Zinc Coordination Is Required for and Regulates Transcription Activation by Epstein-Barr Nuclear Antigen 1

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO2). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO2 and redox potential

The bovine papillomavirus origin of replication requires a binding site for the E2 transcriptional activator.

The bovine papillomavirus type I transcriptional activator E2 is essential for replication of bovine papillomavirus DNA, yet most of the high-affinity binding sites for E2 are dispensable. Here we demonstrate an absolute requirement for a binding site for the E2 polypeptide as a cis-acting replication element, establishing that site-specific binding of E2 to the origin is a prerequisite for bovine papillomavirus replication in vivo. The position and distance of the E2 binding site relative to the other origin of replication components are flexible, but function at a distance requires high-affinity E2 binding sites. Thus, low-affinity binding sites function only when located close to the origin of replication, while activity at greater distances requires multimerized high-affinity E2 binding sites. The requirement for E2, although different in some respects, shows distinct similarities to what has been termed replication enhancers and may provide insight into the function of this class of DNA replication element

Identification of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1.

Expression of the viral polypeptides E1 and E2 is necessary and sufficient for replication of BPV in mouse C127 cells. By providing these factors from heterologous expression vectors we have identified a minimal origin fragment from BPV that contains all the sequences required in cis for replication of BPV in short term replication assays. This same sequence is also required for stable replication in the context of the entire viral genome. The identified region is highly conserved between different papillomaviruses, and is unrelated to the previously identified plasmid maintenance sequences. The minimal ori sequence contains a binding site for the viral polypeptide E1, which we identify as a sequence specific DNA binding protein, but surprisingly, an intact binding site for the viral transactivator E2 at the ori is not required. The isolated origin shows an extended host region for replication and replicates efficiently in both rodent and primate cell lines

Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator.

Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. To address the mechanisms by which the viral DNA is stably propagated in the transformed cells, we have constructed a cell line CH04.15 expressing constitutively the viral proteins E1 and E2, that are required for initiation of viral DNA replication. We show that these viral proteins are necessary and sufficient for stable extrachromosomal replication. Using the cell line CH04.15, we have shown that the bovine papillomavirus-1 (BPV-1) minimal origin of replication (MO) is absolutely necessary, but is not sufficient for stable extrachromosomal replication of viral plasmids. By deletion and insertion analysis, we identified an additional element (minichromosome maintenance element, MME) in the upstream regulatory region of BPV-1 which assures stable replication of the MO-containing plasmids. This element is composed of multiple binding sites for the transcription activator E2. MME appears to function in the absence of replication but requires E1 and E2 proteins for activity. In contrast to, for example, Epstein-Barr virus oriP, stably maintained BPV-1 plasmids are not subject to once-per-cell cycle replication as determined by density labelling experiments. These results indicate that papillomavirus episomal replicators replicate independently of the chromosomal DNA of their hosts

Identification and Tracking of Antiviral Drug Combinations

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status.Peer reviewe