Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.

ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.

A Proteomics-Based Approach Identifies the NEDD4 Adaptor NDFIP2 as an Important Regulator of Ifitm3 Levels.

IFITMs are a family of highly related interferon-induced transmembrane proteins that interfere with the processes of fusion between viral and cellular membranes and are thus endowed with broad antiviral properties. A number of studies have shown how the antiviral potency of IFITMs is highly dependent on their steady-state levels, their intracellular distribution and a complex pattern of post-translational modifications, parameters that are overall tributary of a number of cellular partners. In an effort to identify additional protein partners involved in the biology of IFITMs, we devised a proteomics-based approach based on the piggyback incorporation of IFITM3 partners into extracellular vesicles. MS analysis of the proteome of vesicles bearing or not bearing IFITM3 identified the NDFIP2 protein adaptor protein as an important regulator of IFITM3 levels. NDFIP2 is a membrane-anchored adaptor protein of the E3 ubiquitin ligases of the NEDD4 family that have already been found to be involved in IFITM3 regulation. We show here that NDFIP2 acts as a recruitment factor for both IFITM3 and NEDD4 and mediates their distribution in lysosomal vesicles. The genetic inactivation and overexpression of NDFIP2 drive, respectively, lower and higher levels of IFITM3 accumulation in the cell, overall suggesting that NDFIP2 locally competes with IFITM3 for NEDD4 binding. Given that NDFIP2 is itself tightly regulated and highly responsive to external cues, our study sheds light on a novel and likely dynamic layer of regulation of IFITM3.

Correction: The interferon stimulated gene 20 protein (ISG20) is an innate defense antiviral factor that discriminates self versus non-self translation.

[This corrects the article DOI: 10.1371/journal.ppat.1008093.].

Trim69 is a microtubule regulator that acts as a pantropic viral inhibitor.

Through a screen that combines functional and evolutionary analyses, we identified tripartite motif protein (Trim69), a poorly studied member of the Trim family, as a negative regulator of HIV-1 infection in interferon (IFN)-stimulated myeloid cells. Trim69 inhibits the early phases of infection of HIV-1, but also of HIV-2 and SIVMAC in addition to the negative and positive-strand RNA viruses vesicular stomatitis virus and severe acute respiratory syndrome coronavirus 2, with magnitudes that depend on the combination between cell type and virus. Mechanistically, Trim69 associates directly to microtubules and its antiviral activity is linked to its ability to promote the accumulation of stable microtubules, a program that we uncover to be an integral part of antiviral IFN-I responses in myeloid cells. Overall, our study identifies Trim69 as the antiviral innate defense factor that regulates the properties of microtubules to limit viral spread and highlights the cytoskeleton as an unappreciated battleground in the host-pathogen interactions that underlie viral infections.

A novel domain within the CIL regulates egress of IFITM3 from the Golgi and reveals a regulatory role of IFITM3 on the secretory pathway.

The InterFeron-Induced TransMembrane proteins (IFITMs) are members of the dispanin/CD225 family that act as broad viral inhibitors by preventing viral-to-cellular membrane fusion. In this study, we uncover egress from the Golgi as an important step in the biology of IFITM3 by identifying the domain that regulates this process and that similarly controls the egress of the dispanins IFITM1 and PRRT2, protein linked to paroxysmal kinesigenic dyskinesia. In the case of IFITM3, high levels of expression of wild-type, or mutations in the Golgi egress domain, lead to accumulation of IFITM3 in the Golgi and drive generalized glycoprotein trafficking defects. These defects can be relieved upon incubation with Amphotericin B, compound known to relieve IFITM-driven membrane fusion defects, as well as by v-SNARE overexpression, suggesting that IFITM3 interferes with membrane fusion processes important for Golgi functionalities. The comparison of glycoprotein trafficking in WT versus IFITMs-KO cells indicates that the modulation of the secretory pathway is a novel feature of IFITM proteins. Overall, our study defines a novel domain that regulates the egress of several dispanin/CD225 members from the Golgi and identifies a novel modulatory function for IFITM3.

The interferon stimulated gene 20 protein (ISG20) is an innate defense antiviral factor that discriminates self versus non-self translation.

ISG20 is a broad spectrum antiviral protein thought to directly degrade viral RNA. However, this mechanism of inhibition remains controversial. Using the Vesicular Stomatitis Virus (VSV) as a model RNA virus, we show here that ISG20 interferes with viral replication by decreasing protein synthesis in the absence of RNA degradation. Importantly, we demonstrate that ISG20 exerts a translational control over a large panel of non-self RNA substrates including those originating from transfected DNA, while sparing endogenous transcripts. This activity correlates with the protein's ability to localize in cytoplasmic processing bodies. Finally, these functions are conserved in the ISG20 murine ortholog, whose genetic ablation results in mice with increased susceptibility to viral infection. Overall, our results posit ISG20 as an important defense factor able to discriminate the self/non-self origins of the RNA through translation modulation.

The DNA damage induced by the Cytosine Deaminase APOBEC3A Leads to the production of ROS.

Human apolipoprotein B mRNA-editing catalytic polypeptide-like 3 proteins (APOBEC3s or A3s) are cytosine deaminases that protect cells by introducing promutagenic uraciles in invading retro-elements. However as a drawback of this protective activity, A3s can also target cellular DNA, leading to DNA damage and to the accumulation of somatic mutations that may contribute to tumorigenesis. Among A3s, A3A has been shown to be particularly proficient at mutagenizing cellular DNA, but whether this enzyme exerts additional effects on the cellular physiology remains unclear. Here, we show that A3A editing of cellular DNA leads to reactive oxygen species (ROS) production through Nox-enzymes. ROS production occurs in two distinct model cell lines and it is contingent upon DNA replication and intact enzymatic properties of A3A. For the first time, our results indicate that the editing activity of A3A results in the induction of a pro-inflammatory state that may possibly contribute to the constitution of a tumorigenic-prone environment.

Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs.

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle.

Hydroxyurea synergizes with valproic acid in wild-type p53 acute myeloid leukaemia.

Palliative care in acute myeloid leukaemia (AML) is inadequate. For elderly patients, unfit for intensive chemotherapy, median survival is 2-3 months. As such, there is urgent demand for low-toxic palliative alternatives. We have repositioned two commonly administered anti-leukaemia drugs, valproic acid (VPA) and hydroxyurea (HU), as a combination therapy in AML. The anti-leukemic effect of VPA and HU was assessed in multiple AML cell lines confirming the superior anti-leukemic effect of combination therapy. Mechanistic studies revealed that VPA amplified the ability of HU to slow S-phase progression and this correlated with significantly increased DNA damage. VPA was also shown to reduce expression of the DNA repair protein, Rad51. Interestingly, the tumour suppressor protein p53 was revealed to mitigate cell cycle recovery following combination induced arrest. The efficacy of combination therapy was validated in vivo. Combination treatment increased survival in OCI-AML3 and patient-derived xenograft mouse models of AML. Therapy response was confirmed by optical imaging with multiplexed near-infrared labelled antibodies. The combination of HU and VPA indicates significant potential in preclinical models of AML. Both compounds are widely available and well tolerated. We believe that repositioning this combination could significantly enhance the palliative care of patients unsuited to intensive chemotherapy.

Identification of a New Ribonucleoside Inhibitor of Ebola Virus Replication.

The current outbreak of Ebola virus (EBOV) in West Africa has claimed the lives of more than 15,000 people and highlights an urgent need for therapeutics capable of preventing virus replication. In this study we screened known nucleoside analogues for their ability to interfere with EBOV replication. Among them, the cytidine analogue ß-d-N4-hydroxycytidine (NHC) demonstrated potent inhibitory activities against EBOV replication and spread at non-cytotoxic concentrations. Thus, NHC constitutes an interesting candidate for the development of a suitable drug treatment against EBOV.