Deamination of cytidine residues in single-stranded DNA (ssDNA) is an important mechanism by which apolipoprotein B mRNA-editing, catalytic polypeptide-like (APOBEC) enzymes restrict endogenous and exogenous viruses. The dynamic process underlying APOBEC-induced hypermutation is not fully understood. Here we show that enzymatically active APOBEC3G can be detected in wild-type Vif(+) HIV-1 virions, albeit at low levels. In vitro studies showed that single enzyme-DNA encounters result in distributive deamination of adjacent cytidines. Nonlinear translocation of APOBEC3G, however, directed scattered deamination of numerous targets along the DNA. Increased ssDNA concentrations abolished enzyme processivity in the case of short, but not long, DNA substrates, emphasizing the key role of rapid intersegmental transfer in targeting the deaminase. Our data support a model by which APOBEC3G intersegmental transfer via monomeric binding to two ssDNA segments results in dispersed hypermutation of viral genomes.

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. Newcastle disease virus (NDV) is an avian paramyxovirus that has a potential selective oncolytic effect on human tumors (5,7,13,21,25, 26). NDV's natural host is avian, and while mammalian cells bear the sialic acid receptor for NDV and may be infected by the virus, the virus has limited replication capacity in normal mammalian cells (21). We recently reported the development of an attenuated (lentogenic) isolate of NDV (HUJ) that undergoes only one cycle replication in infected mammalian cells (7,25). NDV-HUJ is a single clone derived from the parental strain NDV Hitchner B1, which contains a mixed viral population. The new virus clone is attenuated due to multiple passages in specific-pathogen-free (SPF) eggs, and its intracerebral pathogenicity index (ICPI) value is low (an ICPI of 0.01 versus an ICPI of 0.93 for the parental NDV Hitchner B1). Sequence analysis of NDV-HUJ indicated 156 changes at the nucleotide sequence level and multiple amino acid changes from the parental B1 virus in all six viral genes (see Fig. S1 in the supplemental material). Although NDV-HUJ is an attenuated virus in chicken, it retains a selective cytotoxic potential for cancer cells, as determined in vitro and in vivo, using murine and human lung carcinomas (25). The oncolytic effect of the virus is apoptosis dependent (25).NDV-HUJ has been applied to treat glioblastoma patients in a phase I/II clinical trials and found to be safe and potentially active (7).The inhibitors of apoptosis proteins (IAPs) are receiving increased attention as key players in the initiation of tumors, their progression, and resistance to chemotherapy treatment (17). To date, eight human IAPs have been identified, including Livin. IAPs are characterized by one or more repeats of a highly conserved 70-amino-acid domain termed the baculovirus IAP repeat (BIR) that can bind and inhibit caspases, some IAPs also contain a conserved sequence termed the RING finger. RING finger proteins might function as E3 ubiquitin ligases; however, the exact nature of the E3 ligase activity of IAPs is still largely unclear.IAPs inhibit apoptosis induced by a variety of stimuli, mainly through their ability to bind and inhibit specific caspases (17). Intense study has shown that the role of IAP in apoptosis regulation is highly diverse, with a prominent role in tumorigenesis and resistance to therapy. Among the human IAPs, XIAP is the best characterized and the m...

Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields’ locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)–T–based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag–specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients’ glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.

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Livin is a member of the Inhibitor of Apoptosis (IAP) protein family that inhibits apoptosis triggered by a variety of stimuli. We previously demonstrated that while Livin inhibits caspase activity, caspases can cleave Livin to produce a truncated protein, tLivin and that this newly formed tLivin paradoxically induces cell death. However to date, the mechanism of tLivin-induced cell death is not fully understood. In this study, we set out to characterize the form of cell death mediated by tLivin. Here we demonstrate that, unlike most death-promoting proteins, tLivin is a flexible inducer of cell death capable of promoting necrosis or apoptosis in different cell lines. The unusual flexibility of tLivin is displayed by its ability to activate an alternative form of cell death when apoptosis is inhibited. Thus, tLivin can promote more than one form of cell death in the same cell type. Interestingly, in cells where tLivin induces necrosis, deletion of the caspase binding BIR domain results in tLivin-induced apoptosis, suggesting the BIR domain can potentially hamper the ability of tLivin to induce apoptosis. We further elucidate that tLivin activates the JNK pathway and both tLivin-induced apoptosis and necrosis are partially mediated by JNK activity. Acquired resistance to apoptosis, common in many tumors, impinges on the efficiency of conventional anti-cancer agents that function primarily by inducing apoptosis. The ability of tLivin to induce death of apoptosis-compromised cells makes it an attractive candidate for targeted cancer therapy.