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Abstract Background We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form. Results Several bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway. Conclusion Our observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.

In eukaryotic cells, the spatial distribution between cytoplasm and nucleus is essential for cell homeostasis. This dynamic distribution is selectively regulated by the nuclear pore complex (NPC), which allows the passive or energy-dependent transport of proteins between these two compartments. Viruses possess many strategies to hijack nucleocytoplasmic shuttling for the benefit of their viral replication. Here, we review how viruses interfere with the karyopherin CRM1 that controls the nuclear export of protein cargoes. We analyze the fact that the viral hijacking of CRM1 provokes are-localization of numerous cellular factors in a suitable place for specific steps of viral replication. While CRM1 emerges as a critical partner for viruses, it also takes part in antiviral and inflammatory response regulation. This review also addresses how CRM1 hijacking affects it and the benefits of CRM1 inhibitors as antiviral treatments.

The stability of intracellular proteins is highly variable, from a few minutes to several hours, and can be tightly regulated to respond to external and internal cellular environment changes. Several techniques can be used to study the stability of a specific protein, including pulse-chase labeling and blocking of translation. Another approach that has gained interest in recent years is fusing a protein of interest to a fluorescent reporter. In this report, the authors present a new version of this approach aimed at optimizing expression and comparison of the two reporter proteins. The authors show that the system works efficiently in various cells and can be useful for studying changes in protein stability and assessing the effects of drugs.

Before the establishment of an adaptive immune response, retroviruses can be targeted by several cellular host factors at different stages of the viral replication cycle. This intrinsic immunity relies on a large diversity of antiviral processes. In the case of HTLV-1 infection, these active innate host defense mechanisms are debated. Among these mechanisms, we focused on an RNA decay pathway called nonsense-mediated mRNA decay (NMD), which can target multiple viral RNAs, including HTLV-1 unspliced RNA, as has been recently demonstrated. NMD is a co-translational process that depends on the RNA helicase UPF1 and regulates the expression of multiple types of host mRNAs. RNA sensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP) composition. HTLV-1 has evolved several means to evade the NMD threat, leading to NMD inhibition. In the early steps of infection, NMD inhibition favours the production of HTLV-1 infectious particles, which may contribute to the survival of the fittest clones despite genome instability; however, its direct long-term impact remains to be investigated.

Many posttranscriptional processes are known to regulate gene expression and some of them can act as an antiviral barrier. The nonsense-mediated mRNA decay (NMD) was first identified as an mRNA quality control pathway that triggers rapid decay of mRNA containing premature stop codons due to mutations. NMD is now considered as a general posttranscriptional regulation pathway controlling the expression of a large set of cellular genes. In addition to premature stop codons, many other features including alternative splicing, 5′ uORF, long 3′ UTR, selenocystein codons, and frameshift are able to promote NMD. Interestingly, many viral mRNAs exhibit some of these features suggesting that virus expression and replication might be sensitive to NMD. Several studies, including recent ones, have shown that this is the case for retroviruses; however, it also appears that retroviruses have developed strategies to overcome NMD in order to protect their genome and ensure a true expression of their genes. As a consequence of NMD inhibition, these viruses also affect the expression of host genes that are prone to NMD, and therefore can potentially trigger pathological effects on infected cells. Here, we review recent studies supporting this newly uncovered function of the NMD pathway as a defense barrier that viruses must overcome in order to replicate. [ABSTRACT FROM AUTHOR]

UPF1 is a central protein in nonsense-mediated mRNA decay (NMD), but contribution of its RNA processivity to NMD is unclear. Here, the authors show how the retroviral Tax protein interacts with and inhibits UPF1, and demonstrate that UPF1’s translocase activity contributes to NMD.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

In eukaryotic cells, the spatial distribution between cytoplasm and nucleus is essential for cell homeostasis. This dynamic distribution is selectively regulated by the nuclear pore complex (NPC), which allows the passive or energy-dependent transport of proteins between these two compartments. Viruses possess many strategies to hijack nucleocytoplasmic shuttling for the benefit of their viral replication. Here, we review how viruses interfere with the karyopherin CRM1 that controls the nuclear export of protein cargoes. We analyze the fact that the viral hijacking of CRM1 provokes are-localization of numerous cellular factors in a suitable place for specific steps of viral replication. While CRM1 emerges as a critical partner for viruses, it also takes part in antiviral and inflammatory response regulation. This review also addresses how CRM1 hijacking affects it and the benefits of CRM1 inhibitors as antiviral treatments.

Telomeric repeat-binding factor 2 (TRF2) is a subunit of the shelterin protein complex, which binds to and protects telomeres from unwanted DNA damage response (DDR) activation. TRF2 expression plays a pivotal role in aging and cancer, being downregulated during cellular senescence and overexpressed during oncogenesis. Cancers overexpressing TRF2 often exhibit a poor prognosis. In cancer cells, TRF2 plays multiple functions, including telomere protection and non-cell autonomous roles, promoting neo-angiogenesis and immunosuppression. We present here an original screening strategy, which enables identification of small molecules that decrease or increase TRF2 expression. By screening a small library of Food and Drug Agency (FDA)-approved drugs, we identified two molecules (AR-A014418 and alexidine·2HCl) that impaired tumor growth, neo-angiogenesis and immunosuppression by downregulating TRF2 expression in a mouse xenograft model. These results support the chemotherapeutic strategy of downregulating TRF2 expression to treat aggressive human tumors and validate this cell-based assay capable of screening for potential anti-cancer and anti-aging molecules by modulating TRF2 expression levels.

Altered expression of the translation factor eIF3e is associated with breast cancer occurrence. We have previously shown that eIF3e deficiency leads to an impaired DNA damage response with a marked decrease in DNA repair by homologous recombination. Here, we explored the possibility to exploit this DNA repair defect in targeted cancer therapy using PARP inhibitors. Surprisingly, eIF3e-deficient breast cancer cells are resistant to these drugs, in contrast to BRCA1-deficient cells. Studying this, we found that eIF3e-depleted cells synthesize lowered amounts of PARP1 protein, due to a weakened translation of the corresponding mRNA, associated with a strong decrease in cellular poly(ADP-ribosyl)ation. Additionally, we discovered that the mTORC1 signaling pathway is aberrantly activated in response to eIF3e suppression. Together, these PARP1 and mTORC1 dysfunctions upon eIF3e depletion are causally linked to induction of cellular senescence associated with a pro-inflammatory secretory phenotype. This study provides mechanistic insights into how eIF3e protects against breast cancer, with potential novel cancer therapeutic opportunities. While PARP inhibitors appear as inappropriate drugs for eIF3e-deficient breast tumors, our findings suggest that such cancers may benefit from senolytic drugs or mTORC1 inhibitors., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Morris et al.)

Before the establishment of an adaptive immune response, retroviruses can be targeted by several cellular host factors at different stages of the viral replication cycle. This intrinsic immunity relies on a large diversity of antiviral processes. In the case of HTLV-1 infection, these active innate host defense mechanisms are debated. Among these mechanisms, we focused on an RNA decay pathway called nonsense-mediated mRNA decay (NMD), which can target multiple viral RNAs, including HTLV-1 unspliced RNA, as has been recently demonstrated. NMD is a co-translational process that depends on the RNA helicase UPF1 and regulates the expression of multiple types of host mRNAs. RNA sensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP) composition. HTLV-1 has evolved several means to evade the NMD threat, leading to NMD inhibition. In the early steps of infection, NMD inhibition favours the production of HTLV-1 infectious particles, which may contribute to the survival of the fittest clones despite genome instability; however, its direct long-term impact remains to be investigated., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study, in the writing of the manuscript, or in the decision to publish the results.

Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5'UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels., (© 2020 Panthu et al.)

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.