AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy.

BACKGROUND: Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma. METHODS: The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro . A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry. RESULTS: Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro . AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 + /CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion. CONCLUSIONS: This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.

LncRNA NKILA inhibits HBV replication by repressing NF-κB signalling activation.

Hepatitis B virus (HBV) infection results in liver cirrhosis and hepatocellular carcinoma (HCC). HBx/nuclear factor (NF)-κB pathway plays a role in HBV replication. However, whether NF-κB-interacting long noncoding RNA (NKILA), a suppressor of NF-κB activation, regulates HBV replication remains largely unknown. In this study, gain-and-loss experiments showed that NKILA inhibited HBV replication by inhibiting NF-κB activity. In turn, HBV infection down-regulated NKILA expression. In addition, expression levels of NKILA were lower in the peripheral blood-derived monocytes (PBMCs) of HBV-positive patients than in healthy individuals, which were correlated with HBV viral loads. And a negative correlation between NKILA expression level and HBV viral loads was observed in blood serum from HBV-positive patients. Lower levels of endogenous NKILA were also observed in HepG2 cells expressing a 1.3-fold HBV genome, HBV-infected HepG2-NTCP cells, stable HBV-producing HepG2.2.15 and HepAD38 â€‹cells, compared to those HBV-negative cells. Furthermore, HBx was required for NKILA-mediated inhibition on HBV replication. NKILA decreased HBx-induced NF-κB activation by interrupting the interaction between HBx and p65, whereas NKILA mutants lack of essential domains for NF-ĸB inhibition, lost the ability to inhibit HBV replication. Together, our data demonstrate that NKILA may serve as a suppressor of HBV replication via NF-ĸB signalling.

Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners.

BACKGROUND: Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated. METHODS: Immunoblotting, real-time polymerase chain reaction, in vivo / in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4 + T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data). RESULTS: The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression ( r  = 0.5110) and CD4 + T-cell counts ( r  = 0.5083) in HIV-1-infected patients. CONCLUSIONS: USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

IFN-ß1b induces OAS3 to inhibit EV71 via IFN-ß1b/JAK/STAT1 pathway.

Enterovirus 71 (EV71) caused hand, foot and mouth disease (HFMD) is a serious threat to the health of young children. Although type I interferon (IFN-I) has been proven to control EV71 replication, the key downstream IFN-stimulated gene (ISG) remains to be clarified and investigated. Recently, we found that 2'-5'-oligoadenylate synthetases 3 (OAS3), as one of ISG of IFN-ß1b, was antagonized by EV71 3C protein. Here, we confirm that OAS3 is the major determinant of IFN-ß1b-mediated EV71 inhibition, which depends on the downstream constitutive RNase L activation. 2'-5'-oligoadenylate (2-5A) synthesis activity deficient mutations of OAS3 D816A, D818A, D888A, and K950A lost resistance to EV71 because they could not activate downstream RNase L. Further investigation proved that EV71 infection induced OAS3 but not RNase L expression by IFN pathway. Mechanically, EV71 or IFN-ß1b-induced phosphorylation of STAT1, but not STAT3, initiated the transcription of OAS3 by directly binding to the OAS3 promoter. Our works elucidate the immune regulatory mechanism of the host OAS3/RNase L system against EV71 replication.

Ångstrom-scale silver particles potently combat SARS-CoV-2 infection by suppressing the ACE2 expression and inflammatory responses.

The SARS-CoV-2 pandemic has become a severe global public health event, and the development of protective and therapeutic strategies is urgently needed. Downregulation of angiotensin converting enzyme 2 (ACE2; one of the important SARS-CoV-2 entry receptors) and aberrant inflammatory responses (cytokine storm) are the main targets to inhibit and control COVID-19 invasion. Silver nanomaterials have well-known pharmaceutical properties, including antiviral, antibacterial, and anticancer properties. Here, based on a self-established metal evaporation-condensation-size graded collection system, smaller silver particles reaching the Ångstrom scale (AgÅPs) were fabricated and coated with fructose to obtain a stabilized AgÅP solution (F-AgÅPs). F-AgÅPs potently inactivated SARS-CoV-2 and prevented viral infection. Considering the application of anti-SARS-CoV-2, a sterilized F-AgÅP solution was produced via spray formulation. In our model, the F-AgÅP spray downregulated ACE2 expression and attenuated proinflammatory factors. Moreover, F-AgÅPs were found to be rapidly eliminated to avoid respiratory and systemic toxicity in this study as well as our previous studies. This work presents a safe and potent anti-SARS-CoV-2 agent using an F-AgÅP spray.

EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication.

The global spread of enteroviruses (EVs) has become more frequent, severe and life-threatening. Intereron (IFN) I has been proved to control EVs by regulating IFN-stimulated genes (ISG) expression. 2'-5'-oligoadenylate synthetases 3 (OAS3) is an important ISG in the OAS/RNase L antiviral system. The relationship between OAS3 and EVs is still unclear. Here, we reveal that OAS3, superior to OAS1 and OAS2, significantly inhibited EV71 replication in vitro. However, EV71 utilized autologous 3C protease (3Cpro) to cleave intracellular OAS3 and enhance viral replication. Rupintrivir, a human rhinovirus 3C protease inhibitor, completely abolished the cleavage of EV71 3Cpro on OAS3. And the proteolytically deficient mutants H40G, E71A, and C147G of EV71 3Cpro also lost the ability of OAS3 cleavage. Mechanistically, the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3Cpro cutting site. Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3 (CVB3), Coxsackievirus A16 (CA16), Enterovirus D68 (EVD68), and Coxsackievirus A6 (CA6) subtypes. Notably, unlike other four subtypes, CA16 3Cpro could not cleave OAS3. Two key amino acids variation Ile36 and Val86 in CA16 3Cpro might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage. Our works elucidate the broad anti-EVs function of OAS3, and illuminate a novel mechanism by which EV71 use 3Cpro to escape the antiviral effect of OAS3. These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.

No Statistically Apparent Difference in Antifungal Effectiveness Observed Among Trimethoprim/Sulfamethoxazole Plus Clindamycin or Caspofungin, and Trimethoprim/Sulfamethoxazole Monotherapy in HIV-Infected Patients with Moderate to Severe Pneumocystis Pneumonia: Results of an Observational Multicenter Cohort Study.

INTRODUCTION: Pneumocystis pneumonia is a common opportunistic infection in patients with HIV/AIDS, and is a leading cause of death in this population. Early selection of effective treatment is therefore critical to reduce mortality. We conducted a clinical trial to compare the effectiveness and safety of three different antifungal treatment regimens in HIV-infected patients with moderate to severe PCP. METHODS: Our study was a multicenter, observational prospective clinical trial. We recruited 320 HIV-infected patients with moderate to severe PCP, and stratified these subjects into a trimethoprim/sulfamethoxazole (TMP-SMX) monotherapy group, a TMP-SMX plus clindamycin group, and a TMP-SMX plus caspofungin group. Patients were invited to participate in 12 weeks of follow-up. Outcomes included the difference in overall mortality and the proportion of overall positive response to treatment in the three groups at weeks 4 and 12, the difference in treatment duration, and the proportion of adverse events among the three groups during the study period. RESULTS: The probability of survival not statistically different among three treatment groups. Mortality in the TMP-SMX monotherapy group (group 1) was 15/115 (13.04%) vs. 20/83 (24.10%) in the TMP-SMX plus clindamycin group (group 2) vs. 24/107 (22.43%) in the TMP-SMX plus caspofungin group (group 3) at week 12 (p = 0.092). The overall positive response rate to treatment in the three groups was 24.14%, 34.94%, and 38.32%, respectively, at week 4, and 33.91%, 38.55%, and 44.86%, respectively, at week 12. No significant difference in the overall positive response rate to treatment at either week 4 or week 12 was noted (p = 0.061, p = 0.246). Rates of changes to therapy were 6.50% (8/123) in group 1, 3.40% (3/87) in group 2, and 2.70% (3/110) in group 3, and did not differ significantly among the three groups (p = 0.376). There were also no significant differences in adverse events among the three treatment groups of patients with moderate to severe PCP. CONCLUSIONS: Our results indicate that there are no significant statistical differences among the three studied treatment regimens in terms of antifungal effectiveness in HIV-infected patients with moderate to severe PCP. TMP-SMX monotherapy is a convenient, cheap, and effective therapeutic drug regimen to treat HIV-infected patients with moderate to severe PCP, and is an appropriate treatment strategy in resource-limited settings. CLINICAL TRIAL REGISTRATION: www.ClinicalTrials.gov , ID: ChiCTR1900021195. Registered on February 1, 2019.

[Retracted] miR­138 inhibits gastric cancer growth by suppressing SOX4.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the colony formation assay data shown in Fig. 2C were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Oncology Reports 38: 1295­1302, 20137; DOI: 10.3892/or.2017.5745].

Specific Deubiquitinating Enzymes Promote Host Restriction Factors Against HIV/SIV Viruses.

Hijacking host ubiquitin pathways is essential for the replication of diverse viruses. However, the role of deubiquitinating enzymes (DUBs) in the interplay between viruses and the host is poorly characterized. Here, we demonstrate that specific DUBs are potent inhibitors of viral proteins from HIVs/simian immunodeficiency viruses (SIVs) that are involved in viral evasion of host restriction factors and viral replication. In particular, we discovered that T cell-functioning ubiquitin-specific protease 8 (USP8) is a potent and specific inhibitor of HIV-1 virion infectivity factor (Vif)-mediated apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3)G (A3G) degradation. Ectopic expression of USP8 inhibited Vif-induced A3G degradation and suppressed wild-type HIV-1 infectivity even in the presence of Vif. In addition, specific DUBs repressed Vpr-, Vpu-, and Vpx-triggered host restriction factor degradation. Our study has revealed a previously unrecognized interplay between the host's DUBs and viral replication. Enhancing the antiviral activity of DUBs therefore represents an attractive strategy against HIVs/SIVs.

Increased BST-2 expression by HBV infection promotes HBV-associated HCC tumorigenesis.

BACKGROUND: The majority of hepatocellular carcinoma (HCC) is closely associated with hepatitis B virus (HBV) infection, while the mechanism of HCC induced by HBV is debatable. Bone marrow stromal cell antigen 2 (BST-2), an N-glycoprotein, has been characterized as an oncogenic factor in several types of cancer. However, whether BST-2 plays an important role in HCC tumorigenesis remains unknown. METHODS: A total of 182 HCC tumorous and adjacent nontumor liver tissues were collected. HepG2, Huh7, L02, HepAD38, and HEK293T cell lines were adopted in this study. Tumor proliferation was detected by CCK8, transwell, wound healing, colony formation assays in vitro, and in vivo tumorigenesis was measured by mouse xenografts. NF-κB activation was determined by luciferase assay and Western blot. Protein expression was detected by Western blot, ELISA, or qPCR. Immunoprecipitation was used to confirm the interaction between BST-2 and Syk. RESULTS: Here, we observed the higher BST-2 expression in HBV-infected HCC than their paired adjacent tissues and HBV-uninfected HCC tissues, particularly more aberrant non-N-glycosylated BST-2 in HBV-infected HCC tumors. We also observed the increased ER degradation-enhancing α-mannosidase-like protein 3 (EDEM3), which is trimming of N-linked glycans by sequential removal of mannose residues, might result in more non-N-glycosylated form of BST-2. Moreover, we demonstrated that BST-2 and non-N-glycosylated BST-2 N65/92A mutant, not only enhanced the tumor characteristics of hepatoma cell lines in vitro, but also enhanced the growth of mouse xenografts in vivo. Mechanically, N65/92A mutant has stronger ability to promote HCC than BST-2 via NF-κB/ERK1/2 but not NF-κB/anti-apoptotic factors pathway. NF-κB inhibitor attenuated BST-2-mediated tumorigenesis of HCC. CONCLUSIONS: Our findings illuminate the novel function of BST-2 as an oncogene of HBV-associated HCC, and highlight the novel relationship of N-glycosylation of BST-2 in regulating HCC tumorigenesis in vitro.

Deubiquitinating Enzyme USP21 Inhibits HIV-1 Replication by Downregulating Tat Expression.

Ubiquitination plays an important role in human immunodeficiency virus 1 (HIV-1) infection. HIV proteins such as Vif and Vpx mediate the degradation of the host proteins APOBEC3 and SAMHD1, respectively, through the proteasome pathway. However, whether deubiquitylating enzymes play an essential role in HIV-1 infection is largely unknown. Here, we demonstrate that the deubiquitinase USP21 potently inhibits HIV-1 production by indirectly downregulating the expression of HIV-1 transactivator of transcription (Tat), which is essential for transcriptional elongation in HIV-1. USP21 deubiquitylates Tat via its deubiquitinase activity, but a stronger ability to reduce Tat expression than a dominant-negative ubiquitin mutant (Ub-KO) showed that other mechanisms may contribute to USP21-mediated inhibition of Tat. Further investigation showed that USP21 downregulates cyclin T1 mRNA levels by increasing methylation of histone K9 in the promoter of cyclin T1, a subunit of the positive transcription elongation factor b (P-TEFb) that interacts with Tat and transactivation response element (TAR) and is required for transcription stimulation and Tat stability. Moreover, USP21 had no effect on the function of other HIV-1 accessory proteins, including Vif, Vpr, Vpx, and Vpu, indicating that USP21 was specific to Tat. These findings improve our understanding of USP21-mediated functional suppression of HIV-1 production. IMPORTANCE Ubiquitination plays an essential role in viral infection. Deubiquitinating enzymes (DUBs) reverse ubiquitination by cleaving ubiquitins from target proteins, thereby affecting viral infection. The role of the members of the USP family, which comprises the largest subfamily of DUBs, is largely unknown in HIV-1 infection. Here, we screened a series of USP members and found that USP21 inhibits HIV-1 production by specifically targeting Tat but not the other HIV-1 accessory proteins. Further investigations revealed that USP21 reduces Tat expression in two ways. First, USP21 deubiquitinates polyubiquitinated Tat, causing Tat instability, and second, USP21 reduces the mRNA levels of cyclin T1 (CycT1), an important component of P-TEFb, that leads to Tat downregulation. Thus, in this study, we report a novel role of the deubiquitinase, USP21, in HIV-1 infection. USP21 represents a potentially useful target for the development of novel anti-HIV drugs.

ATP1B3 Restricts Hepatitis B Virus Replication Via Reducing the Expression of the Envelope Proteins.

Our recent study reported that ATP1B3 inhibits hepatitis B virus (HBV) replication via inducing NF-κB activation. However, ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication, suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression. Here, we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs, MHBs and SHBs, but had no effect on intracellular HBV DNA, RNA levels as well as HBV promoter activities. Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation, demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins. Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination. Immunofluorescence co-localization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together. Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.

NF-κB-Interacting Long Noncoding RNA Regulates HIV-1 Replication and Latency by Repressing NF-κB Signaling.

NF-κB-interacting long noncoding RNA (NKILA) was recently identified as a negative regulator of NF-κB signaling and plays an important role in the development of various cancers. It is well known that NF-κB-mediated activation of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-driven gene expression is required for HIV-1 transcription and reactivation of latency. However, whether NKILA plays essential roles in HIV-1 replication and latency is unclear. Here, by ectopic expression and silencing experiments, we demonstrate that NKILA potently inhibits HIV-1 replication in an NF-κB-dependent manner by suppressing HIV-1 LTR promoter activity. Moreover, NKILA showed broad-spectrum inhibition on the replication of HIV-1 clones with different coreceptor tropisms as well as on LTR activity of various HIV-1 clinical subtypes. Chromatin immunoprecipitation (ChIP) assays revealed that NKILA expression abolishes the recruitment of p65 to the duplicated κB binding sites in the HIV-1 LTR. NKILA mutants disrupting NF-κB inhibition also lost the ability to inhibit HIV-1 replication. Notably, HIV-1 infection or reactivation significantly downregulated NKILA expression in T cells in order to facilitate viral replication. Downregulated NKILA was mainly due to reduced acetylation of histone K27 on the promoter of NKILA by HIV-1 infection, which blocks NKILA expression. Knockdown of NKILA promoted the reactivation of latent HIV-1 upon phorbol myristate acetate (PMA) stimulation, while ectopic NKILA suppressed the reactivation in a well-established clinical model of withdrawal of azidothymidine (AZT) in vitro These findings improve our understanding of the functional suppression of HIV-1 replication and latency by NKILA through NF-κB signaling.IMPORTANCE The NF-κB pathway plays key roles in HIV-1 replication and reactivation of HIV-1 latency. A regulator inhibiting NF-κB activation may be a promising therapeutic strategy against HIV-1. Recently, NF-κB-interacting long noncoding RNA (NKILA) was identified to suppress the development of different human cancers by inhibiting IκB kinase (IKK)-induced IκB phosphorylation and NF-κB pathway activation, whereas the relationship between NKILA and HIV-1 replication is still unknown. Here, our results show that NKILA inhibits HIV-1 replication and reactivation by suppressing HIV-1 long terminal repeat (LTR)-driven transcription initiation. Moreover, NKILA inhibited the replication of HIV-1 clones with different coreceptor tropisms. This project may reveal a target for the development of novel anti-HIV drugs.

SERINC5 Inhibits the Secretion of Complete and Genome-Free Hepatitis B Virions Through Interfering With the Glycosylation of the HBV Envelope.

Serine incorporator 3 (SERINC3) and SERINC5 were recently identified as host intrinsic factors against human immunodeficiency virus (HIV)-1 and counteracted by HIV-1 Nef. However, whether they inhibit hepatitis B virus (HBV), which is a severe health problem worldwide, is unknown. Here, we demonstrate that SERINC5 potently inhibited HBV virion secretion in the supernatant without affecting intracellular core particle-associated DNA and the total RNA, but SERINC3 and SERINC1 did not. Further investigation discovered that SERINC5 increased the non-glycosylation of LHB, MHB, and SHB proteins of HBV and slightly decreased HBs proteins levels, which led to the decreased HBV secretion. Importantly, SERINC5 co-localized with LHB proteins in the Golgi apparatus, which is important for glycan processing and transport. In addition, we determined the functional domain in SERINC5 required for HBV inhibition, which was completely different from that required for HIV-1 restriction, whereas phosphorylation and glycosylation sites in SERINC5 were dispensable for HBV restriction. Taken together, our results demonstrate that SERINC5 suppresses HBV virion secretion through interfering with the glycosylation of HBV proteins, suggesting that SERINC5 might possess broad-spectrum antiviral activity.

The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses.

Human enteroviruses (EVs), including coxsackieviruses, the numbered enteroviruses, and echoviruses, cause a wide range of diseases, such as hand, foot, and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis (AFM), pneumonia, and bronchiolitis. Therefore, broad-spectrum anti-EV drugs are urgently needed to treat EV infection. Here, we demonstrate that FNC (2'-deoxy-2'-ß-fluoro-4'-azidocytidine), a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs, including enterovirus 71 (EV71), coxsackievirus A16 (CA16), CA6, EVD68, and coxsackievirus B3 (CVB3), at the nanomolar level. The antiviral mechanism of FNC involves mainly positive- and negative-strand RNA synthesis inhibition by targeting and competitively inhibiting the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol), as demonstrated through quantitative real-time reverse transcription-PCR (RT-qPCR), in vitro 3Dpol activity, and isothermal titration calorimetry (ITC) experiments. We further demonstrated that FNC treatment every 2 days with 1 mg/kg of body weight in EV71 and CA16 infection neonatal mouse models successfully protected mice from lethal challenge with EV71 and CA16 viruses and reduced the viral load in various tissues. These findings provide important information for the clinical development of FNC as a broad-spectrum inhibitor of human EV pathogens.IMPORTANCE Human enterovirus (EV) pathogens cause various contagious diseases such as hand, foot, and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, and bronchiolitis, which have become serious health threats. However, except for the EV71 vaccine on the market, there are no effective strategies to prevent and treat other EV pathogen infections. Therefore, broad-spectrum anti-EV drugs are urgently needed. In this study, we demonstrated that FNC, a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs at the nanomolar level. Further investigation revealed that FNC inhibits positive- and negative-strand RNA synthesis of EVs by interacting and interfering with the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol). Our findings demonstrate for the first time that FNC is an effective broad-spectrum inhibitor for human EV pathogens.

ATP1B3 cooperates with BST-2 to promote hepatitis B virus restriction.

Increasing evidence indicates ATP1B3, one of the regulatory subunits of Na+ /K+ -ATPase, is involved in numerous viral propagations, such as HIV and EV71. However, the function and mechanism of ATP1B3 on hepatitis B virus (HBV) propagation is unknown. Here, we demonstrated that ATP1B3 overexpression reduced the quantity of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in supernatants of HBV expression plasmids cotransfected HepG2 cells. Correspondingly, small interfering RNA and short hairpin RNA mediated ATP1B3 silencing promoted HBsAg and HBeAg expression in the supernatants of HBV expression plasmids transfected HepG2 cells. Mechanically, we reported that ATP1B3 expression could activate nuclear factor-κB (NF-κB) pathway by inducing the expression, phosphorylation, and nuclear import of P65 for the first time. And NF-κB inhibitor (Bay11) impaired the restraint of ATP1B3 on HBV replication. This counteraction effect of Bay11 proved that ATP1B3-induced NF-κB activation was crucial for HBV restriction. Accordingly, we observed that anti-HBV factors interferon-α (IFN-α) and interleukin-6 (IL-6) production were increased in HepG2 cells after the NF-κB activation. It suggested that ATP1B3 suppressed HBsAg and HBeAg by NF-κB/IFN-α and NF-κB/IL-6 axis. Further experiments proved that ATP1B3 overexpression induced anti-HBV factor BST-2 expression by NF-κB/IFN-α axis in HepG2 cells but not HEK293T cells, and ATP1B3 silencing downregulated BST-2 messenger RNA level in HepG2 cells. As an HBV restriction factor, BST-2 cooperated with ATP1B3 to antagonize HBsAg but not HBeAg in HepG2 cells. Our work identified ATP1B3 as a novel candidate of HBV restrictor with unrevealed mechanism and we highlighted it might serve as a potential therapeutic molecule for HBV infection.

TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity.

SAMHD1 possesses multiple functions, but whether cellular factors regulate SAMHD1 expression or its function remains not well characterized. Here, by investigating why cultured RD and HEK293T cells show different sensitivity to enterovirus 71 (EV71) infection, we demonstrate that SAMHD1 is a restriction factor for EV71. Importantly, we identify TRIM21, an E3 ubiquitin ligase, as a key regulator of SAMHD1, which specifically interacts and degrades SAMHD1 through the proteasomal pathway. However, TRIM21 has no effect on EV71 replication itself. Moreover, we prove that interferon production stimulated by EV71 infection induces increased TRIM21 and SAMHD1 expression, whereas increasing TRIM21 overrides SAMHD1 inhibition of EV71 in cells and in a neonatal mouse model. TRIM21-mediated degradation of SAMHD1 also affects SAMHD1-dependent restriction of HIV-1 and the regulation of interferon production. We further identify the functional domains in TRIM21 required for SAMHD1 binding and the ubiquitination site K622 in SAMHD1 and show that phosphorylation of SAMHD1 at T592 also blocks EV71 restriction. Our findings illuminate how EV71 overcomes SAMHD1 inhibition via the upregulation of TRIM21.

Defective modulation of LINE-1 retrotransposition by cancer-associated SAMHD1 mutants.

Long interspersed nuclear elements (LINE-1) is now considered as the only active autonomous mobile DNA in humans, LINE-1 retrotransposition activities are associated with and fluctuate during cancer initiation and progression; however, the mechanism underlying the increased LINE-1 activity in cancer is poorly understood. SAMHD1 has been reported to be a potent inhibitor of LINE-1 retrotransposition, and SAMHD1 mutations are frequently associated with cancer development. To gain insights on whether cancer-related SAMHD1 mutants affect LINE-1 activity, we explored the biochemical and cellular properties of some human mutants known correlate with the development of cancer. Most of the tested SAMHD1 cancer-related mutations were defective in LINE-1 inhibition. Interestingly we also found that SAMHD1 mutant K288T was defective for dNTPase activity but showed potent activity against LINE-1 retrotransposition. These findings suggest that LINE-1 inhibition does not depend solely on the dNTPase activity of SAMHD1. In contrast, SAMHD1's ability to inhibit ORF2p-mediated LINE-1 RNP reverse transcription was correlated with SAMHD1-mediated LINE-1 inhibition. Together, our data could also facilitate the deeper understanding for the inhibition of endogenous LINE-1 elements by SAMHD1.

Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G.

Although the host restriction factor APOBEC3G (A3G) has broad spectrum antiviral activity, whether A3G inhibits enterovirus 71 (EV71) has been unclear until now. In this study, we demonstrated for the first time that A3G could inhibit EV71 virus replication. Silencing A3G in H9 cells enhanced EV71 replication, and EV71 replication was lower in H9 cells expressing A3G than in Jurkat cells without A3G expression, indicating that the EV71 inhibition was A3G-specific. Further investigation revealed that A3G inhibited the 5'UTR activity of EV71 by competitively binding to the 5'UTR through its nucleic acid binding activity. This binding impaired the interaction between the 5'UTR and the host protein poly(C)-binding protein 1 (PCBP1), which is required for the synthesis of EV71 viral proteins and RNA. On the other hand, we found that EV71 overcame A3G suppression through its non-structural protein 2C, which induced A3G degradation through the autophagy-lysosome pathway. Our research provides new insights into the interplay mechanisms of A3G and single-stranded positive RNA viruses.

Coxsackievirus A6 Induces Cell Cycle Arrest in G0/G1 Phase for Viral Production.

Recent epidemiological data indicate that outbreaks of hand, foot, and mouth disease (HFMD), which can be categorized according to its clinical symptoms as typical or atypical, have markedly increased worldwide. A primary causative agent for typical HFMD outbreaks, enterovirus 71 (EV71), has been shown to manipulate the cell cycle in S phase for own replication; however, it is not clear whether coxsackievirus (CVA6), the main agent for atypical HFMD, also regulates the host cell cycle. In this study, we demonstrate for the first time that CVA6 infection arrests the host cell cycle in G0/G1-phase. Furthermore, synchronization in G0/G1 phase, but not S phase or G2/M phase, promotes viral production. To investigate the mechanism of cell cycle arrest induced by CVA6 infection, we analyzed cell cycle progression after cell cycle synchronization at G0/G1 or G2/M. Our results demonstrate that CVA6 infection promotes G0/G1 phase entry from G2/M phase, and inhibits G0/G1 exit into S phase. In line with its role to arrest cells in G0/G1 phase, the expression of cyclinD1, CDK4, cyclinE1, CDK2, cyclinB1, CDK1, P53, P21, and P16 is regulated by CVA6. Finally, the non-structural proteins of CVA6, RNA-dependent RNA polymerase 3D and protease 3C , are demonstrated to be responsible for the G0/G1-phase arrest. These findings suggest that CVA6 infection arrested cell cycle in G0/G1-phase via non-structural proteins 3D and 3C, which may provide favorable environments for virus production.