How the HIV1 Vpr protein initiates the host cell response leading to cell cycle arrest in G(2) has remained unknown. Here, we show that recruitment of DCAF1/VprBP by Vpr is essential for its cytostatic activity, which can be abolished either by single mutations of Vpr that impair DCAF1 binding, or by siRNA-mediated silencing of DCAF1. Furthermore, DCAF1 bridges Vpr to DDB1, a core subunit of Cul4 ubiquitin ligases. Altogether these results point to a mechanism where Vpr triggers G(2) arrest by hijacking the Cul4/DDB1(DCAF1) ubiquitin ligase. We further show that, Vpx, a non-cytostatic Vpr-related protein acquired by HIV2 and SIV, also binds DCAF1 through a conserved motif. Thus, Vpr from HIV1 and Vpx from SIV recruit DCAF1 with different physiological outcomes for the host cell. This in turn suggests that both proteins have evolved to preserve interaction with the same Cul4 ubiquitin ligase while diverging in the recognition of host substrates targeted for proteasomal degradation.

No abstract

Treatment for chronic hepatitis C virus (HCV) infection has evolved considerably in the last years. The standard of care (SOC) for HCV infection consists in the combination of pegylated interferon (PEG-IFN) plus ribavirin. However, it only induces a sustained virological response (SVR) in half of genotype 1-infected patients. Several viral and host factors have been associated with non-response: steatosis, obesity, insulin resistance, age, male sex, ethnicity and genotypes. Many studies have demonstrated that in non-responders, some interferon-stimulated genes were upregulated before treatment. Those findings associated to clinical, biochemical and histological data may help detect responders before starting any treatment. This is a very important issue because the standard treatment is physically and economically demanding. The future of HCV treatment would probably consist in the addition of specifically targeted antiviral therapy for HCV such as protease and/or polymerase inhibitors to the SOC. In genotype 1 patients, very promising results have been reported when the protease inhibitor telaprevir or boceprevir is added to the SOC. It increases the SVR rates from approximately 50% (PEG-IFN plus ribavirin) to 70% (for patients treated with a combination of PEG-IFN plus ribavirin plus telaprevir). Different elements are associated with non-response: (i) viral factors, (ii) host factors and (iii) molecular mechanisms induced by HCV proteins to inhibit the IFN signalling pathway. The goal of this review is to present the mechanisms of non-response, to overcome it and to identify factors that can help to predict the response to anti-HCV therapy.

ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF ␤TrCP ubiquitin ligase that binds to ␤TrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.ATF4, a member of the ATF/CREB bZIP transcription factor family, plays a crucial role in response to stress, because multiple intracellular stress pathways (endoplasmic reticulum stress, amino acid deprivation, and exposure to oxidant or reactive metals) converge on a single event, phosphorylation of eIF2␣, which leads both to a general inhibition of protein synthesis but also to the translational up-regulation of the mRNA encoding ATF4 (1-4). Thus the targets of ATF4 are of paramount importance in a generalized stress response. In fact, higher eucaryotes have conserved through activation of ATF4 the same fundamental mechanism used by yeast through up-regulation of GCN4 in response to amino acid starvation (5). In addition, ATF4 is important for cell proliferation and differentiation, because ATF4 knock-out mice display abnormal lens formation (6) and defects in cell proliferation in fetal liver, embryonic lens and hair follicles, as well as an overall reduction in size of the animals (7). ATF4 is a critical regulator of osteoblast differentiation and function (8) and bone resorption (9). ATF4 is also involved in long term memory induction (10). Hence ATF4 is a master transcription factor for which temporal expression and activity are under tight cellular control. ATF4 interacts with several general transcription factors such as TBP, TFIIB, and RAP30 (11). The transcriptional selectivity of ATF4 is modulated by the formation of heterodimers with multiple C/EBP bZIP or AP-1 family members (12-14). CBP and p300 acetylate ATF4 in its bZIP domain (15) and enhance its transcriptional activity (11,15).Acetylation of histone and non-histone proteins is emerging as a central process in transcriptional activation. Nuclear histone acetyltransferases (HATs) 5 act as transcriptional co-activators that have been shown to acetylate different transcription factors, including p53, ␤cate-nin, MyoD, E2F-1, ATF4, and SREBP1 (15-19). The consequences of acetylation on protein function vary from one protein to another depending on where within the protein the acetylation takes place. Acetylation has also been reported to modulate protein-protein interactions, to inhibit nuclear export (20), a...

HCV infection is a global health problem that affects 170 million people worldwide. The severity of the disease varies from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma (HCC). Recently, the standard of care for genotype 1 patients has greatly improved with the addition of protease inhibitors (telaprevir or boceprevir) to pegylated interferon (PegIFN) and ribavirin (RBV). The prediction of fibrosis progression and the response to antiviral treatment are two major issues in the management of patients with chronic hepatitis C. Differential expression of mRNAs was first analyzed for both progression of fibrosis and treatment response. Specific polymorphisms, associated with either fibrosis or viral response, were identified thanks to major improvements in genome scanning technologies. Since 2009, several independent genome wide association studies (GWAS) have reported an association between genetic polymorphisms within the IL-28B promoter and both natural and treatment-induced clearance in genotype 1 infected patients. These different studies showed the strong association and the importance of IL-28B polymorphisms in the treatment response. Combining the different genetic factors could improve their predictive value and help identify patients at a high risk of progression of fibrosis as well as those with a lower chance of responding to treatment. The aim of this review was to discuss the genomic factors (mRNAs, miRNAs, and SNPs) and HCV infection with clinical implications for either progression of fibrosis or treatment response. Recent findings on the IL-28B polymorphism and its application in clinical practice will also be discussed.

The Ras-association domain family 1 (RASSF1) gene has seven different isoforms; isoform A is a tumor-suppressor gene (RASSF1A). The promoter of RASSF1A is inactivated in many cancers, whereas the expression of another major isoform, RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A, interacts with BTrCP. Binding of RASSF1C to BTrCP involves serine 18 and serine 19 of the SS 18 GYXS 19 motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by BTrCP; however, surprisingly, the association between RASSF1C and BTrCP does not occur via the BTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the B-catenin oncogene, due to inhibition of its BTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for B-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on B-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis. [Cancer Res 2007;67(3):1054-61]

Viral protein U (Vpu) of HIV-1 has two known functions in replication of the virus: degradation of its cellular receptor CD4 and enhancement of viral particle release. Vpu binds CD4 and simultaneously recruits the βTrCP subunit of the SCFβTrCP ubiquitin ligase complex through its constitutively phosphorylated DS52GXXS56 motif. In this process, Vpu was found to escape degradation, while inhibiting the degradation of βTrCP natural targets such as β-catenin and IκBα. We further addressed this Vpu inhibitory function with respect to the degradation of Emi1 and Cdc25A, two βTrCP substrates involved in cell-cycle progression. In the course of these experiments, we underscored the importance of a novel phosphorylation site in Vpu. We show that, especially in cells arrested in early mitosis, Vpu undergoes phosphorylation of the serine 61 residue, which lies adjacent to the βTrCP-binding motif. This phosphorylation event triggers Vpu degradation by a βTrCP-independent process. Mutation of Vpu S61 in the HIV-1 provirus extends the half-life of the protein and significantly increases the release of HIV-1 particles from HeLa cells. However, the S61 determinant of regulated Vpu turnover is highly conserved within HIV-1 isolates. Altogether, our results highlight a mechanism where differential phosphorylation of Vpu determines its fate as an adaptor or as a substrate of distinct ubiquitin ligases. Conservation of the Vpu degradation determinant, despite its negative effect on virion release, argues for a role in overall HIV-1 fitness.

Staging fibrosis is crucial for the prognosis and to determine the rapid need of treatment in patients with chronic hepatitis B (CHB) and C (CHC). The expression of 13 fibrosis-related microRNAs (miRNAs) (miR-20a, miR-21, miR-27a, miR-27b, miR-29a, miR-29c, miR-92a, miR-122, miR-146a, miR-155, miR-221, miR-222, and miR-224) was analyzed in 194 serums and 177 liver biopsies of patients with either CHB or CHC to develop models to diagnose advanced fibrosis and cirrhosis (Metavir F3-F4). In CHB patients, the model (serum miR-122, serum miR-222, platelet count and alkaline phosphatase) was more accurate than APRI and FIB-4 to discriminate in between mild and moderate fibrosis (F1-F2) and F3-F4 (AUC of CHB model: 0.85 vs APRI: 0.70 and FIB-4: 0.81). In CHC patients, the model (hepatic miR-122, hepatic miR-224, platelet count, albumin and alanine aminotransferase) was more accurate than both APRI and FIB-4 to discriminate in between patients with F3-F4 and F1-F2 (AUC of the CHC model = 0.93 vs APRI: 0.86 and FIB-4: 0.79). Most of the miRNAs tested were differentially expressed in patients with CHB and CHC. In particular, serum miR-122 was 28-fold higher in patients with CHB than in those with CHC. Both CHB and CHC models may help for the diagnosis of advanced fibrosis and cirrhosis (F3-F4).