A glimpse into viral warfare: decoding the intriguing role of highly pathogenic coronavirus proteins in apoptosis regulation.

Coronaviruses employ various strategies for survival, among which the activation of endogenous or exogenous apoptosis stands out, with viral proteins playing a pivotal role. Notably, highly pathogenic coronaviruses such as SARS-CoV-2, SARS-CoV, and MERS-CoV exhibit a greater array of non-structural proteins compared to low-pathogenic strains, facilitating their ability to induce apoptosis via multiple pathways. Moreover, these viral proteins are adept at dampening host immune responses, thereby bolstering viral replication and persistence. This review delves into the intricate interplay between highly pathogenic coronaviruses and apoptosis, systematically elucidating the molecular mechanisms underpinning apoptosis induction by viral proteins. Furthermore, it explores the potential therapeutic avenues stemming from apoptosis inhibition as antiviral agents and the utilization of apoptosis-inducing viral proteins as therapeutic modalities. These insights not only shed light on viral pathogenesis but also offer novel perspectives for cancer therapy.

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.

TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion.

Innate immunity represents one of the main host responses to viral infection.1-3 STING (Stimulator of interferon genes), a crucial immune adapter functioning in host cells, mediates cGAS (Cyclic GMP-AMP Synthase) sensing of exogenous and endogenous DNA fragments and generates innate immune responses.4 Whether STING activation was involved in infection and replication of enterovirus remains largely unknown. In the present study, we discovered that human enterovirus A71 (EV-A71) infection triggered STING activation in a cGAS dependent manner. EV-A71 infection caused mitochondrial damage and the discharge of mitochondrial DNA into the cytosol of infected cells. However, during EV-A71 infection, cGAS-STING activation was attenuated. EV-A71 proteins were screened and the viral protease 2Apro had the greatest capacity to inhibit cGAS-STING activation. We identified TRAF3 as an important factor during STING activation and as a target of 2Apro. Supplement of TRAF3 rescued cGAS-STING activation suppression by 2Apro. TRAF3 supported STING activation mediated TBK1 phosphorylation. Moreover, we found that 2Apro protease activity was essential for inhibiting STING activation. Furthermore, EV-D68 and CV-A16 infection also triggered STING activation. The viral protease 2Apro from EV-D68 and CV-A16 also had the ability to inhibit STING activation. As STING activation prior to EV-A71 infection generated cellular resistance to EV-A71 replication, blocking EV-A71-mediated STING suppression represents a new anti-viral target.

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.

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, 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.

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.

Ectopic Expression of TRIM25 Restores RIG-I Expression and IFN Production Reduced by Multiple Enteroviruses 3Cpro.

Enteroviruses (EVs) 3C proteins suppress type I interferon (IFN) responses mediated by retinoid acid-inducible gene I (RIG-I), while an E3 ubiquitin ligase, tripartite motif protein 25 (TRIM25)-mediated RIG-I ubiquitination is essential for RIG-I antiviral activity. Therefore, whether the effect of EVs 3C on RIG-I is associated with TRIM25 expression is worth to be further investigated. Here, we demonstrate that 3C proteins of EV71 and coxsackievirus B3 (CVB3) reduced not only RIG-I expression but also TRIM25 expression through protease cleavage activity, while overexpression of TRIM25 restored RIG-I expression and IFN-ß production reduced by 3C proteins. Further investigation confirmed that the two amino acids and functional domains in TRIM25 required for RIG-I ubiquitination and TRIM25 structural conformation were essential for the recovery of RIG-I expression. Moreover, we also observed that TRIM25 could rescue RIG-I expression reduced by 3C proteins of CVA6 and EV-D68 but not CVA16. Our findings provide an insightful interpretation of 3C-mediated host innate immune suppression and support TRIM25 as an attractive target against multiple EVs infection.

Unique and complementary suppression of cGAS-STING and RNA sensing- triggered innate immune responses by SARS-CoV-2 proteins.

The emergence of SARS-CoV-2 has resulted in the COVID-19 pandemic, leading to millions of infections and hundreds of thousands of human deaths. The efficient replication and population spread of SARS-CoV-2 indicates an effective evasion of human innate immune responses, although the viral proteins responsible for this immune evasion are not clear. In this study, we identified SARS-CoV-2 structural proteins, accessory proteins, and the main viral protease as potent inhibitors of host innate immune responses of distinct pathways. In particular, the main viral protease was a potent inhibitor of both the RLR and cGAS-STING pathways. Viral accessory protein ORF3a had the unique ability to inhibit STING, but not the RLR response. On the other hand, structural protein N was a unique RLR inhibitor. ORF3a bound STING in a unique fashion and blocked the nuclear accumulation of p65 to inhibit nuclear factor-κB signaling. 3CL of SARS-CoV-2 inhibited K63-ubiquitin modification of STING to disrupt the assembly of the STING functional complex and downstream signaling. Diverse vertebrate STINGs, including those from humans, mice, and chickens, could be inhibited by ORF3a and 3CL of SARS-CoV-2. The existence of more effective innate immune suppressors in pathogenic coronaviruses may allow them to replicate more efficiently in vivo. Since evasion of host innate immune responses is essential for the survival of all viruses, our study provides insights into the design of therapeutic agents against SARS-CoV-2.

Apolipoprotein H drives hepatitis B surface antigen retention and endoplasmic reticulum stress during hepatitis B virus infection.

BACKGROUND: Apolipoprotein H (APOH), also known as beta2-glycoprotein I (beta2-GPI), is an acute phase protein in hepatitis B virus (HBV) infection and binds to hepatitis B surface antigen (HBsAg) with high-affinity. APOH expression is upregulated by HBV and the large surface protein (LHBs), but also elevated in HBV-related hepatoma cells. Previous studies show that intracellular retention of HBsAg induces endoplasmic reticulum (ER) stress, a key driver of hepatocyte damage during chronic liver injury, but the mechanisms are unclear. We hypothesize that APOH mediates HBV-induced ER stress through increased retention of HBsAg. METHODS: VR-APOH-myc and VR-LHBs-flag plasmids were constructed by PCR using pcDNA3.1(-)-APOH or an HBV expression vector, respectively. APOH and ER stress markers were examined at protein and mRNA levels by Western Blot or RT-qPCR. HBsAg titer was assayed by ELISA. RNA-seq was performed to elucidate the transcriptional impact of APOH manipulation in HBV-producing cells (HepG2.2.15 cells). RESULTS: We found that HBV upregulates APOH expression in 293 T cells, and APOH overexpression subsequently inhibits secretion of HBsAg. Next, we show that LHBs overexpression in conjunction with APOH leads to ER stress in 293 T cells, as evidenced by production of the binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), as well as increased splicing of X-box binding protein 1 (XBP1). We further observed that loss of beta2-GPI reduced CHOP expression in HepG2.2.15 cells, while beta2-GPI overexpression enhanced CHOP production. CONCLUSION: The interaction of beta2-GPI and HBV initiates ER stress through driving intracellular retention of HBsAg and activates the UPR.

HIV-2/SIV Vpx targets a novel functional domain of STING to selectively inhibit cGAS-STING-mediated NF-κB signalling.

Innate immunity is the first line of host defence against pathogens. Suppression of innate immune responses is essential for the survival of all viruses. However, the interplay between innate immunity and HIV/SIV is only poorly characterized. We have discovered Vpx as a novel inhibitor of innate immune activation that associates with STING signalosomes and interferes with the nuclear translocation of NF-κB and the induction of innate immune genes. This new function of Vpx could be separated from its role in mediating degradation of the antiviral factor SAMHD1, and is conserved among diverse HIV-2/SIV Vpx. Vpx selectively suppressed cGAS-STING-mediated nuclear factor-κB signalling. Furthermore, Vpx and Vpr had complementary activities against cGAS-STING activity. Since SIVMAC lacking both Vpx and Vpr was less pathogenic than SIV deficient for Vpr or Vpx alone, suppression of innate immunity by HIV/SIV is probably a key pathogenic determinant, making it a promising target for intervention.

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.

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.

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.

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.

Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif.

HIV-1 Vif assembles the Cul5-EloB/C E3 ubiquitin ligase to induce proteasomal degradation of the cellular antiviral APOBEC3 proteins. Detailed structural studies have confirmed critical functional domains in Vif that we have previously identified as important for the interaction of EloB/C, Cul5, and CBFß. However, the mechanism by which Vif recognizes substrates remains poorly understood. Specific regions of Vif have been identified as being responsible for binding and depleting APOBEC3G and APOBEC3F. Interestingly, we have now identified distinct yet overlapping domains that are required for HIV-1 Vif-mediated G2/M-phase cell cycle arrest and APOBEC3H degradation, but not for the inactivation of APOBEC3G or APOBEC3F. Surprisingly, Vif molecules from primary HIV-1 variants that caused G2/M arrest were unable to inactivate APOBEC3H; on the other hand, HIV-1 Vif variants that could inactivate APOBEC3H were unable to induce G2/M arrest. All of these Vif variants still maintained the ability to inactivate APOBEC3G/F. Thus, primary HIV-1 variants have evolved to possess distinct functional activities that allow them to suppress APOBEC3H or cause G2 cell cycle arrest, using mutually exclusive interface domains. APOBEC3H depletion and G2 arrest are apparently evolutionary selected features that cannot co-exist on a single Vif molecule. The existence and persistence of both types of HIV-1 Vif variant suggests the importance of APOBEC3H suppression and cell cycle regulation for HIV-1's survival in vivo.

Dispersed and conserved hydrophobic residues of HIV-1 Vif are essential for CBFß recruitment and A3G suppression.

UNLABELLED: CBFß was recently found to be a key regulator of the ability of human immunodeficiency virus type 1 (HIV-1) Vif to overcome host antiviral APOBEC3 proteins. However, the detailed molecular requirements for the Vif-CBFß interaction are still not clear. Here, we mapped the minimum Vif domain required for CBFß binding. In terms of CBFß binding, the Vif N terminus was very sensitive to deletions. We determined that the Vif fragment from residues 5 to 126 was sufficient to form a stable complex with CBFß in vitro. We also observed that ionic interactions were not the main contributor to the interaction between Vif and CBFß. Instead, hydrophobic interactions were important for maintaining the Vif-CBFß complex, since it could be disrupted by nonionic detergent. Site-directed mutagenesis of conserved hydrophobic amino acids revealed novel residues in Vif that were important for CBFß binding and APOBEC3 inactivation. At least part of the well-characterized HCCH domain (residues 108 to 139) was required to form a stable Vif-CBFß complex. Thus, the HCCH motif may have a dual role in binding both Cul5 and CBFß. Considering the importance of Vif in HIV-1 infection, this unique Vif-CBFß interaction represents an attractive pharmacological intervention target against HIV-1. IMPORTANCE: Vif-induced APOBEC3 protein degradation was the first host antiviral mechanism against HIV-1/simian immunodeficiency virus to be revealed, yet details regarding which proteins are degraded are not fully demonstrated. Recently, host cellular factor CBFß was found to be essential for Vif to function and promote viral infectivity. In this study, we present more critical information on the Vif-CBFß interaction by revealing that hydrophobicity contributes the most to the Vif-CBFß interaction and locating several novel hydrophobic sites (tryptophans and phenylalanines) that are conserved among Vif proteins from different lentiviruses and essential for Vif binding to CBFß. Mutations on these sites result in a reduced/abolished Vif-CBFß interaction, leading to the attenuated potency of Vif on both inducing the degradation of antiviral factors like APOBEC3G and promoting HIV-1 infectivity. Therefore, information from this study will help people to further understand how Vif acts against host antiviral mechanism, which is important for novel anti-HIV-1 drug development.

Differential requirements for HIV-1 Vif-mediated APOBEC3G degradation and RUNX1-mediated transcription by core binding factor beta.

Core binding factor beta (CBFß), a transcription regulator through RUNX binding, was recently reported critical for Vif function. Here, we mapped the primary functional domain important for Vif function to amino acids 15 to 126 of CBFß. We also revealed that different lengths and regions are required for CBFß to assist Vif or RUNX. The important interaction domains that are uniquely required for Vif but not RUNX function represent novel targets for the development of HIV inhibitors.