SARS-CoV-2 and oncolytic EV-D68-encoded proteases differentially regulate pyroptosis.

Pyroptosis, a pro-inflammatory programmed cell death, has been implicated in the pathogenesis of coronavirus disease 2019 and other viral diseases. Gasdermin family proteins (GSDMs), including GSDMD and GSDME, are key regulators of pyroptotic cell death. However, the mechanisms by which virus infection modulates pyroptosis remain unclear. Here, we employed a mCherry-GSDMD fluorescent reporter assay to screen for viral proteins that impede the localization and function of GSDMD in living cells. Our data indicated that the main protease NSP5 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) blocked GSDMD-mediated pyroptosis via cleaving residues Q29 and Q193 of GSDMD. While another SARS-CoV-2 protease, NSP3, cleaved GSDME at residue G370 but activated GSDME-mediated pyroptosis. Interestingly, respiratory enterovirus EV-D68-encoded proteases 3C and 2A also exhibit similar differential regulation on the functions of GSDMs by inactivating GSDMD but initiating GSDME-mediated pyroptosis. EV-D68 infection exerted oncolytic effects on human cancer cells by inducing pyroptotic cell death. Our findings provide insights into how respiratory viruses manipulate host cell pyroptosis and suggest potential targets for antiviral therapy as well as cancer treatment.IMPORTANCEPyroptosis plays a crucial role in the pathogenesis of coronavirus disease 2019, and comprehending its function may facilitate the development of novel therapeutic strategies. This study aims to explore how viral-encoded proteases modulate pyroptosis. We investigated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory enterovirus D68 (EV-D68) proteases on host cell pyroptosis. We found that SARS-CoV-2-encoded proteases NSP5 and NSP3 inactivate gasdermin D (GSDMD) but initiate gasdermin E (GSDME)-mediated pyroptosis, respectively. We also discovered that another respiratory virus EV-D68 encodes two distinct proteases 2A and 3C that selectively trigger GSDME-mediated pyroptosis while suppressing the function of GSDMD. Based on these findings, we further noted that EV-D68 infection triggers pyroptosis and produces oncolytic effects in human carcinoma cells. Our study provides new insights into the molecular mechanisms underlying virus-modulated pyroptosis and identifies potential targets for the development of antiviral and cancer therapeutics.

cGAS-STING-mediated novel nonclassic antiviral activities.

Stimulatorof interferon genes (STING) is an intracellular sensor of cyclic dinucleotides involved in the innate immune response against pathogen- or self-derived DNA. For years, interferon (IFN) induction of cyclic GMP-AMP synthase (cGAS)-STING has been considered as a canonical pattern defending the host from viral invasion. The mechanism of the cGAS-STING-IFN pathway has been well-illustrated. However, other signalling cascades driven by cGAS-STING have emerged in recent years and some of them have been found to possess antiviral ability independent of IFN. Here, we summarize the current progress on cGAS-STING-mediated nonclassic antiviral activities with an emphasis on the nuclear factor-κB and autophagy pathways, which are the most-studied pathways. In addition, we briefly present the primordial function of the cGAS-STING pathway in primitive species to show the importance of IFN-unrelated antiviral activity from an evolutionary angle. Finally, we discuss open questions that need to be solved for further exploitation of this field.

SARS-CoV-2, HIV, and HPV: Convergent evolution of selective regulation of cGAS-STING signaling.

Recognizing aberrant cytoplasmic double-stranded DNA and stimulating innate immunity is essential for the host's defense against viruses and tumors. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that synthesizes the second messenger 2'3'-cGAMP and subsequently activates stimulator of interferon genes (STING)-mediated activation of TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and the production of type I interferon (IFN-I). Both the cGAS-STING-mediated IFN-I antiviral defense and the countermeasures developed by diverse viruses have been extensively studied. However, recent studies have revealed a convergent evolutionary feature of severe acute respiratory syndrome coronavirus 2 and human immunodeficiency virus (HIV) viral proteins in terms of the selective regulation of cGAS-STING-mediated nuclear factor-κB (NF-κB) signaling without any effect on cGAS-STING-mediated TBK1/IRF3 activation and IFN production. The potential beneficial effect of this cGAS-STING-mediated, NF-κB-dependent antiviral effect, and the possible detrimental effect of IFN-I in the pathogenesis of coronavirus disease 2019 and HIV infection deserve more attention and future investigation.

DNA virus oncoprotein HPV18 E7 selectively antagonizes cGAS-STING-triggered innate immune activation.

Cellular infections by DNA viruses trigger innate immune responses mediated by DNA sensors. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signaling pathway has been identified as a DNA-sensing pathway that activates interferons in response to viral infection and, thus, mediates host defense against viruses. Previous studies have identified oncogenes E7 and E1A of the DNA tumor viruses, human papillomavirus 18 (HPV18) and adenovirus, respectively, as inhibitors of the cGAS-STING pathway. However, the function of STING in infected cells and the mechanism by which HPV18 E7 antagonizes STING-induced Interferon beta production remain unknown. We report that HPV18 E7 selectively antagonizes STING-triggered nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation but not IRF3 activation. HPV18 E7 binds to STING in a region critical for NF-κB activation and blocks the nuclear accumulation of p65. Moreover, E7 inhibition of STING-triggered NF-κB activation is related to HPV pathogenicity but not E7-Rb binding. HPV18 E7, severe acute respiratory syndrome coronavirus-2 open reading frame 3a, human immunodeficiency virus-2 viral protein X, and Kaposi's sarcoma-associated herpesvirus KSHV viral interferon regulatory factor 1 selectively inhibited STING-triggered NF-κB or IRF3 activation, suggesting a convergent evolution among these viruses toward antagonizing host innate immunity. Collectively, selective suppression of the cGAS-STING pathway by viral proteins is likely to be a key pathogenic determinant, making it a promising target for treating oncogenic virus-induced tumor diseases.

SARS-CoV-2 ORF3a inhibits cGAS-STING-mediated autophagy flux and antiviral function.

Recognizing aberrant cytoplasmic dsDNA and stimulating cGAS-STING-mediated innate immunity is essential for the host defense against viruses. Recent studies have reported that SARS-CoV-2 infection, responsible for the COVID-19 pandemic, triggers cGAS-STING activation. cGAS-STING activation can trigger IRF3-Type I interferon (IFN) and autophagy-mediated antiviral activity. Although viral evasion of STING-triggered IFN-mediated antiviral function has been well studied, studies concerning viral evasion of STING-triggered autophagy-mediated antiviral function are scarce. In the present study, we have discovered that SARS-CoV-2 ORF3a is a unique viral protein that can interact with STING and disrupt the STING-LC3 interaction, thus blocking cGAS-STING-induced autophagy but not IRF3-Type I IFN induction. This novel function of ORF3a, distinct from targeting autophagosome-lysosome fusion, is a selective inhibition of STING-triggered autophagy to facilitate viral replication. We have also found that activation of bat STING can induce autophagy and antiviral activity despite its defect in IFN induction. Furthermore, ORF3a from bat coronaviruses can block bat STING-triggered autophagy and antiviral function. Interestingly, the ability to inhibit STING-induced autophagy appears to be an acquired function of SARS-CoV-2 ORF3a, since SARS-CoV ORF3a lacks this function. Taken together, these discoveries identify ORF3a as a potential target for intervention against COVID-19.

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.

Contrast of Mastoscopic and Conventional Axillary Lymph Node Dissection of Patients With Breast Cancer: Meta-Analysis.

Mastoscopic axillary lymph node dissection (MALND) is a currently used and safe surgical treatment option for breast cancer. However, the extensive application of MALND is still debatable because of the use of conventional axillary lymph node dissection (CALND). Therefore, in the current study, we aimed to compare the efficacy and safety of MALND and CALND for obtaining evidence-based conclusions about the short-term and long-term outcomes of MALND for patients with breast cancer. PubMed, Web of Science, Cochrane Library, and CNKI were comprehensively searched for articles published between January 1998 and January 2019. Then Newcastle-Ottawa scale was used for quality assessment. The Review Manager software version 5.0 was utilized for generating forest maps and funnel plots. Twelve studies including 2157 patients were selected for the meta-analysis. There were no significant differences in the number of lymph node dissections, tumor recurrence rate, axillary drainage, postoperative hospitalization time, and tumor size between the MALND and CALND groups (P > .05). In the MALND group, the surgery time was longer, while the incidence of intraoperative bleeding was lesser and the duration of drainage was shorter than those in the CALND group (P < .01). The complications in the MALND group were also fewer than those in the CALND group (P < .05). The results of the current study showed that MALND is reliable and feasible for breast cancer owing to the lesser incidence of intraoperative bleeding, shorter drainage duration, and lower incidence of complications compared to CALND.

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.

Emerging roles and therapeutic value of exosomes in cancer metastasis.

Exosomes are cell-derived vesicles of 30 to 150 nm that contain diverse proteins, nucleic acids, and lipids. These vesicles facilitate effective intercellular communication and trigger profound environmental changes. In recent years, many studies have identified diverse roles for exosomes in tumor metastasis, a major cause of cancer-related deaths; furthermore, circulating tumor-derived exosomes can drive the initiation and progression of metastasis and determine the specific target organs affected. Fortunately, our growing understanding of exosomes and relevant modification technology have provided new ideas for potential treatment of tumor metastases. Here we review recent advances concerning the role of exosomes in metastasis, focusing on their regulatory mechanisms and therapeutic targeting in advanced cancer.

Determinants of lentiviral Vpx-CRL4 E3 ligase-mediated SAMHD1 degradation in the substrate adaptor protein DCAF1.

The lentiviral accessory protein Vpx enhances viral replication in macrophages, dendritic cells and resting CD4+ T cells by utilizing the host CRL4-DCAF1 E3 ligase to trigger the degradation of the intrinsic antiviral factor SAMHD1. Distinct from the species-specific recognition of either the N or C-terminus of SAMHD1 by Vpx proteins of different HIV-2 and SIV lineages, Vpx recruits SAMHD1 onto the same CRL4-DCAF1 complex. However, the determinants in DCAF1 that are required for Vpx-mediated SAMHD1 degradation have not been well characterized. Here, we demonstrate that the viral protein Vpx is resistant to suppression by a cellular inhibitor of the CRL4-DCAF1 E3 ligase, Merlin/NF2, through targeting a separate binding region in DCAF1. The Merlin binding-deficient DCAF1 truncation mutant (1-1417) is sufficient for Vpx-CRL4-DCAF1 E3 ligase assembly and SAMHD1 degradation. We found that the carboxyl-terminus ED-rich region (1312-1417) of DCAF1 is required for the nuclear localization of DCAF1 and for the Vpx-DCAF1 interaction. We identified the DCAF1 (1-1311) truncation mutant as a dominant negative mutant of wild-type DCAF1 that inhibits Vpx-mediated SAMHD1 degradation. These results suggest a unique strategy by which Vpx exploits DCAF1 to counteract this host restriction factor.

Vif-CBFß interaction is essential for Vif-induced cell cycle arrest.

Interaction between HIV-1 Vif and host factor CBFß leads to the assembly of the Vif-Cul5-EloB/C ubiquitin ligase (E3 complex). By inducing the formation of E3 complex, Vif depletes host APOBEC3 restriction factors and promotes HIV-1 infection. In addition, Vif is known to arrest host cells at G2/M phase (G2 arrest), benefiting HIV-1 replication and contributing to the depletion of CD4+ T cells. However, whether CBFß is also involved in Vif-induced cell cycle arrest remains unclear. In the present study, we report that CBFß is an essential factor for Vif-induced G2 arrest. Reducing endogenous CBFß expression significantly compromised Vif's potency in cell cycle regulation. In addition, tests with CBFß and Vif mutants indicated that Vif-CBFß interaction is crucial for Vif to induce G2 arrest. Furthermore, suppressors against Vif-hijacked E3 complex or proteasome-mediated proteolysis also abolished Vif's ability to cause G2 arrest. In general, our data indicated that Vif induces G2 arrest through depletion of a yet-unknown cellular factor, where the involvement of CBFß is essential. On the other hand, our data also suggested that, antiviral drugs targeting the Vif-CBFß interaction have the potential to abolish Vif's ability to cause APOBEC3 degradation as well as G2 arrest in host cells, thus reducing both HIV-1 replication and Vif-induced CD4+ T-cell depletion.

Adenovirus oncoprotein E4orf6 triggers Cullin5 neddylation to activate the CLR5 E3 ligase for p53 degradation.

The human adenovirus oncoprotein E4orf6 hijacks intracellular Cullin 5-based E3 ubiquitin ligases (CRL5s) to induce the degradation of host proteins, including p53, that impede efficient viral replication. The complex also relies on another viral protein, E1B55K, to recruit substrates for ubiquitination. However, the determinants of adenoviral E4orf6-CRL5 E3 ligase-mediated p53 degradation in the scaffolding protein Cullin5 remain rarely investigated. Here, we demonstrated that the viral protein E4orf6 triggered relocalization of the Cullin5 protein from the cytoplasm to the nucleus and induced activation of the CRL5 E3 ligase via facilitating neddylation. The expression of the deneddylase SENP8/Den1 was significantly downregulated by E4orf6. We then identified SENP8 as a natural restriction factor for E4orf6-induced p53 degradation. Furthermore, our results indicated that the NEDD8-conjugating E2 enzyme UBE2M was essential for E4orf6-mediated p53 degradation and that its dominant negative mutant UBE2M C111S dramatically blocked E4orf6 functions. The Nedd8-activating enzyme inhibitor MLN4924 decreased E4orf6-induced neddylation of the cullin5 protein and subsequently suppressed p53 degradation. Collectively, our findings illuminate the strategy by which this viral oncoprotein specifically utilizes the neddylation pathway to activate host CRL E3 ligases to degrade host restriction factors. Disrupting this post-translational modification is an attractive pharmacological intervention against human adenoviruses.

Long Noncoding RNA uc002yug.2 Activates HIV-1 Latency through Regulation of mRNA Levels of Various RUNX1 Isoforms and Increased Tat Expression.

The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4+ T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions.IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1.

Divergent Pathogenic Properties of Circulating Coxsackievirus A6 Associated with Emerging Hand, Foot, and Mouth Disease.

Coxsackievirus A6 (CV-A6) is an emerging pathogen associated with hand, foot, and mouth disease (HFMD). Its genetic characterization and pathogenic properties are largely unknown. Here, we report 39 circulating CV-A6 strains isolated in 2013 from HFMD patients in northeast China. Three major clusters of CV-A6 were identified and related to CV-A6, mostly from Shanghai, indicating that domestic CV-A6 strains were responsible for HFMD emerging in northeast China. Four full-length CV-A6 genomes representing each cluster were sequenced and analyzed further. Bootscanning tests indicated that all four CV-A6-Changchun strains were most likely recombinants between the CV-A6 prototype Gdula and prototype CV-A4 or CV-A4-related viruses, while the recombination pattern was related to, yet distinct from, the strains isolated from other regions of China. Furthermore, different CV-A6 strains showed different capabilities of viral replication, release, and pathogenesis in a mouse model. Further analyses indicated that viral protein 2C contributed to the diverse pathogenic abilities of CV-A6 by causing autophagy and inducing cell death. To our knowledge, this study is the first to report lethal and nonlethal strains of CV-A6 associated with HFMD. The 2C protein region may play a key role in the pathogenicity of CV-A6 strains.IMPORTANCE Hand, foot, and mouth disease (HFMD) is a major and persistent threat to infants and children. Besides the most common pathogens, such as enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16), other enteroviruses are increasingly contributing to HFMD. The present study focused on the recently emerged CV-A6 strain. We found that CV-A6 strains isolated in Changchun City in northeast China were associated with domestic origins. These Changchun viruses were novel recombinants of the CV-A6 prototype Gdula and CV-A4. Our results imply that measures to control CV-A6 transmission are urgently needed. Further analyses revealed differing pathogenicities in strains isolated in a neonatal mouse model. One of the possible causes has been narrowed down to the viral protein 2C, using phylogenetic studies, viral sequences, and direct tests on cultured human cells. Thus, the viral 2C protein is a promising target for antiviral drugs to prevent CV-A6-induced tissue damage.

PD-1 high expression predicts lower local disease control in stage IV M0 nasopharyngeal carcinoma.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) play a critical role in tumor immune surveillance and immune suppression. Understanding tumor infiltrating T cell subset density, location and PD-1/PD-L1 expression might provide insight for the prediction of tumor therapeutic response and clinical outcome. The purpose of this study was to evaluate the expression and localization of CD8, FoxP3, PD-1, and PD-L1 in primary tumor tissues and their effects on prognosis of stage IV M0 locally advanced nasopharyngeal carcinoma (NPC) patients. METHODS: Sixty NPC patients with stage IV M0 locally advanced disease were treated with definitive chemoradiation. Tumor biopsies from primary lesion were analyzed for the expression and localization of CD8, FoxP3, PD-1, and PD-L1 by immunohistochemistry. Their associations with local disease control and survival of NPC were analyzed. RESULTS: The average follow-up time was 43 months (range from 14 to 61 months). High expression of CD8+, FoxP3+, PD-1+ and PD-L1+ was observed in 60, 86.7, 56.7, and 91.7% of patients, respectively. There was no correlation between clinicopathological features and the expression of these immune markers. High PD-1 expression was found to be associated with lower local disease control (5-year LRFS 23.2% vs 96.8%, p < 0.001) and unfavorable clinical outcome (5-year OS 47.4% vs 73.3%, p = 0.014). In multivariate analysis, PD-1 expression was also an adverse prognostic factor for 5-year OS (HR: 3.68, P = 0.023) and LRFS (HR: 16.89, 1.27-11.84, P = 0.007). Those with PD-1 distribution in both stroma and tumor region had the poorest prognosis. However, PD-1 expression has no significant correlation with 5-year RRFS (p = 0.980) and DMFS (p = 0.865). Patients with both PD-1 and PD-L1 high expression had significant poor local disease control (5-year LRFS 96.0% vs 43.0%, p < 0.001) and overall survival (5-year OS 80.8% vs 45.1%, p < 0.001) compared with the others. Other immune markers were not found having corrections with disease control and survival. CONCLUSIONS: PD-1 high expression, especially with PD-L1 co-expression, is associated with high local recurrence and unfavorable clinical outcome for stage IV M0 NPC patients, and might be a potential target for immunotherapy.

Aicardi-Goutières syndrome protein TREX1 suppresses L1 and maintains genome integrity through exonuclease-independent ORF1p depletion.

Maintaining genome integrity is important for cells and damaged DNA triggers autoimmunity. Previous studies have reported that Three-prime repair exonuclease 1(TREX1), an endogenous DNA exonuclease, prevents immune activation by depleting damaged DNA, thus preventing the development of certain autoimmune diseases. Consistently, mutations in TREX1 are linked with autoimmune diseases such as systemic lupus erythematosus, Aicardi-Goutières syndrome (AGS) and familial chilblain lupus. However, TREX1 mutants competent for DNA exonuclease activity are also linked to AGS. Here, we report a nuclease-independent involvement of TREX1 in preventing the L1 retrotransposon-induced DNA damage response. TREX1 interacted with ORF1p and altered its intracellular localization. Furthermore, TREX1 triggered ORF1p depletion and reduced the L1-mediated nicking of genomic DNA. TREX1 mutants related to AGS were deficient in inducing ORF1p depletion and could not prevent L1-mediated DNA damage. Therefore, our findings not only reveal a new mechanism for TREX1-mediated L1 suppression and uncover a new function for TREX1 in protein destabilization, but they also suggest a novel mechanism for TREX1-mediated suppression of innate immune activation through maintaining genome integrity.

Role of APOBEC3F Gene Variation in HIV-1 Disease Progression and Pneumocystis Pneumonia.

Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant (p. I231V, with allele (V) frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) (RH = 0.53, 95% CI: 0.37-0.76). A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression.

Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production.

The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55Gag compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55Gag interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55Gag, which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4+ T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55Gag interaction provides a potential target for the future development of antiviral strategies.IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55Gag) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55Gag although HIV-1 Vif proteins bind Pr55Gag less efficiently than those of BIV Vif. Our research not only sheds new light on this feature of these conserved lentiviral Vif proteins but also provides a formerly unrecognized target for the development of antiviral strategies. Since increasing the Vif-Pr55Gag interaction could potentially suppress virus proliferation, this approach could offer a new strategy for the development of HIV inhibitors.

Disruption of MDA5-Mediated Innate Immune Responses by the 3C Proteins of Coxsackievirus A16, Coxsackievirus A6, and Enterovirus D68.

Coxsackievirus A16 (CV-A16), CV-A6, and enterovirus D68 (EV-D68) belong to the Picornaviridae family and are major causes of hand, foot, and mouth disease (HFMD) and pediatric respiratory disease worldwide. The biological characteristics of these viruses, especially their interplay with the host innate immune system, have not been well investigated. In this study, we discovered that the 3Cpro proteins from CV-A16, CV-A6, and EV-D68 bind melanoma differentiation-associated gene 5 (MDA5) and inhibit its interaction with MAVS. Consequently, MDA5-triggered type I interferon (IFN) signaling in the retinoic acid-inducible gene I-like receptor (RLR) pathway was blocked by the CV-A16, CV-A6, and EV-D68 3Cpro proteins. Furthermore, the CV-A16, CV-A6, and EV-D68 3Cpro proteins all cleave transforming growth factor ß-activated kinase 1 (TAK1), resulting in the inhibition of NF-κB activation, a host response also critical for Toll-like receptor (TLR)-mediated signaling. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed novel mechanisms to subvert host innate immune responses by targeting key factors in the RLR and TLR pathways. Blocking the ability of 3Cpro proteins from diverse enteroviruses and coxsackieviruses to interfere with type I IFN induction should restore IFN antiviral function, offering a potential novel antiviral strategy.IMPORTANCE CV-A16, CV-A6, and EV-D68 are emerging pathogens associated with hand, foot, and mouth disease and pediatric respiratory disease worldwide. The pathogenic mechanisms of these viruses are largely unknown. Here we demonstrate that the CV-A16, CV-A6, and EV-D68 3Cpro proteins block MDA5-triggered type I IFN induction. The 3Cpro proteins of these viruses bind MDA5 and inhibit its interaction with MAVS. In addition, the CV-A16, CV-A6, and EV-D68 3Cpro proteins cleave TAK1 to inhibit the NF-κB response. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed a mechanism to subvert host innate immune responses by simultaneously targeting key factors in the RLR and TLR pathways. These findings indicate the potential merit of targeting the CV-A16, CV-A6, and EV-D68 3Cpro proteins as an antiviral strategy.

Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly.

The lentiviral accessory proteins Vpx and Vpr are known to utilize CRL4 (DCAF1) E3 ligase to induce the degradation of the host restriction factor SAMHD1 or host helicase transcription factor (HLTF), respectively. Selective disruption of viral CRL4 (DCAF1) E3 ligase could be a promising antiviral strategy. Recently, we have determined that posttranslational modification (neddylation) of Cullin-4 is required for the activation of Vpx-CRL4 (DCAF1) E3 ligase. However, the mechanism of Vpx/Vpr-CRL4 (DCAF1) E3 ligase assembly is still poorly understood. Here, we report that zinc coordination is an important regulator of Vpx-CRL4 E3 ligase assembly. Residues in a conserved zinc-binding motif of Vpx were essential for the recruitment of the CRL4 (DCAF1) E3 complex and Vpx-induced SAMHD1 degradation. Importantly, altering the intracellular zinc concentration by treatment with the zinc chelator N,N,N'-tetrakis-(2'-pyridylmethyl)ethylenediamine (TPEN) potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac (simian immunodeficiency virus of macaques) infection of myeloid cells, even in the presence of Vpx. TPEN selectively inhibited Vpx and DCAF1 binding but not the Vpx-SAMHD1 interaction or Vpx virion packaging. Moreover, we have shown that zinc coordination is also important for the assembly of the HIV-1 Vpr-CRL4 E3 ligase. In particular, Vpr zinc-binding motif mutation or TPEN treatment efficiently inhibited Vpr-CRL4 (DCAF1) E3 ligase assembly and Vpr-mediated HLTF degradation or Vpr-induced G2 cell cycle arrest. Collectively, our study sheds light on a conserved strategy by the viral proteins Vpx and Vpr to recruit host CRL4 (DCAF1) E3 ligase, which represents a target for novel anti-human immunodeficiency virus (HIV) drug development.IMPORTANCE The Vpr and its paralog Vpx are accessory proteins encoded by different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) lentiviruses. To facilitate viral replication, Vpx has evolved to induce SAMHD1 degradation and Vpr to mediate HLTF degradation. Both Vpx and Vpr perform their functions by recruiting CRL4 (DCAF1) E3 ligase. In this study, we demonstrate that the assembly of the Vpx- or Vpr-CRL4 E3 ligase requires a highly conserved zinc-binding motif. This motif is specifically required for the DCAF1 interaction but not for the interaction of Vpx or Vpr with its substrate. Selective disruption of Vpx- or Vpr-CRL4 E3 ligase function was achieved by zinc sequestration using N,N,N'-tetrakis-(2'-pyridylmethyl)ethylenediamine (TPEN). At the same time, zinc sequestration had no effect on zinc-dependent cellular protein functions. Therefore, information obtained from this study may be important for novel anti-HIV drug development.