Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.     

Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity

FEN1, a key participant in DNA replication and repair, is the major human flap endonuclease that recognizes and cleaves flap DNA structures. Deficiencies in FEN1 function or deletion of the fen1 gene have profound biological effects, including the suppression of repair of DNA damage incurred from the action of various genotoxic agents. Given the importance of FEN1 in resolving abnormal DNA structures, inhibitors of the enzyme carry a potential as enhancers of DNA-interactive anticancer drugs. To facilitate the studies of FEN1 activity and the search for novel inhibitors, we developed a pair of complementary-readout homogeneous assays utilizing fluorogenic donor/quencher and AlphaScreen chemiluminescence strategies. A previously reported FEN1 inhibitor 3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione displayed equal potency in the new assays, in agreement with its published IC₅₀. The assays were optimized to a low 4 µl volume and used to investigate a set of small molecules, leading to the identification of previously-unreported FEN1 inhibitors, among which aurintricarboxylic acid and NSC-13755 (an arylstibonic derivative) displayed submicromolar potency (average IC₅₀ of 0.59 and 0.93 µM, respectively). The availability of these simple complementary assays obviates the need for undesirable radiotracer-based assays and should facilitate efforts to develop novel inhibitors for this key biological target.     

The role of ITCH protein in human T-cell leukemia virus type 1 release

Human T-cell leukemia virus type 1 (HTLV-1) has two late domain (LD) motifs, PPPY and PTAP, which are important for viral budding. Mutations in the PPPY motif are more deleterious for viral release than changes in the PTAP motif. Several reports have shown that the interaction of PPPY with the WW domains of a Nedd4 (neuronal precursor cell-expressed developmentally down-regulated-4) family ubiquitin ligase (UL) is a critical event in virus release. We tested nine members of the Nedd4 family ULs and found that ITCH is the main contributor to HTLV-1 budding. ITCH overexpression strongly inhibited release and infectivity of wild-type (wt) HTLV-1, but rescued the release of infectious virions with certain mutations in the PPPY motif. Electron microscopy showed either fewer or misshapen virus particles when wt HTLV-1 was produced in the presence of overexpressed ITCH, whereas mutants with changes in the PPPY motif yielded normal looking particles at wt level. The other ULs had significantly weaker or no effects on HTLV-1 release and infectivity except for SMURF-1, which caused enhanced release of wt and all PPPY(-) mutant particles. These particles were poorly infectious and showed abnormal morphology by electron microscopy. Budding and infectivity defects due to overexpression of ITCH and SMURF-1 were correlated with higher than normal ubiquitination of Gag. Only silencing of ITCH, but not of WWP1, WWP2, and Nedd4, resulted in a reduction of HTLV-1 budding from 293T cells. The binding efficiencies between the HTLV-1 LD and WW domains of different ULs as measured by mammalian two-hybrid interaction did not correlate with the strength of their effect on HTLV-1 budding.     

Expression and purification of recombinant Kaposi's sarcoma-associated herpesvirus DNA polymerase using a Baculovirus vector system

The DNA polymerase (POL) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA-cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.     

Diverse Small Molecule Inhibitors of Human Apurinic/Apyrimidinic Endonuclease APE1 Identified from a Screen of a Large Public Collection

The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER) pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment.     

A Comprehensive Strategy to Discover Inhibitors of the Translesion Synthesis DNA Polymerase κ

Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N ²-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N ²-dG DNA–DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.     

Identification and Characterization of FAM124B as a Novel Component of a CHD7 and CHD8 Containing Complex

Background

Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry.

Principle findings

The hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues.

Conclusion

Our results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders.