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Steinitz M.,Lautenberg Center for General and Tumor Immunology | Steinitz M.,Hebrew University of Jerusalem
Methods in Molecular Biology | Year: 2014

Epstein-Barr virus (EBV) is a herpes virus which in vitro efficiently immortalizes nearly all human B lymphocytes. The lymphoblastoid diploid cell lines (LCL's) thus generated preserve the characteristics of the cells initially infected by the virus: the cells produce and secrete immunoglobulins and also express these molecules on their surface. A selection of specific antibody-producing cells (i.e., antigen-committed cells) before EBV-infection or when LCL's have already been established, enables isolation of monoclonal cell lines that secrete specific antibodies. If selection of antigen-committed cells is not feasible, secretion of specific antibodies by cloned LCL's in limiting dilution cultures enables isolation of the desired cell lines. The method allows the production of human IgM, IgG, IgA, and IgE monoclonal antibodies from any individual. Monoclonal antibodies produced by the EBV method resemble the antibody repertoire of the donor of the lymphocytes. Human monoclonal antibodies are promising reagents for passive immunization. © 2014 Springer Science+Business Media, LLC.

Nowarski R.,Lautenberg Center for General and Tumor Immunology | Nowarski R.,Yale University | Prabhu P.,Lautenberg Center for General and Tumor Immunology | Kenig E.,Lautenberg Center for General and Tumor Immunology | And 3 more authors.
Journal of Molecular Biology | Year: 2014

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′ + 5′ ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. © 2014 Elsevier Ltd.

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