FoxO4 negatively controls Tat-mediated HIV-1 transcription through the post-transcriptional suppression of Tat encoding mRNA.

The connection between the repression of human immunodeficiency virus type 1(HIV-1) transcription and the resting CD4+ T cell state suggests that the host transcription factors involved in the active maintenance of lymphocyte quiescence are likely to repress the viral transactivator, Tat, thereby restricting HIV-1 transcription. In this study, we analysed the interplay between Tat and the forkhead box transcription factors, FoxO1 and FoxO4. We show that FoxO1 and FoxO4 antagonize Tat-mediated transactivation of HIV-1 promoter through the repression of Tat protein expression. No effect was observed on the expression of two HIV-1 accessory proteins, Vif and Vpr. Unexpectedly, we found that FoxO1 and FoxO4 expression causes a strong dose-dependent post-transcriptional suppression of Tat mRNA, indicating that FoxO should effectively inhibit HIV-1 replication by destabilizing Tat mRNA and suppressing Tat-mediated HIV-1 transcription. In accordance with this, we observed that the Tat mRNA half-life is reduced by FoxO4 expression. The physiological relevance of our findings was validated using the J-Lat 10.6 model of latently infected cells. We demonstrated that the overexpression of a constitutively active FoxO4-TM mutant antagonized HIV-1 transcription reactivation in response to T cell activators, such as TNF-α or PMA. Altogether, our findings demonstrate that FoxO factors can control HIV-1 transcription and provide new insights into their potential role during the establishment of HIV-1 latency.

TRIM22 inhibits influenza A virus infection by targeting the viral nucleoprotein for degradation.

Tripartite motif (TRIM) protein superfamily members are emerging as important effectors of the innate immune response against viral infections. In particular, TRIM22 was reported to exert antiviral activity against RNA viruses, such as hepatitis B virus (HBV), encephalomyocarditis virus (ECMV), and human immunodeficiency virus type 1 (HIV-1). We demonstrate here, for the first time, that TRIM22 is upregulated by influenza A virus (IAV) infection at both mRNA and protein levels in human alveolar epithelial A549 cells. Conversely, TRIM22 potently restricted IAV replication, in that prevention of TRIM22 expression by means of short hairpin RNA led to a 10-fold enhancement of IAV replication in these cells. Depletion of TRIM22 also reduced the anti-IAV activity of alpha interferon (IFN-α), suggesting that TRIM22 is an important IFN-stimulated gene that is required for maximal suppression of IAV by type I IFN. Furthermore, the IAV infectious titer decreased up to 100-fold in MDCK cells expressing exogenous human TRIM22. Restriction of IAV replication was accounted for by the interaction between TRIM22 and the viral nucleoprotein (NP), resulting in its polyubiquitination and degradation in a proteasome-dependent manner. Thus, TRIM22 represents a novel restriction factor upregulated upon IAV infection that curtails its replicative capacity in epithelial cells.

The human T-cell leukemia virus type 1 oncoprotein tax controls forkhead box O4 activity through degradation by the proteasome.

Activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway by the viral Tax oncoprotein plays a pivotal role in clonal expansion of human T-cell leukemia virus type 1 (HTLV-1)-infected cells. As the Forkhead box O (FoxO) tumor suppressors act as downstream effectors of PI3K/Akt, they represent good candidate targets whose dysregulation by Tax might be involved in HTLV-1-mediated activation and transformation of infected cells. In this report, we provide evidence showing that Tax induces a dose-dependent degradation of FoxO4 by the ubiquitin-proteasome pathway. Consistent with that, we demonstrate that Tax expression increases the interaction between FoxO4 and Mdm2 E3 ligase, leading to a strong FoxO4 polyubiquitination. These processes require the phosphorylation of FoxO4 by Akt, since a mutant of FoxO4 with mutations on its three Akt phosphorylation sites appears to be resistant to Tax-mediated degradation and ubiquitination. In addition, we show that Tax expression is associated with degradation and phosphorylation of endogenous FoxO4 in Jurkat T cells. Finally, we demonstrate that Tax represses FoxO4 transcriptional activity. Our study demonstrates that Tax can control FoxO4 protein stability and transcriptional activity and provides new insight into the subversion of cell signaling pathways during HTLV-1 infection.

Mutations Conferring Increased Sensitivity to Tripartite Motif 22 Restriction Accumulated Progressively in the Nucleoprotein of Seasonal Influenza A (H1N1) Viruses between 1918 and 2009.

Influenza A viruses (IAVs) can cause zoonotic infections with pandemic potential when most of the human population is immunologically naive. After a pandemic, IAVs evolve to become seasonal in the human host by acquiring adaptive mutations. We have previously reported that the interferon (IFN)-inducible tripartite motif 22 (TRIM22) protein restricts the replication of seasonal IAVs by direct interaction with the viral nucleoprotein (NP), leading to its polyubiquitination and proteasomal degradation. Here we show that, in contrast to seasonal H1N1 IAVs, the 2009 pandemic H1N1 strain as well as H1N1 strains from the 1930s are resistant to TRIM22 restriction. We demonstrate that arginine-to-lysine substitutions conferring an increased sensitivity to TRIM22-dependent ubiquitination accumulated progressively in the NP of seasonal influenza A (H1N1) viruses between 1918 and 2009. Our findings suggest that during long-term circulation and evolution of IAVs in humans, adaptive mutations are favored at the expense of an increased sensitivity to some components of the innate immune response.IMPORTANCE We have uncovered that long-term circulation of seasonal influenza A viruses (IAV) in the human population resulted in the progressive acquisition of increased sensitivity to a component of the innate immune response: the type I interferon-inducible TRIM22 protein, which acts as a restriction factor by inducing the polyubiquitination of the IAV nucleoprotein (NP). We show that four arginine residues present in the NP of the 1918 H1N1 pandemic strain and early postpandemic strains were progressively substituted for by lysines between 1918 and 2009, rendering NP more susceptible to TRIM22-mediated ubiquitination. Our observations suggest that during long-term evolution of IAVs in humans, variants endowed with increased susceptibility to TRIM22 restriction emerge, highlighting the complexity of selection pressures acting on the NP.

Control of FoxO4 Activity and Cell Survival by TRIM22 Directs TLR3-Stimulated Cells Toward IFN Type I Gene Induction or Apoptosis.

Activation of innate immune response, induced after the recognition of double-stranded RNA (dsRNA), formed during replication of most viruses, results in intracellular signaling cascades ultimately culminating in the expression of type I interferon (IFN). In this study, we provide the first evidence that FoxO4 triggers the activation of the innate immune signaling pathway in coupling stimulation of TLR3 and RIG-like receptors by the synthetic dsRNA analog, poly(I:C), to IFN-ß and IFN-induced gene induction, whereas knockdown of FoxO4 had opposite effects. Similar effects of FoxO4 were observed during paramyxovirus-mediated IFN-ß transcriptional induction. We further found that knockdown of FoxO4 did not affect IRF3 and NF-κB activation by poly(I:C), suggesting that FoxO4 would act downstream in the signaling pathway. In addition, we show that the IFN-induced TRIM22 ubiquitin ligase targets FoxO4 and antagonizes its activity through an unrelated ubiquitin/autophagosomic-lysosomal pathway. Unexpectedly, TRIM22 knockdown strongly sensitizes cells to dsRNA-induced caspase-dependent apoptosis, as early as 2 h after poly(I:C) stimulation, concomitantly to the inhibition of the expression of the antiapoptotic protein, Bcl-2, indicating that TRIM22 might be a key factor for controlling the cell survival after TLR3 stimulation. Taken together, our data demonstrate that the regulation of FoxO4 protein expression and cell survival by TRIM22 controls TLR3-mediated IFN type I gene induction, preventing excessive antiviral response through dsRNA-induced apoptosis.

TRIM22 E3 ubiquitin ligase activity is required to mediate antiviral activity against encephalomyocarditis virus.

The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C(PRO)) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses.

The viral RNA recognition sensor RIG-I is degraded during encephalomyocarditis virus (EMCV) infection.

RNA helicase-like receptors MDA-5 but not RIG-I has been shown to be essential for triggering innate immune responses against picornaviruses. However, virus-host co-evolution has selected for viruses capable of replicating despite host cells antiviral defences. In this report, we demonstrate that RIG-I is degraded during encephalomyocarditis virus (EMCV) infection. This effect is mediated by both the viral-encoded 3C protease and caspase proteinase. In addition, we show that RIG-I overexpression confers IFN-beta promoter activation during EMCV infection, in MDA-5 knockout (MDA-5(-/-)) mouse embryo fibroblasts. This induction is followed by a strong inhibition reflecting the ability of EMCV to disrupt RIG-I signalling. Taken together, our data strongly suggest that during evolution RIG-I has been involved for triggering innate immune response to picornavirus infections.