Mechanism of resistance of hepatitis C virus replicons to structurally distinct cyclophilin inhibitors.

The current standard of care for hepatitis C virus (HCV) infection, pegylated alpha interferon in combination with ribavirin, has a limited response rate and adverse side effects. Drugs targeting viral proteins are in clinical development, but they suffer from the development of high viral resistance. The inhibition of cellular proteins that are essential for viral amplification is thought to have a higher barrier to the emergence of resistance. Three cyclophilin inhibitors, the cyclosporine analogs DEBIO-025, SCY635, and NIM811, have shown promising results for the treatment of HCV infection in early clinical trials. In this study, we investigated the frequency and mechanism of resistance to cyclosporine (CsA), NIM811, and a structurally unrelated cyclophilin inhibitor, SFA-1, in replicon-containing Huh7 cells. Cross-resistance between all clones was observed. NIM811-resistant clones were selected only after obtaining initial resistance to either CsA or SFA-1. The time required to select resistance against cyclophilin inhibitors was significantly longer than that required for resistance selection against viral protein inhibitors, and the achievable resistance level was substantially lower. Resistance to cyclophilin inhibitors was mediated by amino acid substitutions in NS3, NS5A, and NS5B, with NS5A mutations conferring the majority of resistance. Mutation D320E in NS5A mediated most of the resistance conferred by NS5A. Taken together, the results indicate that there is a very low frequency and level of resistance to cyclophilin-binding drugs mediated by amino acid substitutions in three viral proteins. The interaction of cyclophilin with NS5A seems to be the most critical, since the NS5A mutations have the largest impact on resistance.

Is there a role for SV40 in human cancer?

The question of whether Simian Virus 40 (SV40) can cause human tumors has been one of the most highly controversial topics in cancer research during the last 50 years. The longstanding debate began with the discovery of SV40 as a contaminant in poliovirus vaccine stocks that were used to inoculate approximately 100 million children and adults in the United States between 1955 and 1963, and countless more throughout the world. Concerns regarding the potential health risk of SV40 exposure were reinforced by studies demonstrating SV40's potential to transform human cells and promote tumor growth in animal models. Many studies have attempted to assess the relationship between the potential exposure of humans to SV40 and cancer incidence. Reports of the detection of SV40 DNA in a variety of cancers have raised serious concerns as to whether the inadvertent inoculation with SV40 has led to the development of cancer in humans. However, inconsistent reports linking SV40 with various tumor types has led to conflicting views regarding the potential of SV40 as a human cancer virus. Several recent studies suggest that older detection methodologies were flawed, and the limitations of these methods could account for most, if not all, of the positive correlations of SV40 in human tumors to date. Although many people may have been exposed to SV40 by polio vaccination, there is inadequate evidence to support widespread SV40 infection in the population, increased tumor incidence in those individuals who received contaminated vaccine, or a direct role for SV40 in human cancer.

The carboxyl-terminal domain of large T antigen rescues SV40 host range activity in trans independent of acetylation.

The host range activity of SV40 has been described as the inability of mutant viruses with deletions in the C terminal region of large T Ag to replicate in certain types of African green monkey kidney cells. We constructed new mutant viruses expressing truncated T Ag proteins and found that these mutant viruses exhibited the host range phenotype. The host range phenotype was independent of acetylation of T Ag at lysine 697. Co-expression of the C terminal domain of T Ag (aa 627-708) in trans increased both T Ag and VP1 mRNA as well as protein levels for host range mutant viruses in the restrictive cell type. In addition, the T Ag 627-708 fragment promoted the productive lytic infection of host range mutant viruses in the nonpermissive cell type. The carboxyl-terminal region of T Ag contains a biological function essential for the SV40 viral life cycle.

p53 targets simian virus 40 large T antigen for acetylation by CBP.

Simian virus 40 (SV40) large T antigen (T Ag) interacts with the tumor suppressor p53 and the transcriptional coactivators CBP and p300. Binding of these cellular proteins in a ternary complex has been implicated in T Ag-mediated transformation. It has been suggested that the ability of CBP/p300 to modulate p53 function underlies p53's regulation of cell proliferation and tumorigenesis. In this study, we provide further evidence that CBP activity may be mediated through its synergistic action with p53. We demonstrate that SV40 T Ag is acetylated in vivo in a p53-dependent manner and T Ag acetylation is largely mediated by CBP. The acetylation of T Ag is dependent on its interaction with p53 and on p53's interaction with CBP. We have mapped the site of acetylation on T Ag to the C-terminal lysine residue 697. This acetylation site is conserved between the T antigens of the human polyomaviruses JC and BK, which are also known to interact with p53. We show that both JC and BK T antigens are also acetylated at corresponding sites in vivo. While other proteins are known to be acetylated by CBP/p300, none are known to depend on p53 for acetylation. T Ag acetylation may provide a regulatory mechanism for T Ag binding to a cellular factor or play a role in another aspect of T Ag function.

Favorable interactions between enfuvirtide and 1-beta-D-2,6-diaminopurine dioxolane in vitro.

We evaluated the in vitro anti-human immunodeficiency virus type 1 (HIV-1) interactions between 1-beta-D-2,6-diaminopurine dioxolane (DAPD) and enfuvirtide (T-20) against clinical isolates sensitive and resistant to reverse transcriptase and protease inhibitors. Interactions between T-20 and DAPD were synergistic to nearly additive, with combination index values ranging from 0.53 to 1.06 at 95% inhibitory concentrations. These studies suggest that a combination of T-20 and DAPD might be useful in the treatment of antiretroviral drug-experienced patients.