Interferon-induced MXB protein restricts vimentin-dependent viral infection.

Type I interferon (IFN) inhibits a wide spectrum of viruses through stimulating the expression of antiviral proteins. As an IFN-induced protein, myxovirus resistance B (MXB) protein was reported to inhibit multiple highly pathogenic human viruses. It remains to be determined whether MXB employs a common mechanism to restrict different viruses. Here, we find that IFN alters the subcellular localization of hundreds of host proteins, and this IFN effect is partially lost upon MXB depletion. The results of our mechanistic study reveal that MXB recognizes vimentin (VIM) and recruits protein kinase B (AKT) to phosphorylate VIM at amino acid S38, which leads to reorganization of the VIM network and impairment of intracellular trafficking of virus protein complexes, hence causing a restriction of virus infection. These results highlight a new function of MXB in modulating VIM-mediated trafficking, which may lead towards a novel broad-spectrum antiviral strategy to control a large group of viruses that depend on VIM for successful replication.

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.

CX-6258 hydrochloride hydrate: A potential non-nucleoside inhibitor targeting the RNA-dependent RNA polymerase of norovirus.

Human norovirus (HuNoV), a primary cause of non-bacterial gastroenteritis, currently lacks approved treatment. RdRp is vital for virus replication, making it an attractive target for therapeutic intervention. By application of structure-based virtual screening procedure, we present CX-6258 hydrochloride hydrate as a potent RdRp non-nucleoside inhibitor, effectively inhibiting HuNoV RdRp activity with an IC50 of 3.61 µM. Importantly, this compound inhibits viral replication in cell culture, with an EC50 of 0.88 µM. In vitro binding assay validate that CX-6258 hydrochloride hydrate binds to RdRp through interaction with the "B-site" binding pocket. Interestingly, CX-6258-contacting residues such as R392, Q439, and Q414 are highly conserved among major norovirus GI and GII variants, suggesting that it may be a general inhibitor of norovirus RdRp. Given that CX-6258 hydrochloride hydrate is already utilized as an orally efficacious pan-Pim kinase inhibitor, it may serve as a potential lead compound in the effort to control HuNoV infections.

Immunogenicity and effectiveness of an mRNA therapeutic vaccine for HPV-related malignancies.

Human papillomavirus (HPV) infections account for several human cancers. There is an urgent need to develop therapeutic vaccines for targeting preexisting high-risk HPV (such as HPV 16 and 18) infections and lesions, which are insensitive to preventative vaccines. In this study, we developed a lipid nanoparticle-formulated mRNA-based HPV therapeutic vaccine (mHTV), mHTV-02, targeting the E6/E7 of HPV16 and HPV-18. mHTV-02 dramatically induced antigen-specific cellular immune response and robust memory T-cell immunity in mice, besides significant CD8+ T-cell infiltration and cytotoxicity in TC-1 tumors expressing HPV E6/E7, resulting in tumor regression and prolonged survival in mice. Moreover, evaluation of routes of administration found that intramuscular or intratumoral injection of mHTV-02 displayed significant therapeutic effects. In contrast, intravenous delivery of the vaccine barely showed any benefit in reducing tumor size or improving animal survival. These data together support mHTV-02 as a candidate therapeutic mRNA vaccine via specific administration routes for treating malignancies caused by HPV16 or HPV18 infections.

A cell-based assay to discover inhibitors of Zika virus RNA-dependent RNA polymerase.

Zika virus (ZIKV) belongs to Flaviviridae, the Flavivirus genus. Its infection causes congenital brain abnormalities and Guillain-Barré syndrome. However, there are no effective vaccines, no FDA-approved drugs to manage ZIKV infection. The non-structural protein NS5 of ZIKV has been recognized as a valuable target of antivirals because of its RNA-dependent RNA polymerase (RdRp) and methyltransferase (MTase) activities essential for viral RNA synthesis. Here, we report a cell-based assay for discovering inhibitors of ZIKV NS5 and found that 5-Azacytidine potently inhibits ZIKV NS5, with EC50 of 4.9 µM. Furthermore, 5-Azacytidine suppresses ZIKV replication by inhibiting NS5-mediated viral RNA transcription. Therefore, we have developed a cell-based ZIKV NS5 assay which can be deployed to discover ZIKV NS5 inhibitors and demonstrated the potential of 5-Azacytidine for further development as a ZIKV NS5 inhibitor.

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.

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.

Interferon-inducible SAMHD1 restricts viral replication through downregulation of lipid synthesis.

Background: Type I interferon (IFN) inhibits virus infection through multiple processes. Recent evidence indicates that IFN carries out its antiviral activity through readjusting of the cellular metabolism. The sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1), as an interferon-stimulated gene (ISG), has been reported to inhibit a number of retroviruses and DNA viruses, by depleting dNTPs indispensable for viral DNA replication. Here we report a new antiviral activity of SAMHD1 against RNA viruses including HCV and some other flaviviruses infection. Methods: Multiple cellular and molecular biological technologies have been used to detect virus infection, replication and variation of intracellular proteins, including western blotting, qRT-PCR, Gene silencing, immunofluorescence, etc. Besides, microarray gene chip technology was applied to analyze the effects of SAMHD1 overexpression on total expressed genes. Results: Our data show that SAMHD1 down-regulates the expression of genes related to lipid bio-metabolic pathway, accompanied with impaired lipid droplets (LDs) formation, two events important for flaviviruses infection. Mechanic study reveals that SAMHD1 mainly targets on HCV RNA replication, resulting in a broad inhibitory effect on the infectivity of flaviviruses. The C-terminal domain of SAMHD1 is showed to determine its antiviral function, which is regulated by the phosphorylation of T592. Restored lipid level by overexpression of SREBP1 or supplement with LDs counteracts with the antiviral activity of SAMHD1, providing evidence supporting the role of SAMHD1-mediated down-regulation of lipid synthesis in its function to inhibit viral infection. Conclusion: SAMHD1 plays an important role in IFN-mediated blockade of flaviviruses infection through targeting lipid bio-metabolic pathway.

Repurposing of berbamine hydrochloride to inhibit Ebola virus by targeting viral glycoprotein.

Ebola virus (EBOV) infection leads to staggeringly high mortality rate. Effective and low-cost treatments are urgently needed to control frequent EBOV outbreaks in Africa. In this study, we report that a natural compound called berbamine hydrochloride strongly inhibits EBOV replication in vitro and in vivo. Our work further showed that berbamine hydrochloride acts by directly binding to the cleaved EBOV glycoprotein (GPcl), disrupting GPcl interaction with viral receptor Niemann-Pick C1, thus blocking the fusion of viral and cellular membranes. Our data support the probability of developing anti-EBOV small molecule drugs by targeting viral GPcl. More importantly, since berbamine hydrochloride has been used in clinic to treat leukopenia, it holds great promise of being quickly repurposed as an anti-EBOV drug.

SARS-CoV-2 hijacks cellular kinase CDK2 to promote viral RNA synthesis.

The coronavirus disease 2019 (COVID-19) pandemic has devastated global health. Identifying key host factors essential for SARS-CoV-2 RNA replication is expected to unravel cellular targets for the development of broad-spectrum antiviral drugs which have been quested for the preparedness of future viral outbreaks. Here, we have identified host proteins that associate with nonstructural protein 12 (nsp12), the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using a mass spectrometry (MS)-based proteomic approach. Among the candidate factors, CDK2 (Cyclin-dependent kinase 2), a member of cyclin-dependent kinases, interacts with nsp12 and causes its phosphorylation at T20, thus facilitating the assembly of the RdRp complex consisting of nsp12, nsp7 and nsp8 and promoting efficient synthesis of viral RNA. The crucial role of CDK2 in viral RdRp function is further supported by our observation that CDK2 inhibitors potently impair viral RNA synthesis and SARS-CoV-2 infection. Taken together, we have discovered CDK2 as a key host factor of SARS-CoV-2 RdRp complex, thus serving a promising target for the development of SARS-CoV-2 RdRp 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.

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.

Repurposing of HIV/HCV protease inhibitors against SARS-CoV-2 3CLpro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen that caused the global COVID-19 outbreak. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a key role in virus replication and has become an ideal target for antiviral drug design. In this work, we have employed bioluminescence resonance energy transfer (BRET) technology to establish a cell-based assay for screening inhibitors against SARS-CoV-2 3CLpro, and then applied the assay to screen a collection of known HIV/HCV protease inhibitors. Our results showed that the assay is capable of quantification of the cleavage efficiency of 3CLpro with good reproducibility (Z' factor is 0.59). Using the assay, we found that 9 of 26 protease inhibitors effectively inhibited the activity of SARS-CoV-2 3CLpro in a dose-dependent manner. Among them, four compounds exhibited the ability to bind to 3CLproin vitro. HCV protease inhibitor simeprevir showed the most potency against 3CLpro with an EC50 vale of 2.6 µM, bound to the active site pocket of 3CLpro in a predicted model, and importantly, exhibited a similar activity against the protease containing the mutations P132H in Omicron variants. Taken together, this work demonstrates the feasibility of using the cell-based BRET assay for screening 3CLpro inhibitors and supports the potential of simeprevir for the development of 3CLpro inhibitors.

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.

MxB Disrupts Hepatitis C Virus NS5A-CypA Complex: Insights From a Combined Theoretical and Experimental Approach.

The human myxovirus resistance B (MxB) protein is an interferon-induced restriction factor that fights a wide range of viruses. We previously demonstrated that MxB binds to hepatitis C virus (HCV)-encoded non-structural protein 5A (NS5A) and inhibits HCV infection by impairing the formation of cyclophilin A (CypA)-NS5A complex. However, the molecular details about how the presence of MxB diminishes the binding of NS5A to CypA remain uncovered. In this study, through molecular dynamic simulations and biochemical assays, we characterized that MxB binds to NS5A domain I through its N-terminal and GTPase domains. Specifically, amino acids (aa.) 189-191 and aa. 330-334 within MxB, together with NS5A residues aa. 71-73, are crucial for MxB-NS5A interaction. Furthermore, we predicted the CypA:NS5A and CypA:NS5A:MxB complexes and calculated the per-residue energy decomposition for identified key residues of the CypA-NS5A interface. A 28% decrease in CypA-NS5A binding affinity was observed in the presence of MxB, suggesting a weakened CypA-NS5A association upon binding of MxB to NS5A, which may contribute to the MxB-mediated inhibitory effect on the formation of CypA-NS5A complex. This work provides information for the antiviral mechanism of MxB and may facilitate the discovery of new strategies to combat CypA-dependent viruses.

An anti-influenza A virus microbial metabolite acts by degrading viral endonuclease PA.

The emergence of new highly pathogenic and drug-resistant influenza strains urges the development of novel therapeutics for influenza A virus (IAV). Here, we report the discovery of an anti-IAV microbial metabolite called APL-16-5 that was originally isolated from the plant endophytic fungus Aspergillus sp. CPCC 400735. APL-16-5 binds to both the E3 ligase TRIM25 and IAV polymerase subunit PA, leading to TRIM25 ubiquitination of PA and subsequent degradation of PA in the proteasome. This mode of action conforms to that of a proteolysis targeting chimera which employs the cellular ubiquitin-proteasome machinery to chemically induce the degradation of target proteins. Importantly, APL-16-5 potently inhibits IAV and protects mice from lethal IAV infection. Therefore, we have identified a natural microbial metabolite with potent in vivo anti-IAV activity and the potential of becoming a new IAV therapeutic. The antiviral mechanism of APL-16-5 opens the possibility of improving its anti-IAV potency and specificity by adjusting its affinity for TRIM25 and viral PA protein through medicinal chemistry.

5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

2-((1H-indol-3-yl)thio)-N-phenyl-acetamides: SARS-CoV-2 RNA-dependent RNA polymerase inhibitors.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus Disease 2019 (COVID-19) pandemic. Despite intensive and global efforts to discover and develop novel antiviral therapies, only Remdesivir has been approved as a treatment for COVID-19. Therefore, effective antiviral therapeutics are still urgently needed to combat and halt the pandemic. Viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 demonstrates high potential as a reliable target for the development of antivirals. We previously developed a cell-based assay to assess the efficiency of compounds that target SARS-CoV-2 RdRp, as well as their tolerance to viral exoribonuclease-mediated proof-reading. In our previous study, we discovered that 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides specifically targets the RdRp of both respiratory syncytial virus (RSV) and influenza A virus. Thus, we hypothesize that 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides may also have the ability to inhibit SARS-CoV-2 replication by targeting its RdRp activity. In this research, we test a compound library containing 103 of 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides against SARS-CoV-2 RdRp, using our cell-based assay. Among these compounds, the top five candidates strongly inhibit SARS-CoV-2 RdRp activity while exhibiting low cytotoxicity and resistance to viral exoribonuclease. Compound 6-72-2a is the most promising candidate with the lowest EC50 value of 1.41 µM and highest selectivity index (CC50/EC50) (above 70.92). Furthermore, our data suggests that 4-46b and 6-72-2a also inhibit the replication of HCoV-OC43 and HCoV-NL63 virus in a dose-dependent manner. Compounds 4-46b and 6-72-2a exhibit EC50 values of 1.13 µM and 0.94 µM, respectively, on HCoV-OC43 viral replication. However, higher concentrations of these compounds are needed to effectively block HCoV-NL63 replication. Together, our findings successfully identified 4-46b and 6-72-2a as promising inhibitors against SARS-CoV-2 RdRp.

Quinoline and Quinazoline Derivatives Inhibit Viral RNA Synthesis by SARS-CoV-2 RdRp.

Coronavirus disease 2019 (COVID-19) is a fatal respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of potential drugs is urgently needed to control the pandemic. RNA dependent RNA polymerase (RdRp) is a conserved protein within RNA viruses and plays a crucial role in the viral life cycle, thus making it an attractive target for development of antiviral drugs. In this study, 101 quinoline and quinazoline derivatives were screened against SARS-CoV-2 RdRp using a cell-based assay. Three compounds I-13e, I-13h, and I-13i exhibit remarkable potency in inhibiting RNA synthesis driven by SARS-CoV-2 RdRp and relatively low cytotoxicity. Among these three compounds, I-13e showed the strongest inhibition upon RNA synthesis driven by SARS-CoV-2 RdRp, the resistance to viral exoribonuclease activity and the inhibitory effect on the replication of CoV, thus holding potential of being drug candidate for treatment of SARS-CoV-2.