MOV10 recruits DCP2 to decap human LINE-1 RNA by forming large cytoplasmic granules with phase separation properties.

Long interspersed element 1 (LINE-1) is the only active autonomous mobile element in the human genome. Its transposition can exert deleterious effects on the structure and function of the host genome and cause sporadic genetic diseases. Tight control of LINE-1 mobilization by the host is crucial for genetic stability. In this study, we report that MOV10 recruits the main decapping enzyme DCP2 to LINE-1 RNA and forms a complex of MOV10, DCP2, and LINE-1 RNP, exhibiting liquid-liquid phase separation (LLPS) properties. DCP2 cooperates with MOV10 to decap LINE-1 RNA, which causes degradation of LINE-1 RNA and thus reduces LINE-1 retrotransposition. We here identify DCP2 as one of the key effector proteins determining LINE-1 replication, and elucidate an LLPS mechanism that facilitates the anti-LINE-1 action of MOV10 and DCP2.

Schlafen 5 suppresses human immunodeficiency virus type 1 transcription by commandeering cellular epigenetic machinery.

Schlafen-5 (SLFN5) is an interferon-induced protein of the Schlafen family, which are involved in immune responses and oncogenesis. To date, little is known regarding its anti-HIV-1 function. Here, the authors report that overexpression of SLFN5 inhibits HIV-1 replication and reduces viral mRNA levels, whereas depletion of endogenous SLFN5 promotes HIV-1 replication. Moreover, they show that SLFN5 markedly decreases the transcriptional activity of HIV-1 long terminal repeat (LTR) via binding to two sequences in the U5-R region, which consequently represses the recruitment of RNA polymerase II to the transcription initiation site. Mutagenesis studies show the importance of nuclear localization and the N-terminal 1-570 amino acids fragment in the inhibition of HIV-1. Further mechanistic studies demonstrate that SLFN5 interacts with components of the PRC2 complex, G9a and Histone H3, thereby promoting H3K27me2 and H3K27me3 modification leading to silencing HIV-1 transcription. In concert with this, they find that SLFN5 blocks the activation of latent HIV-1. Altogether, their findings demonstrate that SLFN5 is a transcriptional repressor of HIV-1 through epigenetic modulation and a potential determinant of HIV-1 latency.

An integrative genomic analysis of transcriptional profiles identifies characteristic genes and patterns in HIV-infected long-term non-progressors and elite controllers.

BACKGROUND: Despite that most HIV-infected individuals experience progressive CD4+ T cell loss and develop AIDS, a minority of HIV-infected individuals remain asymptomatic and maintain high level CD4+ T cell counts several years after seroconversion. Efforts have been made to understand the determinants of the nonprogressive status, exemplified by the clinical course of elite controllers (ECs) who maintain an undetectable viremia and viremic nonprogressors (VNPs) who have a normal CD4+ count in spite of circulating viral load. However, the intrinsic mechanism underlying nonprogression remained elusive. In this study, we performed an integrative analysis of transcriptional profiles to pinpoint the underlying mechanism for a naturally occurring viral control. METHODS: Three microarray datasets, reporting mRNA expression of the LTNPs or ECs in HIV-infected patients, were retrieved from Gene Expression Ominbus (GEO) or Arrayexpress databases. These datasets, profiled on the same type of microarray chip, were selected and merged by a bioinformatic approach to build a meta-analysis derived transcriptome (MADNT). In addition, we investigated the different transcriptional pathways and potential biomarkers in CD4+ and CD8+ cells in ECs and whole blood in VNPs compared to HIV progressors. The combined transcriptome and each subgroup was subject to gene set enrichment analysis and weighted co-expression network analysis to search potential transcription patterns related to the non-progressive status. RESULTS: 30 up-regulated genes and 83 down-regulated genes were identified in lymphocytes from integrative meta-analysis of expression data. The interferon response and innate immune activation was reduced in both CD4+ and CD8+ T cells from ECs. Several characteristic genes including CMPK1, CBX7, EIF3L, EIF4A and ZNF395 were indicated to be highly correlated with viremic control. Besides that, we indicated that the reduction of ribosome components and blockade of translation facilitated AIDS disease progression. Most interestingly, among VNPs who have a relatively high viral load, we detected a two gene-interaction networks which showed a strong correlation to immune control even with a rigorous statistical threshold (p value = 2-e4 and p value = 0.004, respectively) by WGCNA. CONCLUSIONS: We have identified differentially expressed genes and transcriptional patterns in ECs and VNPs compared to normal chronic HIV-infected individuals. Our study provides new insights into the pathogenesis of HIV and AIDS and clues for the therapeutic strategies for anti-retroviral administration.

2-thio-6-azauridine inhibits Vpu mediated BST-2 degradation.

BACKGROUD: BST-2 is an interferon-induced host restriction factor that inhibits the release of diverse mammalian enveloped viruses from infected cells by physically trapping the newly formed virions onto the host cell surface. Human Immunodeficiency Virus-1 (HIV-1) encodes an accessory protein Vpu that antagonizes BST-2 by down-regulating BST-2 from the cell surface. RESULTS: Using a cell-based ELISA screening system, we have discovered a lead compound, 2-thio-6-azauridine, that restores cell surface BST-2 level in the presence of Vpu. This compound has no effect on the expression of BST-2 and Vpu, but inhibits Vpu-mediated BST-2 down-regulation and exerts no effect on Vpu-induced down-regulation of CD4 or KSHV K5 protein induced BST-2 down-regulation. 2-thio-6-azauridine suppresses HIV-1 production in a BST-2-dependent manner. Further results indicate that 2-thio-6-azauridine does not interrupt the interaction of BST-2 with Vpu and ß-TrCP2, but decreases BST-2 ubiquitination. CONCLUSION: Our study demonstrates the feasibility of using small molecules to target Vpu function and sensitize wild type HIV-1 to BST-2-mediated host restriction.

Citron kinase enhances ubiquitination of HIV-1 Gag protein and intracellular HIV-1 budding.

Assembly and budding of human immunodeficiency virus type 1 (HIV-1) particles is a complex process involving a number of host proteins. We have previously reported that the RhoA effector citron kinase enhances HIV-1 production. However, the underlying mechanism is not clear. In this study, we found that citron kinase interacted with HIV-1 Gag protein via its zinc finger and leucine zipper domains. Electron microscopy analysis revealed that citron kinase induced viral particle assembly in multivesicular bodies (MVBs). Citron kinase enhanced ubiquitination of HIV-1 Gag protein. Knockdown of Nedd4L, a member of the HECT ubiquitin E3 ligase family, partly decreased the ability of citron kinase to enhance HIV-1 production and reduced ubiquitination of HIV-1 Gag. Interestingly, the function of citron kinase to promote HIV-1 budding was severely impaired when endogenous ALIX was knocked down. Overexpression of the AAA-type ATPase VPS4 eliminated citron-kinase-mediated enhancement of HIV-1 production. Our results suggest that citron kinase interacts with HIV-1 Gag and enhances HIV-1 production by promoting Gag ubiquitination and inducing viral release via the MVB pathway.

[The sensitivity of transmitted/founder HIV-1 to anti-HIV drugs].

The majority of mucosal HIV-1 infection is initially established by a few HIV-1 viral variants, followed by the development of overt systemic infection, and these viral variants are known as transmitted/ founder viruses(T/F viruses). Investigation of the sensitivity of T/F virus to different anti-HIV-1 drugs will provide the best strategies of pre-exposure prophylaxis(Pr EP) for high-risk groups of HIV-infected patients. Herein we constructed for the first time, a luciferase reporter system for HIV-1 T/F viruses, and then compared the drug sensitivity between T/F viruses and chronic infection virus. The result showed that the 50% inhibitory concentration (IC(50)) of nucleoside reverse transcriptase inhibitors(NRTIs), integrase inhibitors(INIs) and protease inhibitors(PIs) were not significantly different between the T/F viruses and chronic infection viruses of the same subtype(P < 0.05), while non-nucleoside reverse transcriptase inhibitors(NNRTIs) showed a moderate resistance to T/F viruses, with a significant increase in IC50(P < 0.05). The conclusion suggests that when patients are in high-risk or in the acute infection of HIV-1, NNRTIs should be avoided in the first-line antiretroviral therapy regimens.

[Establishment and application of a drug screening model for anti-prostate cancer agents targeting androgen receptor].

Androgen receptor(AR) plays an important role in the maintenance of prostate function and development of prostate cancer. AR is the key target in the therapy of prostate cancer. In this study, a cell-based screening assay was established by dual-luciferase reporter system to analyze the activity of AR. In the screening assay, we detected the anti-prostate cancer activities of rhodiola root extract, wild kiwifruit root extract and tripterygium wilfordii root extract, which may provide a new strategy for the treatment of prostate cancer.

Progress and challenges in the use of latent HIV-1 reactivating agents.

Highly active antiretroviral therapy (HAART) can effectively suppress the replication of human immunodeficiency virus-1 (HIV-1) and block disease progression. However, chronic HIV-1 infection remains incurable due to the persistence of a viral reservoir, including the transcriptionally silent provirus in CD4(+) memory T cells and the sanctuary sites that are inaccessible to drugs. Reactivation and the subsequent elimination of latent virus through virus-specific cytotoxic effects or host immune responses are critical strategies for combating the disease. Indeed, a number of latency reactivating reagents have been identified through mechanism-directed approaches and large-scale screening, including: (1) histone deacetylase inhibitors (HDACi); (2) cytokines and chemokines; (3) DNA methyltransferase inhibitors (DNMTI); (4) histone methyltransferase inhibitors (HMTI); (5) protein kinase C (PKC) activators; (6) P-TEFb activators; and (7) unclassified agents, such as disulfram. They have proved to be efficacious in latent cell line models and CD4(+) T lymphocytes from HIV-1-infected patients. This review comprehensively summarizes the recent progress and relative challenges in this field.

[Identification and characterization of HIV-1 transmitted /founder viruses].

During the spread of human immunodeficiency virus type 1 (HIV-1) in the mucosa, the entire genetic diversity of the viruses is significantly reduced. The vast majority of HIV-1 mucosal infections are established by one or a few viruses and ultimately develop into systemic infections, thus the initial virus is called transmitted/founder virus (T/F virus). The study of T/F virus will benefit understanding its key characteristics resulting in successful viral replication in the new host body, which may provide novel strategies for the development of AIDS vaccines, pre-exposure prophylaxis and other therapeutic interventions. In this review, we summarize the discovery and evolutionary characteristics of T/F virus as well as early immune response after HIV-1 infection, which will establish the basis to explore the features of T/F viruses.

Genetic variance in the HIV-1 founder virus Vpr affects its ability to induce cell cycle G2arrest and cell apoptosis.

In the event of acute infection, only a few HIV-1 viral variants can establish the initial productive clinical infection, and these viral variants are known as transmitted/founder viruses (T/F viruses). As one of the accessory proteins of HIV-1, viral protein R (Vpr) plays an important role in viral replication. Therefore, the characterization of T/F virus Vpr is beneficial to understand how virus replicates in a new host. In this study, flow cytometry was used to analyze the effect of G2arrest and cell apoptosis induced by the T/F virus Vpr and the chronic strain MJ4 Vpr. The results showed that the ability of T/F virus ZM246 Vpr and ZM247 Vpr inducing G2arrest and cell apoptosis are more potent than the MJ4 Vpr. The comparison of protein sequences indicated that the amino acids of 77, 85 and 94 contain high freqency mutations, suggesting that these sites may be involved in inducing G2arrest and cell apoptosis. Taken together, our work suggests that in acute infections, T/F viruses increase the capacity of G2arrest and cell apoptosis and promote viral replication and transmission in a new host by Vpr genetic mutation.

A novel peptide to disrupt the interaction of BST-2 and Vpu.

Bone marrow stromal cell antigen 2 (BST-2) inhibits the release of HIV-1 and other enveloped viruses from the cell surface. HIV-1 Vpu binds to BST-2 through an interaction between transmembrane domains (TMD) of the two proteins and induces the downregulation of cell surface BST-2, thereby counteracting its antiviral activity. In this study, we designed and prepared a modified peptide BST2-TM-P1, which include the sequence of BST-2 TMD, keeping its property competing with BST-2 to bind with Vpu. Biological assay results indicate BST2-TM-P1 could increase the BST-2 level at the cell surface in Vpu dependent manner and significantly inhibit the replication of HIV-1 virion. Our studies indicate that blocking the interaction of Vpu and BST-2 is an effective way to combat HIV-1 infection.

Construction and characterization of an infectious cDNA clone of enterovirus type 71 subgenotype C4.

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease and induces fatal neurological complications. In recent years, this virus has become a major threat to public health in the Asia-Pacific region, while no effective antiviral therapies and vaccines are currently available. In this study, we constructed and characterized for the first time an infectious full-length EV71 cDNA clone derived from the SHZH98 strain, which was the first subgenotype C4 strain isolated in China. Our data demonstrate that the rescued EV71 viruses exhibited growth kinetics in vitro and morphologies similar to those of the BrCr-TR strain and reached a maximum titer of 10(7.5) TCID50/ml. Although the rescued viruses were able to infect suckling mice, no typical symptoms of EV71 infection were observed for up to 18 days post-inoculation. Taken together our research provides an important tool to study the epidemic strains of EV71 in the Asia-Pacific region and promote the development of vaccines.

[Total trans-oral endoscopic thyroidectomy and cervical lymphadenectomy: a human cadavers surgery study].

OBJECTIVE: To find an approach for trans-oral endoscopic thyroidectomy (TOET) and cervical lymphadenectomy using conventional endoscopic surgical instruments on frozen fresh cadavers. METHODS: Six frozen fresh cadavers were used in three groups of trans-oral trocar installation experiments: oral vestibule installation, sublingual region installation, and combined bi-vestibular and sublingual installation. TOET (with pretrachealis method to thyroid fixation removal) and cervical lymphadenectomy were performed experiments on another 6 frozen fresh cadavers using the best access approach found in the aforementioned experiments. RESULTS: In oral vestibule trocar installations, the trocars caused large lacerated wound and damaged air tightness. In sublingual installations, only one trocar could be installed in the sublingual area because the space in sublingual area was limited. In combined bi-vestibular and sublingual installations, no gland, vessel or nerve was damaged. Combined bi-vestibular and sublingual access were selected as the surgical approach on the basic of analysis the merits of each approach. TOET and cervical lymphadenectomy in area III, IV, VI, VII were performed without making any accessory damage through combined bi-vestibular and sublingual access approach. CONCLUSIONS: TOET is feasible. Combined bi-vestibular and sublingual approach is available for TOET. Part of the cervical lymph nodes could be resected. Pretrachealis approach to thyroid fixation removal can still be used.

Triptolide reduces PD-L1 through the EGFR and IFN-γ/IRF1 dual signaling pathways.

As the principal ligand of programmed death 1 (PD-1), PD-L1 can induce the exhaustion of effector T cells and the escape of cancer cells through interacting with PD-1 in many solid malignancies. Therefore, targeting the PD-1/PD-L1 axis has become an attractive strategy in cancer immunotherapy. However, at present, no small-molecule agents targeting PD1/PD-L1 pathways have been successfully used in clinical applications. Here, we first found that the natural product Triptolide could significantly reduce the PD-L1 expression on the surface of NSCLC cells. This down-regulation is related to the activity of EGFR signaling pathway. Moreover, the reduction of PD-L1 caused by Triptolide could be substantially rescued by IFN-γ. Furthermore, our findings suggest that Triptolide significantly inhibits the activity of the IFN-γ-JAK-STAT-IRF1 signaling axis, as evidenced by the noticeable reduction in both basal and phosphorylated levels of STAT3. Thus, in NSCLC cells, Triptolide reduces PD-L1 expression both through the EGFR and IFN-γ/JAK1/JAK2/STAT1/STAT3/IRF1 signaling pathways. The results provide new insights into the application of Triptolide in the immune checkpoints treatment of NSCLCs.

Teicoplanin derivatives block spike protein mediated viral entry as pan-SARS-CoV-2 inhibitors.

The rapid emergence of highly transmissible SARS-CoV-2 variants poses serious threat to the efficacy of vaccines and neutralizing antibodies. Thus, there is an urgent need to develop new and effective inhibitors against SARS-CoV-2 and future outbreaks. Here, we have identified a series of glycopeptide antibiotics teicoplanin derivatives that bind to the SARS-CoV-2 spike (S) protein, interrupt its interaction with ACE2 receptor and selectively inhibit viral entry mediated by S protein. Computation modeling predicts that these compounds interact with the residues in the receptor binding domain. More importantly, these teicoplanin derivatives inhibit the entry of both pseudotyped SARS-CoV-2 Delta and Omicron variants. Our study demonstrates the feasibility of developing small molecule entry inhibitors by targeting the interaction of viral S protein and ACE2. Together, considering the proven safety and pharmacokinetics of teicoplanin as a glycopeptide antibiotic, the teicoplanin derivatives hold great promise of being repurposed as pan-SARS-CoV-2 inhibitors.

Schlafen-5 inhibits LINE-1 retrotransposition.

Long interspersed element 1 (LINE-1) is the only currently known active autonomous transposon in humans, and its retrotransposition may cause deleterious effects on the structure and function of host cell genomes and result in sporadic genetic diseases. Host cells therefore developed defense strategies to restrict LINE-1 mobilization. In this study, we demonstrated that IFN-inducible Schlafen5 (SLFN5) inhibits LINE-1 retrotransposition. Mechanistic studies revealed that SLFN5 interrupts LINE-1 ribonucleoprotein particle (RNP) formation, thus diminishing nuclear entry of the LINE-1 RNA template and subsequent LINE-1 cDNA production. The ability of SLFN5 to bind to LINE-1 RNA and the involvement of the helicase domain of SLFN5 in its inhibitory activity suggest a mechanism that SLFN5 binds to LINE-1 RNA followed by dissociation of ORF1p through its helicase activity, resulting in impaired RNP formation. These data highlight a new mechanism of host cells to restrict LINE-1 mobilization.

Identification of Ziyuglycoside II from a Natural Products Library as a STING Agonist.

Given the emerging pivotal roles of stimulator of interferon genes (STING) in host pathogen defense and immune-oncology, STING is regarded as a promising target for drug development. Cyclic dinucleotides (CDNs) are the first-generation STING agonists. However, their poor metabolic stability and membrane permeability limits their therapeutic application. In contrast, small-molecule STING agonists show superior properties such as molecular weight, polar character, and delivery diversity. The quest for a potent small-molecular agonist of human STING remains ongoing. In our study, through an IRF/IFN pathway-targeted cell-based screen of a natural products library, we identified a small-molecular STING agonist, Ziyuglycoside II, termed ST12, with potent stimulation of the IRF/IFN and NF-κB pathways. Furthermore, its binding to the C-terminal domain of human STING, detected by bio-layer interferometry, indicates that ST12 is a human STING agonist. Further Tanimoto similarity analysis with existing small-molecule STING agonists indicates that ST12 is a lead compound with a novel core structure for the further optimization.

Design, synthesis and biological evaluation of protease inhibitors containing morpholine cores with remarkable potency against both HIV-1 subtypes B and C.

By following up on the design vector of optimizing amine-based HIV-1 protease inhibitors, we have designed and biologically evaluated a novel class of inhibitors with the free nitrogen or sulphone in morpholine cores as P2 ligands in combination with diverse substituted phenylsulfonamide P2' ligands. As it turns out, a majority of these inhibitors exhibit prominent enzymatic inhibitory activity in low nanomolar ranges with relatively low cytotoxicity. Particularly, inhibitor 1e containing a morpholine carboxamide P2 ligand and a 4-hydroxyphenylsulfonamide P2' ligand illustrates a robust enzyme inhibitory IC50 value of 90 pM. Furthermore, 1e demonstrates impressive in vivo antiviral activity with EC50 value of 89 nM and a degree of inhibitory potency against the DRV-resistant variant. More importantly, 1e exhibits remarkable activity with EC50 values of 13.59 nM and 8.23 nM against subtype C HIV-1 strains ZM246 and Indie, respectively. Furthermore, the in silico studies provide molecular insights into binding features of inhibitors with HIV-1 protease, and furnish a valuable forecast on further process.

The Zinc-Finger protein ZCCHC3 inhibits LINE-1 retrotransposition.

Long-interspersed element 1 (LINE-1) is an autonomous non-LTR retrotransposon. Its replication can cause mutation and rearrangement of host genomic DNA, which may result in serious genetic diseases. Host cells therefore developed defense strategies to restrict LINE-1 mobilization. In this study, we reported that CCHC-type zinc-finger protein ZCCHC3 can repress LINE-1 retrotransposition, and this activity is closely related to its zinc-finger domain. Further studies show that ZCCHC3 can post-transcriptionally diminish the LINE-1 RNA level. The association of ZCCHC3 with both LINE-1 RNA and ORF1 suggests that ZCCHC3 interacts with LINE-1 RNP and consequently causes its RNA degradation. These data demonstrate collectively that ZCCHC3 contributes to the cellular control of LINE-1 replication.

A High Throughput Cell-Based Screen Assay for LINE-1 ORF1p Expression Inhibitors Using the In-Cell Western Technique.

Long interspersed nuclear element 1 (LINE-1) is a dominant autonomous retrotransposon in human genomes which plays a role in affecting the structure and function of somatic genomes, resulting in human disorders including genetic disease and cancer. LINE-1 encoded ORF1p protein which possesses RNA-binding and nucleic acid chaperone activity, and interacts with LINE-1 RNA to form a ribonucleoprotein particle (RNP). ORF1p can be detected in many kinds of tumors and its overexpression has been regarded as a hallmark of histologically aggressive cancers. In this study, we developed an In-Cell Western (ICW) assay in T47D cells to screen the compounds which can decrease the expression of ORF1p. Using this assay, we screened 1,947 compounds from the natural products library of Target Mol and Selleckchem, among which three compounds, Hydroxyprogesterone, 2,2':5',2″-Terthiophene and Ethynyl estradiol displayed potency in diminishing LINE-1 ORF1p expression level. Further mechanistic studies indicated the compounds act by affecting LINE-1 RNA transcription. Notably, we demonstrated that the compounds have an inhibitory effect on the proliferation of several lung and breast cancer cell lines. Taken together, we established a high throughput screening system for ORF1p expression inhibitors and the identified compounds provide some clues to the development of a novel anti-tumor therapeutic strategy by targeting ORF1p.