Discovery and Synthesis of Heterobifunctional Degraders of Rearranged during Transfection (RET) Kinase.

We describe the design, synthesis, and structure-activity relationship (SAR) of heterobifunctional RET ligand-directed degraders (LDDs) derived from three different second-generation RET inhibitors. These LDDs are composed of a target binding motif (TBM) that binds to the RET protein, a linker, and a cereblon binding motif (CBM) as the E3 ligase recognition unit. This led to the identification of a series of pyrazolopyridine-based heterobifunctional LDDs, as exemplified by compound 39. LDD 39 demonstrated high in vitro inhibitory and degradation potency against both RET wild-type and the two representative mutants, V804M and G810R. Importantly, in PK/PD studies, 39 exhibited a differentiated and favorable in vivo profile compared to the corresponding tyrosine kinase inhibitor (TKI), compound 3. Robust and sustained degradation of total-RET (tRET) protein and inhibition of phospho-RET (pRET) signaling were observed in TPC-1 xenograft tumors driven by RET and the RET/G810R mutant following a single dose of LDD 39 at 15 and 75 mg/kg, respectively.

Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration.

Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a hostile tumor microenvironment that renders T cells dysfunctional. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment. Using a transplant mouse model with spontaneously formed liver metastasis, a genetically engineered KPC mouse model that spontaneously develops PDAC, and a human patient-derived xenograft model, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses. This study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either intratumoral or systemic treatment of STING agonist was associated with increased expression of CTLA-4 on tumor-infiltrating T cells. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. The mouse pancreatic and liver orthotopic model of human patient-derived xenograft reconstituted with PBMC also showed that antitumor and abscopal effects of both intratumoral and intramuscular STING agonist are equivalent. Taken together, this study supports the clinical development of innate agonists via systemic administration for treating PDAC.

DGKα/ζ inhibition lowers the TCR affinity threshold and potentiates antitumor immunity.

Diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG) signaling by converting DAG to phosphatidic acid, thereby suppressing pathways downstream of T cell receptor signaling. Using a dual DGKα/ζ inhibitor (DGKi), tumor-specific CD8 T cells with different affinities (TRP1high and TRP1low), and altered peptide ligands, we demonstrate that inhibition of DGKα/ζ can lower the signaling threshold for T cell priming. TRP1high and TRP1low CD8 T cells produced more effector cytokines in the presence of cognate antigen and DGKi. Effector TRP1high- and TRP1low-mediated cytolysis of tumor cells with low antigen load required antigen recognition, was mediated by interferon-γ, and augmented by DGKi. Adoptive T cell transfer into mice bearing pancreatic or melanoma tumors synergized with single-agent DGKi or DGKi and antiprogrammed cell death protein 1 (PD-1), with increased expansion of low-affinity T cells and increased cytokine production observed in tumors of treated mice. Collectively, our findings highlight DGKα/ζ as therapeutic targets for augmenting tumor-specific CD8 T cell function.

DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

Discovery of Potent, Dual-Inhibitors of Diacylglycerol Kinases Alpha and Zeta Guided by Phenotypic Optimization.

We describe a phenotypic screening and optimization strategy to discover compounds that block intracellular checkpoint signaling in T-cells. We identified dual DGKα and ζ inhibitors notwithstanding the modest similarity between α and ζ relative to other DGK isoforms. Optimized compounds produced cytokine release and T-cell proliferation consistent with DGK inhibition and potentiated an immune response in human and mouse T-cells. Additionally, lead inhibitor BMS-502 demonstrated dose-dependent immune stimulation in the mouse OT-1 model, setting the stage for a drug discovery program.

A multi-omic single cell sequencing approach to develop a CD8 T cell specific gene signature for anti-PD1 response in solid tumors.

Immune checkpoint blockade (ICB) has led to durable clinical responses in multiple cancer types. However, biomarkers that identify which patients are most likely to respond to ICB are not well defined. Many putative biomarkers developed from a small number of samples often fail to maintain their predictive status in larger validation cohorts. We show across multiple human malignancies and syngeneic murine tumor models that neither pretreatment T cell receptor (TCR) clonality nor changes in clonality after ICB correlate with response. Dissection of tumor infiltrating lymphocytes pre- and post-ICB by paired single-cell RNA sequencing and single-cell TCR sequencing reveals conserved and distinct transcriptomic features in expanded TCR clonotypes between anti-PD1 responder and nonresponder murine tumor models. Overall, our results indicate a productive anti-tumor response is agnostic of TCR clonal expansion. Further, we used single-cell transcriptomics to develop a CD8+ T cell specific gene signature for a productive anti-tumor response and show the response signature to be associated with overall survival (OS) on nivolumab monotherapy in CheckMate-067, a phase 3 clinical trial in metastatic melanoma. These results highlight the value of leveraging single-cell assays to dissect heterogeneous tumor and immune subsets and define cell-type specific transcriptomic biomarkers of ICB response.

DGKζ exerts greater control than DGKα over CD8+ T cell activity and tumor inhibition.

Two isoforms of diacylglycerol kinases (DGKs), DGKα and DGKζ, are primarily responsible for terminating DAG-mediated activation of Ras and PKCθ pathways in T cells. A direct comparison of tumor growth between mice lacking each isoform has not been undertaken. We evaluated the growth of three syngeneic tumor cell lines in mice lacking either DGKα or DGKζ in the presence or absence of treatment with anti-PD1 and determined that (i) mice deficient in DGKζ conferred enhanced control of tumor relative to mice deficient in DGKα and (ii) deficiency of DGKζ acted additively with anti-PD1 in tumor control. Consistent with this finding, functional and RNA-sequencing analyses revealed greater changes in stimulated DGKζ-deficient T cells compared with DGKα-deficient T cells, which were enhanced relative to wildtype T cells. DGKζ also imparted greater regulation than DGKα in human T cells. Together, these data support targeting the ζ isoform of DGKs to therapeutically enhance T cell anti-tumor activity.

Prospective prediction of plasma pharmacokinetics of a novel immune-modulating agent in cancer patients after intra-tumoral administration: translation from non-clinical species to humans.

Intra-tumoral (I-TUMOUR) delivery is being widely explored for novel anti-cancer agents. This route is anticipated to result in high tumour concentrations leading to better efficacy and safety. Prediction of human systemic pharmacokinetics (PK) from non-clinical species facilitates understanding of pharmacokinetic-pharmacodynamic relationships, efficient dose selection, and risk assessment of novel drugs. However, there is limited knowledge on the predictability of human pharmacokinetics following I-TUMOUR delivery.In this publication, we present a case study wherein human systemic PK of a novel agent administered intra-tumourally was prospectively predicted and compared with observed human PK.Simple allometry was used to project the human clearance (10.5 mL/min/kg) and steady-state volume of distribution (1.4 L/kg) after intravenous (IV) dosing. Using these IV PK parameters and assuming rapid absorption and complete I-TUMOUR bioavailability, human plasma PK profile was simulated. The projected 30 min concentrations and AUC(0-6h) were within 1.9 to 2.5-fold and 1 to 1.4-fold of the observed PK indicating a reasonable concordance between predicted and observed PK.To our knowledge, this is the first article that prospectively projected human pharmacokinetics after I-TUMOUR dosing. The results from this study indicate that similar approaches can be used to project the human PK of other I-TUMOUR agents.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

Interrogating the immune-modulating roles of radiation therapy for a rational combination with immune-checkpoint inhibitors in treating pancreatic cancer.

BACKGROUND: Radiation therapy (RT) has the potential to enhance the efficacy of immunotherapy, such as checkpoint inhibitors, which has dramatically altered the landscape of treatments for many cancers, but not yet for pancreatic ductal adenocarcinoma (PDAC). Our prior studies demonstrated that PD ligand-1 and indoleamine 2,3-dioxygenase 1 (IDO1) were induced on tumor epithelia of PDACs following neoadjuvant therapy including RT, suggesting RT may prime PDAC for PD-1 blockade antibody (αPD-1) or IDO1 inhibitor (IDO1i) treatments. In this study, we investigated the antitumor efficacy of the combination therapies with radiation and PD-1 blockade or IDO1 inhibition or both. METHODS: We developed and used a mouse syngeneic orthotopic model of PDAC suitable for hypofractionated RT experiments. RESULTS: The combination therapy of αPD-1 and RT improved survival. The dual combination of RT/IDO1i and triple combination of RT/αPD-1/IDO1i did not improve survival compared with RT/αPD-1, although all of these combinations offer similar local tumor control. RT/αPD-1 appeared to result in the best systemic interferon-γ response compared with other treatment groups and the highest local expression of immune-activation genes, including Cd28 and Icos. CONCLUSION: Our RT model allows examining the immune-modulatory effects of RT alone and in combination with immune-checkpoint inhibitors in the pancreas/local microenvironment. This study highlights the importance of choosing the appropriate immune-modulatory agents to be combined with RT to tip the balance toward antitumor adaptive immune responses.

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti-PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti-PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti-PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. SIGNIFICANCE: These findings show that pan-inhibition of TAM receptors in combination with anti-PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

Safety and efficacy of anti-PD-L1 therapy in the woodchuck model of HBV infection.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.

High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

A novel small molecule inhibitor of hepatitis C virus entry.

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

Ultrasensitive genotypic detection of antiviral resistance in hepatitis B virus clinical isolates.

Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other l-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy.

Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naïve patients is rare through 5 years of therapy.

UNLABELLED: Patients with chronic hepatitis B virus (HBV) infection who develop antiviral resistance lose benefits of therapy and may be predisposed to further resistance. Entecavir (ETV) resistance (ETVr) results from HBV reverse transcriptase substitutions at positions T184, S202, or M250, which emerge in the presence of lamivudine (LVD) resistance substitutions M204I/V +/- L180M. Here, we summarize results from comprehensive resistance monitoring of patients with HBV who were continuously treated with ETV for up to 5 years. Monitoring included genotypic analysis of isolates from all patients at baseline and when HBV DNA was detectable by polymerase chain reaction (> or = 300 copies/mL) from Years 1 through 5. In addition, genotyping was performed on isolates from patients experiencing virologic breakthrough (> or = 1 log(10) rise in HBV DNA). In vitro phenotypic ETV susceptibility was determined for virologic breakthrough isolates, and for HBV containing novel substitutions emerging during treatment. The results over 5 years of therapy showed that in nucleoside-naïve patients, the cumulative probability of genotypic ETVr and genotypic ETVr associated with virologic breakthrough was 1.2% and 0.8%, respectively. In contrast, a reduced barrier to resistance was observed in LVD-refractory patients, as the LVD resistance substitutions, a partial requirement for ETVr, preexist, resulting in a 5-year cumulative probability of genotypic ETVr and genotypic ETVr associated with breakthrough of 51% and 43%, respectively. Importantly, only four patients who achieved < 300 copies/mL HBV DNA subsequently developed ETVr. CONCLUSION: Long-term monitoring showed low rates of resistance in nucleoside-naïve patients during 5 years of ETV therapy, corresponding with potent viral suppression and a high genetic barrier to resistance. These findings support ETV as a primary therapy that enables prolonged treatment with potent viral suppression and minimal resistance.

Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies.

APOBEC3G is an antiviral host factor capable of inhibiting the replication of both exogenous and endogenous retroviruses as well as hepatitis B, a DNA virus that replicates through an RNA intermediate. To gain insight into the mechanism whereby APOBEC3G restricts retroviral replication, we investigated the subcellular localization of the protein. Herein, we report that APOBEC3G localizes to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of nontranslating mRNAs. Biochemical analysis revealed that APOBEC3G localizes to a ribonucleoprotein complex with other P-body proteins which have established roles in cap-dependent translation (eIF4E and eIF4E-T), translation suppression (RCK/p54), RNA interference-mediated post-transcriptional gene silencing (AGO2), and decapping of mRNA (DCP2). Similar analysis with other APOBEC3 family members revealed a potential link between the localization of APOBEC3G and APOBEC3F to a common ribonucleoprotein complex and P-bodies with potent anti-HIV-1 activity. In addition, we present evidence suggesting that an important role for HIV-1 Vif, which subverts both APOBEC3G and APOBEC3F antiviral function by inducing their degradation, could be to selectively remove these proteins from and/or restrict their localization to P-bodies. Taken together, the results of this study reveal a novel link between innate immunity against retroviruses and P-bodies suggesting that APOBEC3G and APOBEC3F could function in the context of P-bodies to restrict HIV-1 replication.

Analysis of HIV-1 viral infectivity factor-mediated proteasome-dependent depletion of APOBEC3G: correlating function and subcellular localization.

To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and nonfunctional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with gamma-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23. When co-expressed, Vif exhibited more pronounced localization to the cytoplasm and reduced the total cellular levels of APOBEC3G but rarely co-localized with APOBEC3G at cytoplasmic bodies. On the contrary, Vif(C114S), which is inactive but continues to interact with APOBEC3G, stably associated with APOBEC3G in the cytoplasm, resulting in complete co-localization at cytoplasmic bodies and a dose-dependent exclusion of Vif(C114S) from the nucleus. Following proteasome inhibition, cytoplasmic APOBEC3G levels increased, and both proteins co-accumulated nonspecifically into a vimentin-encaged aggresome. Furthermore in the presence or absence of APOBEC3G, Vif localization was significantly altered by proteasome inhibition, suggesting that aberrant localization may also contribute to the loss of Vif function. Finally mutations at Vif Ile(9) disrupted the ability of Vif or Vif(C114S) to coimmunoprecipitate and to co-localize with APOBEC3G, suggesting that the N terminus of Vif mediates interactions with APOBEC3G. Taken together, these results demonstrate that cytoplasmic Vif-APOBEC3G interactions are required but are not sufficient for Vif to modulate APOBEC3G and can be monitored by co-localization in vivo.

Biosynthesis of glycosylphosphatidylinositol is essential to the survival of the protozoan parasite Toxoplasma gondii.

The PIGA gene from Toxoplasma gondii has been cloned and characterized. Like mammalian PIGA, the transmembrane and C-terminal domains are sufficient to direct localization to the parasite endoplasmic reticulum. A functional copy of PIGA is required for tachyzoite viability, demonstrating that glycosylphosphatidylinositol biosynthesis is an essential process in T. gondii.

Clostridium septicum alpha-toxin is active against the parasitic protozoan Toxoplasma gondii and targets members of the SAG family of glycosylphosphatidylinositol-anchored surface proteins.

As is the case with many other protozoan parasites, glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of Toxoplasma gondii tachyzoites. The mechanisms by which T. gondii GPI-anchored proteins are synthesized and transported through the unusual triple-membrane structure of the parasite pellicle to the plasma membrane remain largely unknown. As a first step in developing tools to study these processes, we show here that Clostridium septicum alpha-toxin, a pore-forming toxin that targets GPI-anchored protein receptors on the surface of mammalian cells, is active against T. gondii tachyzoites (50% effective concentration, 0.2 nM). Ultrastructural studies reveal that a tight physical connection between the plasma membrane and the underlying membranes of the inner membrane complex is locally disrupted by toxin treatment, resulting in a massive outward extension of the plasma membrane and ultimately lysis of the parasite. Toxin treatment also causes swelling of the parasite endoplasmic reticulum, providing the first direct evidence that alpha-toxin is a vacuolating toxin. Alpha-toxin binds to several parasite GPI-anchored proteins, including surface antigen 3 (SAG3) and SAG1. Interestingly, differences in the toxin-binding profiles between the virulent RH and avirulent P strain were observed. Alpha-toxin may prove to be a powerful experimental tool for molecular genetic analysis of GPI anchor biosynthesis and GPI-anchored protein trafficking in T. gondii and other susceptible protozoa.