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.

Development of an mRNA-based therapeutic vaccine mHTV-03E2 for high-risk HPV-related malignancies.

Human papillomavirus (HPV) 16 and 18 infections are related to many human cancers. Despite several preventive vaccines for high-risk (hr) HPVs, there is still an urgent need to develop therapeutic HPV vaccines for targeting pre-existing hrHPV infections and lesions. In this study, we developed a lipid nanoparticle (LNP)-formulated mRNA-based HPV therapeutic vaccine (mHTV)-03E2, simultaneously targeting the E2/E6/E7 of both HPV16 and HPV18. mHTV-03E2 dramatically induced antigen-specific cellular immune responses, leading to significant CD8+ T cell infiltration and cytotoxicity in TC-1 tumors derived from primary lung epithelial cells of C57BL/6 mice expressing HPV E6/E7 antigens, mediated significant tumor regression, and prolonged animal survival, in a dose-dependent manner. We further demonstrated significant T cell immunity against HPV16/18 E6/E7 antigens for up to 4 months post-vaccination in immunological and distant tumor rechallenging experiments, suggesting robust memory T cell immunity against relapse. Finally, mHTV-03E2 synergized with immune checkpoint blockade to inhibit tumor growth and extend animal survival, indicating the potential in combination therapy. We conclude that mHTV-03E2 is an excellent candidate therapeutic mRNA vaccine for treating malignancies caused by HPV16 or HPV18 infections.

MxB inhibits long interspersed element type 1 retrotransposition.

Long interspersed element type 1 (LINE-1, also L1 for short) is the only autonomously transposable element in the human genome. Its insertion into a new genomic site may disrupt the function of genes, potentially causing genetic diseases. Cells have thus evolved a battery of mechanisms to tightly control LINE-1 activity. Here, we report that a cellular antiviral protein, myxovirus resistance protein B (MxB), restricts the mobilization of LINE-1. This function of MxB requires the nuclear localization signal located at its N-terminus, its GTPase activity and its ability to form oligomers. We further found that MxB associates with LINE-1 protein ORF1p and promotes sequestration of ORF1p to G3BP1-containing cytoplasmic granules. Since knockdown of stress granule marker proteins G3BP1 or TIA1 abolishes MxB inhibition of LINE-1, we conclude that MxB engages stress granule components to effectively sequester LINE-1 proteins within the cytoplasmic granules, thus hindering LINE-1 from accessing the nucleus to complete retrotransposition. Thus, MxB protein provides one mechanism for cells to control the mobility of retroelements.

SERINC5 restricts influenza virus infectivity.

SERINC5 is a multi-span transmembrane protein that is incorporated into HIV-1 particles in producing cells and inhibits HIV-1 entry. Multiple retroviruses like HIV-1, equine infectious anemia virus and murine leukemia virus are subject to SERINC5 inhibition, while HIV-1 pseudotyped with envelope glycoproteins of vesicular stomatitis virus and Ebola virus are resistant to SERINC5. The antiviral spectrum and the underlying mechanisms of SERINC5 restriction are not completely understood. Here we show that SERINC5 inhibits influenza A virus infection by targeting virus-cell membrane fusion at an early step of infection. Further results show that different influenza hemagglutinin (HA) subtypes exhibit diverse sensitivities to SERINC5 restriction. Analysis of the amino acid sequences of influenza HA1 strains indicates that HA glycosylation sites correlate with the sensitivity of influenza HA to SERINC5, and the inhibitory effect of SERINC5 was lost when certain HA glycosylation sites were mutated. Our study not only expands the antiviral spectrum of SERINC5, but also reveals the role of viral envelope glycosylation in resisting SERINC5 restriction.

Design, synthesis, and antiviral activity of 1-aryl-4-arylmethylpiperazine derivatives as Zika virus inhibitors with broad antiviral spectrum.

Zika virus (ZIKV) disease has been given attention due to the risk of congenital microcephaly and neurodevelopmental disorders after ZIKV infection in pregnancy, but no vaccine or antiviral drug is available. Based on a previously reported ZIKV inhibitor ZK22, a series of novel 1-aryl-4-arylmethylpiperazine derivatives was designed, synthesized, and investigated for antiviral activity by quantify cellular ZIKV RNA amount using RT-qPCR method in ZIKV-infected human venous endothelial cells (HUVECs) assay. Structure-activity relationship (SAR) analysis demonstrated that anti-ZIKV activity of 1-aryl-4-arylmethylpiperazine derivatives is not correlated with molecular hydrophobicity, multiple new derivatives with pyridine group to replace the benzonitrile moiety of ZK22 showed stronger antiviral activity, higher ligand lipophilicity efficiency as well as lower cytotoxicity. Two active compounds 13 and 33 were further identified as novel ZIKV entry inhibitors with the potential of oral available. Moreover, both ZK22 and newly active derivatives also possess of obvious inhibition on the viral replication of coronavirus and influenza A virus at low micromolar level. In summary, this work provided better candidates of ZIKV inhibitor for preclinical study and revealed the promise of 1-aryl-4-arylmethylpiperazine chemotype in the development of broad-spectrum antiviral agents.

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.

N6-adenosine methylation and the regulatory mechanism on LINE-1.

Long interspersed elements-1(LINE-1) is the only autonomous transposon in human genome，and its retrotransposition results in change of cellular genome structure and function, leading occurrence of various severe diseases. As a central key intermediated component during life cycle of LINE-1 retrotransposition, the host modification of LINE-1 mRNA affects the LINE-1 transposition directly. N6-adenosine methylation(m6A), the most abundant epigenetic modification on eukaryotic RNA, is dynamically reversible. m6A modification is also found on LINE-1 mRNA, and it participants regulation of the whole LINE-1 replication cycle, with affecting LINE-1 retrotransposition as well as its adjacent genes expression, followed by influencing genomic stability, cellular self-renewal, and differentiation potential, which plays important roles in human development and diseases. In this review, we summarize the research progress in LINE-1 m6A modification, including its modification positions, patterns and related mechanisms, hoping to provide a new sight on the mechanism research and treatment of related diseases.

Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides.

Triterpenoids are a large and medicinally important group of natural products with a wide range of biological and pharmacological effects. Among them, malabaricane-type triterpenoids are a rare group of terpenoids with a 6,6,5-tricyclic ring system, and a few malabaricane triterpene synthases have been characterized to date. Here, an arabidiol synthase AmAS for the formation of the malabaricane-type 6,6,5-tricyclic triterpenoid skeleton in astramalabaricosides biosynthesis was characterized from Astragalus membranaceus. Multiple sequence alignment, site-directed mutagenesis, and molecular docking of AmAS reveal that residues Q256 and Y258 are essential for AmAS activity, and the triad motif IIH725-727 was the critical residue necessary for its product specificity. Mutation of IIH725-727 with VFN led to the formation of seven tricyclic, tetracyclic, and pentacyclic triterpenoids (1-7). Glycosylation of malabaricane-type triterpenoids in the biosynthesis of astramalabaricosides was also explored. Three triterpenoids (1, 5, and 6) displayed potent inhibitory effects against influenza A virus in vitro. These findings provide insights into malabaricane-type triterpenoids biosynthesis in A. membranaceus and access to diverse bioactive triterpenoids for drug discovery.

Identification of an N-phenylsulfonyl-2-(piperazin-1-yl)methyl-benzonitrile derivative as Zika virus entry inhibitor.

Zika virus (ZIKV) infection could cause severe neurological complications such as neonatal microcephaly, Guillain-Barré syndrome, and myelitis in adults. No vaccine or therapeutic drug is available for prevention and control of ZIKV infection yet. Based on previously reported anti-ZIKV hit compound 1, a series of novel N-benzoyl or phenylsulfonyl substituted 2-(piperazin-1-yl)methyl-benzonitrile (PMBN) derivatives was designed, synthesized, and investigated for the antiviral activity against ZIKV replication in different cell-based phenotypic assays. The results indicated that N-phenylsulfonyl-PMBN derivative 24 displayed the comparable antiviral activity and higher oral availability than hit compound 1. Meanwhile, mechanism of action study confirmed that compound 24 acts on the early entry stage of ZIKV life cycle. The identification of this new ZIKV entry inhibitor chemotype provided a promising lead for further optimization to develop new drug for ZIKV infection.

Progress on viral and host determinants of influenza A virus species specificity.

Influenza A viruses have a wide range of hosts and are highly infectious, which can cause zoonotic diseases and pose a serious public health threat to human safety. An influenza pandemic could outbreak if new strains gain the ability of human-to-human transmission, either by genetic mutation or by gene reassortment. It is an urgent issue for the scientific community to reveal the genetic basis and molecular mechanisms underlying the interspecies transmission of influenza viruses, which will provide important implications for the effective monitoring and prevention of potential influenza pandemics. In this review, we summarize the molecular determinants of influenza viruses for host adaptation, and highlight the advances in the gene mutations of the virus itself and the interaction between virus and host factors. This will help to make a theoretical reserve for the next influenza pandemic and find new strategies to fight against influenza.

IMB-BZ as an Inhibitor Targeting ESX-1 Secretion System to Control Mycobacterial Infection.

BACKGROUND: Resistance to anti-tuberculosis (TB) drugs is a major issue in TB control, and demands the discovery of new drugs targeting the virulence factor ESX-1. METHODS: We first established a high-throughput screen (HTS) assay for the discovery of ESX-1 secretion inhibitors. The positive hits were then evaluated for the potency of diminishing the survival of virulent mycobacteria and reducing bacterial virulence. We further investigated the probability of inducing drug resistance and the underlying mechanism using mycobacterial protein fragment complementation. RESULTS: A robust HTS assay was developed to identify small molecules that inhibit ESX-1 secretion without impairing bacterial growth in vitro. A hit named IMB-BZ specifically inhibits the secretion of CFP-10 and reduces virulence in an ESX-1-dependent manner, therefore resulting in significant reduction in intracellular and in vivo survival of mycobacteria. Blocking the CFP-10-EccCb1 interaction directly or indirectly underlies the inhibitory effect of IMB-BZ on the secretion of CFP-10. Importantly, our finding shows that the ESX-1 inhibitors pose low risk of drug resistance development by mycobacteria in vitro as compared with traditional anti-TB drugs, and exhibit high potency against chronic mycobacterial infection. CONCLUSIONS: Targeting ESX-1 may lead to the development of novel therapeutics for tuberculosis. IMB-BZ holds the potential for future development into a new anti-TB drug.

Design, synthesis, and anti-influenza A virus activity evaluation of novel indole containing derivatives of triazole.

We designed and synthesized 18 substituted indole derivatives containing a triazole scaffold as novel anti-influenza A virus candidates using a bio-isosteric and scaffold-hopping strategy from the lead compound 4-32-2. Most of the target compounds (eg: 6, 7a, 7d, 7f-j, 7l, 7m, 7o, 7q) exhibited potent anti-influenza A virus activity and low cytotoxicity in vitro. In particular, 7a exhibited the most potent anti-IAV activity (IC50: 1.34 ± 0.13 µM) with low cytotoxicity (CC50: > 100 µM), and high selectivity index (SI: > 74.63), which provides a new chemical scaffold for the development of novel anti-IAV drug.

A kind of HIV-1 protease inhibitors containing phenols with antiviral activity against DRV-resistant variants.

Based upon the preliminary design of enhancing genetic barrier to drug-resistant viral mutants by maximizing hydrogen-bonding or other van der Waals contacts, we have designed, synthesized and biologically evaluated a new class of HIV-1 protease inhibitors with phenol derived P2 ligands and nitro or halogens in P2' ligands. Results indicate that a majority of inhibitors exhibit robust enzyme inhibitory with IC50 values in picomolar or single digit nanomolar ranges. Among which, compound 17d displays potency with IC50 value of 21 pM and high protease selectivity. Of note, 17d exhibits greater antiviral activity against the DRV-resistant variant than the efficacy against the wild type virus. Furthermore, the molecular modeling studies demonstrate important interactions between 17d and the active sites of both the wild-type and DRV-resistant HIV-1 protease, as well as furnish insights for further optimization of new inhibitors.

Repurposing of the antihistamine mebhydrolin napadisylate for treatment of Zika virus infection.

Zika virus (ZIKV) infection can lead to severe neurological disorders and fetal defects, which has become a public health problem worldwide. However, effective clinical treatment for ZIKV infection was still arduous. Antihistamines are attractive candidates for drug repurposing due to their low price and widespread availability. Here we screened FDA-approved antihistamine drugs to obtain anti-ZIKV candidate compounds and identified mebhydrolin napadisylate (MHL) that exhibits the potent inhibition of ZIKV infection in various cell lines in a histamine H1 receptor-independent manner. Mechanistic studies suggest that MHL acts as a ZIKV NS5 RNA-dependent RNA polymerase (RdRp) inhibitor, supported by a structure-activity relationship (SAR) analysis showing the correlation between the inhibitory effect upon viral RNA synthesis and ZIKV infectivity. Furthermore, MHL was shown to bind ZIKV NS5 RdRp in vitro and predicted to interact with key residues at the active site of ZIKV NS5 RdRp by molecular docking analysis. Our data together suggest that MHL suppresses ZIKV infection through the inhibition of ZIKV NS5 RdRp activity. This study highlights that MHL might be a promising clinical anti-ZIKV therapeutic.

The mechanism and pharmacodynamics of 2-((1H-indol-3-yl)thio/sulfinyl)-N-pheny acetamide derivative as a novel inhibitor against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection worldwide. Until now, there are no licenced vaccines or effective antiviral drugs against RSV infections. In our previous work, we found 2-((1H-indol-3-yl)thio/sulfinyl)-N-pheny acetamide derivatives (4-49 C and 1-HB-63) being a novel inhibitor against RSV in vitro. Here, we explored the underlying mechanism of 2-((1H-indol-3-yl)thio/sulfinyl)-N-pheny acetamide derivatives to inhibit RSV replication in vitro and disclosed that 4-49 C worked as the inhibitor of membrane fusion and 1-HB-63 functioned at the stage of RSV genome replication/transcription. Yet, both of them could not inhibit RSV infection of BALB/c mice by using RSV-Luc, in vivo imaging and RT-qPCR analyses, for which it may be due to the fast metabolism in vivo. Our work suggests that further structural modification and optimisation of 2-((1H-indol-3-yl) thio/sulfinyl)-N-pheny acetamide derivative are needed to obtain drug candidates with effective anti-RSV activities in vivo.

Design and Evaluation of Novel HIV-1 Protease Inhibitors Containing Phenols or Polyphenols as P2 Ligands with High Activity against DRV-Resistant HIV-1 Variants.

With the increasing prevalence of drug-resistant variants, novel potent HIV-1 protease inhibitors with broad-spectrum antiviral activity against multidrug-resistant causative viruses are urgently needed. Herein, we designed and synthesized a new series of HIV-1 protease inhibitors with phenols or polyphenols as the P2 ligands and a variety of sulfonamide analogs as the P2' ligands. A number of these new inhibitors showed superb enzymatic inhibitory activity and antiviral activity. In particular, inhibitors 15d and 15f exhibited potent enzymatic inhibitory activity in the low picomolar range, and the latter showed excellent activity against the Darunavir-resistant HIV-1 variant. Furthermore, the molecular modeling studies provided insight into the ligand-binding site interactions between inhibitors and the enzyme cavity, and they sparked inspiration for the further optimization of potent inhibitors.

Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro.

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 µM and 3.5 µM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

Pro-515 of the dynamin-like GTPase MxB contributes to HIV-1 inhibition by regulating MxB oligomerization and binding to HIV-1 capsid.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505-527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515-519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

Clinicopathologic features, tumor immune microenvironment and genomic landscape of Epstein-Barr virus-associated intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about Epstein-Barr virus (EBV)-associated intrahepatic cholangiocarcinoma (EBVaICC) because of its rarity. We aimed to comprehensively investigate the clinicopathology, tumor immune microenvironment (TIME) and genomic landscape of this entity in southern China. METHODS: We evaluated 303 intrahepatic cholangiocarcinomas (ICCs) using in situ hybridization for EBV. We compared clinicopathological parameters between EBVaICC and nonEBVaICC, and we analyzed EBV infection status, tumor-infiltrating lymphocytes (TILs) and genomic features of EBVaICC by immunohistochemistry, double staining, nested PCR, multiplex immunofluorescence staining, fluorescence in situ hybridization and whole-exome sequencing. RESULTS: EBVaICC accounted for 6.6% of ICCs and was associated with EBV latency type I infection and clonal EBV isolates. Patients with EBVaICC were more often female and younger, with solitary tumors, higher HBV infection rates and less frequent cirrhosis; the lymphoepithelioma-like (LEL) subtype was more common in EBVaICC. EBVaICC was associated with a significantly larger TIME component than nonEBVaICC. The LEL subtype of EBVaICC - associated with a significantly increased density and proportion of CD20+ B cells and CD8+ T cells - was associated with significantly higher 2-year survival rates than conventional EBVaICC and nonEBVaICC. Both PD-1 and PD-L1 in TILs, and PD-L1 in tumor cells, were overexpressed in EBVaICC. High PD-L1 expression in tumor cells and high CD8+ TIL densities were significantly more common in EBVaICC than in nonEBVaICC. Seven genes (MUC4, DNAH1, GLI2, LIPE, MYH7, RP11-766F14.2 and WDR36) were mutated in at least 3 patients. EBVaICC had a different mutational pattern to liver fluke-associated cholangiocarcinoma and HBV-associated ICC. CONCLUSIONS: EBVaICC, as a subset of ICC, has unique etiological, clinicopathological and genetic characteristics, with a significantly larger TIME component. Paradoxically, patients with EBVaICC could be candidates for immune checkpoint therapy. LAY SUMMARY: Epstein-Barr virus (EBV) is associated with a subtype of intrahepatic cholangiocarcinoma, with unique clinicopathological and genetic characteristics. The tumor immune microenvironment is also different in this tumor subtype and patients with EBV-associated intrahepatic cholangiocarcinoma may respond well to immune checkpoint inhibitors.

Non-volatile acylphloroglucinol components from Eucalyptus robusta inhibit Zika virus by impairing RdRp activity of NS5.

Eucalyptus is a large genus of the Myrtaceae family with high value in various fields of industry. Recently, attention has been focused on the functional properties of Eucalyptus extracts. These extracts have been traditionally used to combat various infectious diseases, and volatile oils are usually considered to play a major role. But the positive effects of non-volatile acylphloroglucinols, a class of specialized metabolites with relatively high content in Eucalyptus, should not be neglected. Herein, non-volatile acylphloroglucinols from leaves of Eucalyptus robusta were evaluated for their abilities to inhibit Zika virus (ZIKV) which is associated with severe neurological damage and complications. The results showed eucalyprobusone G, a new symmetrical acylphloroglucinol dimer, possessed the significant ability to inhibit ZIKV without inducing cytotoxicity. The EC50 values of eucalyprobusone G against the African lineage (MR766) and Asian lineage (SZ-WIV01) of ZIKV were 0.43 ± 0.08 and 10.10 ± 3.84 µM which were 110 times and 5.8 times better than those of the reference compound ribavirin, respectively. Further action mode research showed that eucalyprobusone G impairs the viral binding and RdRp activity of NS5. The results broaden the functional properties of Eucalyptus robusta and indicate acylphloroglucinol dimers could be developed as anti-ZIKV agents.