFs even though their C proteins bind Stat1 and prevent its activity) is also unable to decrease bulk Stat1 levels or to increase the level of ubiquinated Stat1.

Proteolytic Processing and Translation Initiation

“…In particular, all four have been shown to interact with Stat1, an interaction that blocks type 1 interferon signaling to reporter genes carrying interferon-stimulated response elements. However, whereas binding to either CЈ or C leads to proteasomemediated Stat1 turnover and hence a subsequent block on the interferon-induced antiviral state, binding to the Y proteins does not destabilize Stat1, and the antiviral state can be observed (36,45). Our own work suggests that both CЈ and C expression but not Y is detrimental to cell viability.…”

“…6A), and assays have yet to discern a clear functional difference between the two. Both inhibit viral genome expression (7,8) and bind Stat1 to induce its turnover (10,36). However, we reasoned that the additional amino acids on CЈ may serve as a targeting signal that could direct at least a fraction of the protein to a particular cellular location.…”

“…Why Two Mechanisms to Produce the Same Polypeptide Chain?-CЈ, C, Y1, and Y2 form an N-terminal nested set of proteins that impinge on both the readout of the viral genome and the host cell response to viral infection (7,8,36). In particular, all four have been shown to interact with Stat1, an interaction that blocks type 1 interferon signaling to reporter genes carrying interferon-stimulated response elements.…”

“…It may also be a means of inactivating part of the CЈ function at a particular intracellular location while conserving those activities associated with the Y module. Indeed a number of important functions map to the N-terminal extension of C and CЈ that are not present in the Y proteins (8,10,36). Alternatively the small N-terminal cleavage product itself (34 amino acids at Y1 and 40 amino acids at Y2) may exercise a specific and unique function in the cell.…”

Roles of nuclear trafficking in infection by cytoplasmic negative-strand RNA viruses: paramyxoviruses and beyond

“…While intranuclear functions of other paramyxovirus M proteins have not been described, recent characterization of the interactome of the NiV, HeV, SeV and NDV M proteins (Pentecost et al, 2015) identified a number of nuclear proteins, suggesting that M proteins may have several roles in the nucleus, which the new proteomic data should help to elucidate. (Garcin et al, 2002) *Excludes components of the nuclear transport machinery (see Table 2 …”