Andrew J. Rennekamp

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell–cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell–cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell–cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.

DC-SIGN and DC-SIGNR Interact with the Glycoprotein of Marburg Virus and the S Protein of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.

Folate Receptor Alpha and Caveolae Are Not Required for Ebola Virus Glycoprotein-Mediated Viral Infection

ABSTRACT Folate receptor alpha (FRα) has been described as a factor involved in mediating Ebola virus entry into cells (6). Furthermore, it was suggested that interaction with FRα results in internalization and subsequent viral ingress into the cytoplasm via caveolae (9). Descriptions of cellular receptors for Ebola virus and its entry mechanisms are of fundamental importance, particularly with the advent of vectors bearing Ebola virus glycoprotein (GP) being utilized for gene transfer into cell types such as airway epithelial cells. Thus, the ability of FRα to mediate efficient entry of viral pseudotypes carrying GP was investigated. We identified cell lines and primary cell types such as macrophages that were readily infected by GP pseudotypes despite lacking detectable surface FRα, indicating that this receptor is not essential for Ebola virus infection. Furthermore, we find that T-cell lines stably expressing FRα are not infectible, suggesting that FRα is also not sufficient to mediate entry. T-cell lines lack caveolae, the predominant route of FRα-mediated folate metabolism. However, the coexpression of FRα with caveolin-1, the major structural protein of caveolae, was not able to rescue infectivity in a T-cell line. In addition, other cell types lacking caveolae are fully infectible by GP pseudotypes. Finally, a panel of ligands to and soluble analogues of FRα were unable to inhibit infection on a range of cell lines, questioning the role of FRα as an important factor for Ebola virus entry.

15 years of zebrafish chemical screening

In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies.

Chromatin Profiling of Epstein-Barr Virus Latency Control Region

ABSTRACT Epstein-Barr virus (EBV) escapes host immunity by the reversible and epigenetic silencing of immunogenic viral genes. We previously presented evidence that a dynamic chromatin domain, which we have referred to as the latency control region (LCR), contributes to the reversible repression of EBNA2 and LMP1 gene transcription. We now explore the protein-DNA interaction profiles for a few known regulatory factors and histone modifications that regulate LCR structure and activity. A chromatin immunoprecipitation assay combined with real-time PCR analysis was used to analyze protein-DNA interactions at ∼500-bp intervals across the first 60,000 bp of the EBV genome. We compared the binding patterns of EBNA1 with those of the origin recognition complex protein ORC2, the chromatin boundary factor CTCF, the linker histone H1, and several histone modifications. We analyzed three EBV-positive cell lines (MutuI, Raji, and LCL3459) with distinct transcription patterns reflecting different latency types. Our findings suggest that histone modification patterns within the LCR are complex but reflect differences in each latency type. The most striking finding was the identification of CTCF sites immediately upstream of the Qp, Cp, and EBER transcription initiation regions in all three cell types. In transient assays, CTCF facilitated EBNA1-dependent transcription activation of Cp, suggesting that CTCF coordinates interactions between different chromatin domains. We also found that histone H3 methyl K4 clustered with CTCF and EBNA1 at sites of active transcription or DNA replication initiation. Our findings support a model where CTCF delineates multiple domains within the LCR and regulates interactions between these domains that correlate with changes in gene expression.

Highly Conserved Regions within the Spike Proteins of Human Coronaviruses 229E and NL63 Determine Recognition of Their Respective Cellular Receptors

ABSTRACT We have recently demonstrated that the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin converting enzyme 2 (ACE2) also mediates cellular entry of the newly discovered human coronavirus (hCoV) NL63. Here, we show that expression of DC-SIGN augments NL63 spike (S)-protein-driven infection of susceptible cells, while only expression of ACE2 but not DC-SIGN is sufficient for entry into nonpermissive cells, indicating that ACE2 fulfills the criteria of a bona fide hCoV-NL63 receptor. As for SARS-CoV, murine ACE2 is used less efficiently by NL63-S for entry than human ACE2. In contrast, several amino acid exchanges in human ACE2 which diminish SARS-S-driven entry do not interfere with NL63-S-mediated infection, suggesting that SARS-S and NL63-S might engage human ACE2 differentially. Moreover, we observed that NL63-S-driven entry was less dependent on a low-pH environment and activity of endosomal proteases compared to infection mediated by SARS-S, further suggesting differences in hCoV-NL63 and SARS-CoV cellular entry. NL63-S does not exhibit significant homology to SARS-S but is highly related to the S-protein of hCoV-229E, which enters target cells by engaging CD13. Employing mutagenic analyses, we found that the N-terminal unique domain in NL63-S, which is absent in 229E-S, does not confer binding to ACE2. In contrast, the highly homologous C-terminal parts of the NL63-S1 and 229E-S1 subunits in conjunction with distinct amino acids in the central regions of these proteins confer recognition of ACE2 and CD13, respectively. Therefore, despite the high homology of these sequences, they likely form sufficiently distinct surfaces, thus determining receptor specificity.

Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt

ABSTRACT The genetic elements of herpesvirus origins of lytic replication have been characterized in detail; however, much remains to be elucidated concerning their functional role in replication initiation. In the case of the Epstein-Barr virus (EBV), we have found that in addition to the two well-defined critical elements required for lytic replication (the upstream and downstream essential elements, UEE and DEE), the origin of lytic replication (OriLyt) also requires the presence of a GC-rich RNA in cis. The BHLF1 transcript is similar to the essential K5 transcript identified at the Kaposis sarcoma-associated herpesvirus OriLyt. We have found that truncation of the BHLF1 transcript or deletion of the TATA box, but not the putative ATG initiation codon, reduce OriLyt function to background levels. By using an antibody specific for RNA-DNA hybrid molecules, we found the BHLF1 RNA stably annealed to its DNA template during the early steps of lytic reactivation. Furthermore, expression of human RNase H1, which degrades RNA in RNA-DNA hybrids, drastically reduces OriLyt-dependent DNA replication as well as recruitment of the viral single-stranded DNA binding protein BALF2 to OriLyt. These studies suggest that a GC-rich OriLyt transcript is an important component of gammaherpesvirus lytic origins and is required for initial strand separation and loading of core replication proteins.

Epstein-Barr Virus Immediate-Early Protein Zta Co-Opts Mitochondrial Single-Stranded DNA Binding Protein To Promote Viral and Inhibit Mitochondrial DNA Replication

ABSTRACT Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. To better understand these functions and identify cellular collaborators of Zta, we purified an epitope-tagged version of Zta in cells capable of supporting lytic replication. FLAG-tagged Zta was purified from a nuclear fraction using FLAG antibody immunopurification and peptide elution. Zta-associated proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. The Zta-associated proteins included members of the HSP70 family and various single-stranded DNA and RNA binding proteins. The nuclear replication protein A subunits (RPA70 and RPA32) and the human mitochondrial single-stranded DNA binding protein (mtSSB) were confirmed by Western blotting to be specifically enriched in the FLAG-Zta immunopurified complex. mtSSB coimmunoprecipitated with endogenous Zta during reactivation of EBV-positive Burkitt lymphoma and lymphoblastoid cell lines. Small interfering RNA depletion of mtSSB reduced Zta-induced lytic replication of EBV but had only a modest effect on transcription activation function. A point mutation in the Zta DNA binding domain (C189S), which is known to reduce lytic cycle replication, eliminated mtSSB association with Zta. The predominantly mitochondrial localization of mtSSB was shifted to partly nuclear localization in cells expressing Zta. Mitochondrial DNA synthesis and genome copy number were reduced by Zta-induced EBV lytic replication. We conclude that Zta interaction with mtSSB serves the dual function of facilitating viral and blocking mitochondrial DNA replication.

Behavioral barcoding in the cloud: embracing data-intensive digital phenotyping in neuropharmacology

For decades, studying the behavioral effects of individual drugs and genetic mutations has been at the heart of efforts to understand and treat nervous system disorders. High-throughput technologies adapted from other disciplines (e.g., high-throughput chemical screening, genomics) are changing the scale of data acquisition in behavioral neuroscience. Massive behavioral datasets are beginning to emerge, particularly from zebrafish labs, where behavioral assays can be performed rapidly and reproducibly in 96-well, high-throughput format. Mining these datasets and making comparisons across different assays are major challenges for the field. Here, we review behavioral barcoding, a process by which complex behavioral assays are reduced to a string of numeric features, facilitating analysis and comparison within and across datasets.

Different host cell proteases activate the SARS-coronavirus spike-protein for cell–cell and virus–cell fusion

Abstract Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus–cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus–virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell–cell fusion was independent of cathepsin L, a protease essential for virus–cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus–cell and cell–cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.