Nucleolin Malonylation as a Nuclear-Cytosol Signal Exchange Mechanism to Drive Cell Proliferation in Hepatocarcinoma by Enhancing AKT Translation.

Cancer cells undergo metabolic reprogramming that is intricately linked to malignancy. Protein acylations are especially responsive to metabolic changes, influencing signal transduction pathways and fostering cell proliferation. However, as a novel type of acylations, the involvement of malonylation in cancer remains poorly understood. In this study, we observed a significant reduction in malonyl-CoA levels in hepatocellular carcinoma (HCC), which correlated with a global decrease in malonylation. Subsequent nuclear malonylome analysis unveiled nucleolin (NCL) malonylation, which was notably enhanced in HCC biopsies. we demonstrated that NCL undergoes malonylation at lysine residues 124 and 398. This modification triggers the translocation of NCL from the nucleolus to nucleoplasm and cytoplasm, binding to AKT mRNA, and promoting AKT translation in HCC. Silencing AKT expression markedly attenuated HCC cell proliferation driven by NCL malonylation. These findings collectively highlight nuclear signaling in modulating AKT expression, suggesting NCL malonylation as a novel mechanism through which cancer cells drive cell proliferation.

Palmitoylation of SARS-CoV-2 Envelope protein is central to virus particle formation.

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles.IMPORTANCEIn this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors.

Antiviral role of IFITM3 in prototype foamy virus infection.

BACKGROUND: Foamy viruses (FVs) are retroviruses with unique replication strategies that cause lifelong latent infections in their hosts. FVs can also produce foam-like cytopathic effects in vitro. However, the effect of host cytokines on FV replication requires further investigation. Although interferon induced transmembrane (IFITMs) proteins have become the focus of antiviral immune response research due to their broad-spectrum antiviral ability, it remains unclear whether IFITMs can affect FV replication. METHOD: In this study, the PFV virus titer was characterized by measuring luciferase activity after co-incubation of PFVL cell lines with the cell culture supernatants (cell-free PFV) or the cells transfected with pcPFV plasmid/infected with PFV (cell-associated PFV). The foam-like cytopathic effects of PFV infected cells was observed to reflect the virus replication. The total RNA of PFV infected cells was extracted, and the viral genome was quantified by Quantitative reverse transcription PCR to detect the PFV entry into target cells. RESULTS: In the present study, we demonstrated that IFITM1-3 overexpression inhibited prototype foamy virus (PFV) replication. In addition, an IFITM3 knockdown by small interfering RNA increased PFV replication. We further demonstrated that IFITM3 inhibited PFV entry into host cells. Moreover, IFITM3 also reduced the number of PFV envelope proteins, which was related to IFITM3 promoted envelope degradation through the lysosomal pathway. CONCLUSIONS: Taken together, these results demonstrate that IFITM3 inhibits PFV replication by inhibiting PFV entry into target cells and reducing the number of PFV envelope.

Interferon activates promoter of Nmi gene via interferon regulator factor-1.

N-Myc interactor (Nmi) is reported to participate in many activities, such as signaling transduction, transcription regulation, and antiviral responses. As Nmi may play important roles in interferon (IFN)-induced responses, we investigated the mechanism how Nmi protein is regulated. We identified and cloned the promoter of Nmi gene. Sequence analysis and luciferase assays shown that an IFN-stimulated response element (ISRE) and a GC box in the promoter were essential for the basal transcription activity of Nmi gene. We also found that interferon regulatory factor 1 (IRF-1) could activate transcription of Nmi by binding to the ISRE in the promoter. Knockdown of IRF-1 decreases IFN-induced Nmi transcription. These results revealed that IRF-1 is involved in the IFN-inducible expression of Nmi.

Malonate differentially affects cell survival and confers chemoresistance in cancer cells via the induction of p53-dependent autophagy.

Metabolic network intertwines with cancerous signaling and drug responses. Malonate is a prevailing metabolite in cancer and a competitive inhibitor of succinate dehydrogenase (SDH). Recent studies showed that malonate induced reactive oxygen species (ROS)-dependent apoptosis in neuroblastoma cells, but protected cells from ischemia-reperfusion injury. We here revealed that malonate differentially regulated cell death and survival in cancer cells. While high-dose malonate triggered ROS-dependent apoptosis, the low-dose malonate induced autophagy and conferred resistance to multiple chemotherapeutic agents. Mechanistically, our results showed that malonate increased p53 stability and transcriptionally up-regulated autophagy modulator DRAM (damage-regulated autophagy modulator), thus promoting autophagy. We further proved that autophagy is required for malonate-associated chemoresistance. Collectively, our findings suggest that malonate plays a double-edge function in cancer response to stressors, and highlights a pro-cancer impact of p53-induced autophagy in response to malonate.

Effect of Different Nuclear Localization Signals on the Subcellular Localization and Anti-HIV-1 Function of the MxB Protein.

Interferon exerts its antiviral activity by stimulating the expression of antiviral proteins. These interferon stimulate genes (ISGs) often target a group of viruses with unique molecular mechanisms. One such ISG is myxovirus resistance B (MxB) that has been reported to inhibit human immunodeficiency virus type 1 (HIV-1) by targeting viral capsid and impairing nuclear import of viral DNA. The antiviral specificity of MxB is determined by its N-terminal 25 amino acids sequence which has the nuclear localization activity, therefore functions as a nuclear localization signal (NLS). In this study, we report that the bipartite NLS, but not the classic NLS, the PY-NLS, nor the arginine-rich NLS, when used to replace the N-terminal sequence of MxB, drastically suppress HIV-1 gene expression and virus production, thus creates a new anti-HIV-1 mechanism. MxB preserves its anti-HIV-1 activity when its N-terminal sequence is replaced by the arginine-rich NLS. Interestingly, the arginine-rich NLS allows MxB to inhibit HIV-1 CA mutants that are otherwise resistant to wild type MxB, which suggests sequence specific targeting of viral capsid. Together, these data implicate that it is not the nuclear import function itself, but rather the sequence and the mechanism of action of the NLS which define the antiviral property of MxB.

Palmitoylation of the Bovine Foamy Virus Envelope Glycoprotein Is Required for Viral Replication.

Membrane proteins of enveloped viruses have been reported to undergo palmitoylation, a post-translational modification often having a critical role in the function of these viral proteins and hence viral replication. In this study, we report that the foamy virus (FV) envelope (Env) glycoprotein is palmitoylated. Specifically, we found that bovine foamy virus (BFV) Env (BEnv) is palmitoylated at amino acid positions C58 and C59 by BDHHC3 and BDHHC20 in a DHHC motif-dependent manner. In addition, mutations C58S and C58/59S significantly decrease cell surface expression of BEnv, subviral particle (SVP) egress, and its membrane fusion activity, thus ultimately inhibiting BFV replication. The C59S mutation exerts a minor effect in this regard. Taken together, these data demonstrate that the function of BEnv in the context of BFV replication is under the regulation of palmitoylation.

Prototype foamy virus downregulates RelB expression to facilitate viral replication.

Foamy viruses (FVs) are classified in the subfamily Spumaretrovirinae and bridge the gap between Orthoretrovirinae and Hepadnaviridae. FVs have strong cytopathic effects against cells cultured in vitro. However, they establish lifelong latent infections without evident pathology in the host. The roles of cellular factors in FV replication are poorly understood. To better understand this area, we determined the transcriptomes of HT1080 cells infected with prototype foamy virus (PFV) to measure the effect of PFV infection on the expression of cellular genes. We found that the level of RelB mRNA, a member of the nuclear factor-κB (NF-κB) protein family, was significantly decreased as a result of PFV infection, and this was further confirmed with real-time PCR. Interestingly, overexpression of RelB reduced PFV replication, whereas its depletion using small interfering RNA increased PFV replication. This inhibitory effect of RelB results from diminished transactivation of the viral long terminal repeat (LTR) promoter and an internal promoter (IP) by viral Tas protein. Together, these data demonstrate that PFV infection downregulates the viral inhibitory host factor RelB, which otherwise restricts viral gene expression.

HIV-1 resists MxB inhibition of viral Rev protein.

The interferon-inducible myxovirus resistance B (MxB) protein has been reported to inhibit HIV-1 and herpesviruses by blocking the nuclear import of viral DNA. Here, we report a new antiviral mechanism in which MxB restricts the nuclear import of HIV-1 regulatory protein Rev, and as a result, diminishes Rev-dependent expression of HIV-1 Gag protein. Specifically, MxB disrupts the interaction of Rev with the nuclear transport receptor, transportin 1 (TNPO1). Supporting this, the TNPO1-independent Rev variants become less restricted by MxB. In addition, HIV-1 can overcome this inhibition by MxB through increasing the expression of multiply spliced viral RNA and hence Rev protein. Therefore, MxB exerts its anti-HIV-1 function through interfering with the nuclear import of both viral DNA and viral Rev protein.

[Identification of the Transcriptional Activity Domain of EV71 3D(pol)].

Enterovirus 71（EV71）is one of the major pathogens of hand, foot and mouth disease (HFMD). The EV71 genome encodes an RNA-dependent RNA polymerase（RdRp）,3D(pol),which is critical for genome transcription and translation. However, how the 3D(pol) interacts with the host remains unclear. Yeast two-hybrid systems provide an effective approach for detecting protein-protein interactions. In this report, we inserted the DNA sequence of 3D(pol) into the pGBKT7 vector as the bait plasmid for the yeast two-hybrid experiment and transformed the plasmid into the yeast AH109 strain. We detected the expression,cytotoxicity and self-activity of 3D(pol).The 3D(pol) expressed well without affecting cell growth but exhibited strong transcriptional activation in yeast cells. We further constructed a series of pGBKT7-3D(pol) deletion mutants and identified the shortest transcriptional activation domain（1-94aa）using a self-activation assay. The results provide a molecular basis for screening the host proteins that interact with 3D(pol) using the yeast two hybrid system.