A Replicating Cytomegalovirus-Based Vaccine Encoding a Single Ebola Virus Nucleoprotein CTL Epitope Confers Protection against Ebola Virus

Human outbreaks of hemorrhagic disease caused by Ebola virus (EBOV) are a serious health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans. Candidate EBOV vaccines do not spread from the initial vaccinee. In addition to being highly immunogenic, vaccines based on the cytomegalovirus (CMV) platform have the unique potential to re-infect and disseminate through target populations. To explore the utility of CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a region of nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NPCTL). MCMV/ZEBOV-NPCTL induced high levels of long-lasting CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. The absence of ZEBOV neutralizing and only low, sporadic levels of total anti-ZEBOV IgG antibodies in protected animals prior to ZEBOV challenge indicate a role, albeit perhaps not exclusive, for CD8+ T cells in mediating protection. This study demonstrates the ability of a CMV-based vaccine approach to protect against ZEBOV, and provides a ‘proof-of-concept’ for the potential for a ‘disseminating’ CMV-based EBOV vaccine to prevent EBOV transmission in wild animal populations

A Novel Role of the Interferon-inducible Protein IFI16 as Inducer of Proinflammatory Molecules in Endothelial Cells *

The human IFI16 gene is an interferon-inducible gene implicated in the regulation of endothelial cell proliferation and tube morphogenesis. Immunohistochemical analysis has demonstrated that this gene is highly expressed in endothelial cells in addition to hematopoietic tissues. In this study, gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 revealed an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. Consistent with these observations, IFI16 triggered expression of adhesion molecules such as ICAM-1 and E-selectin or chemokines such as interleukin-8 or MCP-1. Treatment of cells with short hairpin RNA targeting IFI16 significantly inhibited ICAM-1 induction by interferon (IFN)-gamma demonstrating that IFI16 is required for proinflammatory gene stimulation. Moreover, functional analysis of the ICAM-1 promoter by deletion- or site-specific mutation demonstrated that NF-kappaB is the main mediator of IFI16-driven gene induction. NF-kappaB activation appears to be triggered by IFI16 through a novel mechanism involving suppression of IkappaBalpha mRNA and protein expression. Support for this finding comes from the observation that IFI16 targeting with specific short hairpin RNA down-regulates NF-kappaB binding activity to its cognate DNA and inhibits ICAM-1 expression induced by IFN-gamma. Using transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrate indeed that activation of the NF-kappaB response is mediated by IFI16-induced block of Sp1-like factor recruitment to the promoter of the IkappaBalpha gene, encoding the main NF-kappaB inhibitor. Activation of NF-kappaB accompanied by induction of proinflammatory molecules was also observed when IkappaBalpha expression was down-regulated by specific small interfering RNA, resulting in an outcome similar to that observed with IFI16 overexpression. Taken together, these data implicate IFI16 as a novel regulator of endothelial proinflammatory activity and provide new insights into the physiological functions of the IFN-inducible gene IFI16

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies

A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγcnull-humanized mouse model. The UL133-UL138NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations

Virology under the microscope—a call for rational discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology

Phosphorothioate-Modified Oligodeoxynucleotides Inhibit Human Cytomegalovirus Replication by Blocking Virus Entry▿

Studies in animal models have provided evidence that Toll-like receptor 9 (TLR9) agonists, such as synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory deoxycytidyl-deoxyguanosine (CpG) motifs (CpG ODNs), protect against a wide range of viral pathogens. This antiviral activity has been suggested to be indirect and secondary to CpG-induced cytokines and inflammatory responses triggered through TLR9 activation. However, few studies have addressed the potential of CpG ODNs as direct antiviral agents. Here, we report on the ability of some CpG ODNs to directly suppress, almost completely, human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells. Murine CMV replication was inhibited as well, whereas no inhibition was observed for herpes simplex virus type 1, adenovirus, or vesicular stomatitis virus. The antiviral activity of these ODNs was significantly reduced when they were added after virus adsorption, indicating that their action may be primarily targeted to the very early phases of the HCMV cycle. In fact, the B-class prototype CpG ODN 2006 effectively prevented the nuclear localization of pp65 and input viral DNA, which suggests that it inhibits HCMV entry. Moreover, a CpG 2006 control, ODN 2137 without CpG motifs, also showed a potent inhibitory activity on the HCMV entry phase, indicating that the anticytomegaloviral activity is independent of the CpG motif. In contrast, a phosphodiester version of CpG 2006 showed reduced antiviral activity, indicating that the inhibitory activity is dependent on the phosphorothioate backbone of the ODN. These results suggest that this yet-unrecognized activity of CpG ODNs may be of interest in the development of novel anticytomegaloviral molecules