Ebola and Marburg Viruses: Molecular and Cellular Biology Chapter Abstracts
Chapter 1
Genome Organization, Replication, and Transcription of Filoviruses
Elke Mühlberger
Filoviruses possess a nonsegmented negative-strand RNA genome. Four proteins, NP, VP35, VP30, and L, are tightly associated with the genome forming the nucleocapsid complex. For Marburg as well as Ebola viruses, NP, VP35, and L are sufficient to mediate replication in a reconstituted replication and transcription system. However, the transcription strategy of both viruses seems to be different. Thus, for Marburg virus transcription, NP, VP35, and L are sufficient, whereas Ebola virus transcription is strongly dependent on the presence of the fourth nucleocapsid protein, VP30. It has been suggested that Ebola virus VP30 acts as an early antitermination protein, presumably by inhibiting pausing of the transcription complex at the transcription start signal of the first gene. VP30-dependent transcription activation is regulated by different mechanisms as protein phosphorylation and RNA secondary structure formation. A second difference in the transcription strategy of Marburg and Ebola virus is that Ebola virus performs mRNA editing to generate a full-length open reading frame of the GP gene. With Marburg virus, mRNA editing has not been observed.
Chapter 2
Structure of Viral Proteins
Winfried Weissenhorn
Filoviruses enter cells by employing their glycoprotein complex Gp1/2, which interacts with cellular receptor(s) and mediates endocytosis of viral particles. The endosomal low-pH environment then triggers fusion of viral and cellular membranes, a process that establishes an infection. The crystal structure of Ebola virus GP2 reveals a rod-like molecule, which is similar to a number of viral fusion protein structures, suggesting a common mechanism for fusion of viral and cellular membranes. Upon replication and transcription of viral structural proteins in the cytoplasm, including the matrix protein VP40 and the nucleoprotein NP, assembly takes place at cellular membranes and viral particles bud off the plasma membrane. NP specifically packs viral RNA and forms polymers with helical or ring-like structures when expressed on its own, possibly resembling the in vivo genome packing mechanism. The matrix protein VP40 is essential for assembly and its crystal structure shows that it is composed of two structurally similar ß-sandwich domains. The monomeric form of VP40 can be induced to change its conformation into oligomeric ring-like structures, which may play an important role in virus assembly and budding.
Chapter 3
Structural and Functional Polymorphism of the Glycoproteins of Filoviruses
Viktor E. Volchkov, Valentina A. Volchkova, Olga Dolnik, Heinz Feldmann, Hans-Dieter Klenk
The surface glycoprotein GP of Marburg virus (MBGV) is the only expression product encoded by gene 4 of the viral genome. In contrast, with Ebola virus (EBOV) 2 proteins are derived from this gene: a soluble glycoprotein sGP as the primary transcription product and GP which requires transcriptional editing for expression. sGP as well as GP undergo a series of co- and posttranslational modifications including N-glycosylation, O-glycosylation, and acylation. In addition, both glycoproteins are processed by cellular proteases. The GP precursor preGP is cleaved by the pro-proteinconvertase furin into the disulfide-linked fragments GP1 and GP2 forming the GP1,2 complex. GP1,2 is converted into GP1,2D by proteolytic removal of its membrane anchor by the metalloprotease TACE. Release of the disulfide bond between GP1 and GP2 gives rise to soluble GP1. sGP is derived from pre-sGP by furin-mediated proteolytic cleavage of the carboxyterminal D peptide. GP1,2 mediates virus entry by binding to cell receptors and by inducing membrane fusion. It is a major viral antigen and gives rise to neutralizing and protective antibodies. There is evidence that GP1,2 is involved in immunosuppression and has cytotoxic effects in particular on endothelial cells. The soluble glycoproteins sGP and GP1,2D may act as decoys and, thus, also modulate the immune response, but their general role in pathogenesis is still poorly understood.
Chapter 4
Molecular Mechanisms of Filovirus Entry
Stephen Y. Chan and Mark A. Goldsmith
The mechanisms by which the Marburg (MBG) and Ebola (EBO) filoviruses bind to and enter target cells are essential both for initiation of the viral replication cycle and for development of hemorrhagic disease. Recent availability of molecular tools for filovirus study outside biosafety level 4 (BSL4) containment has allowed for advancement in the understanding of both viral determinants of entry and the interacting host cell factors. Envelope glycoproteins (GP) mediate host cell binding and membrane fusion, and a growing understanding of their biosynthesis, maturation, and structure has afforded insight into the entry processes as well as the pathogenic consequences of such events. Furthermore, four groups of cell surface factors have been identified to date that play significant yet somewhat unclear roles in filovirus entry: the asialoglycoprotein receptor, the ß1 integrin receptors, the recently characterized DC-SIGN and DC-SIGNR factors, and the folate receptor-a. As a result, a conceptual model for filovirus entry can be constructed which should prove valuable in future elaboration of the entry process and pathogenesis as well as in future identification of suitable therapeutic targets.
The method(s) by which any virus binds to a host cell to initiate entry is a central determinant of viral pathogenesis. Enveloped viruses typically attach to a target cell by binding to a surface receptor(s) via envelope glycoprotein (GP) spikes expressed on the virion surface. Subsequently, receptor binding and/or endocytosis activate a fusogenic domain of the glycoprotein via conformational changes and trigger fusion of the viral envelope with host membranes, thereby allowing entry. Like other enveloped viruses, filoviruses are packaged by an envelope derived from the host cell plasma membrane. Construction of a detailed picture of entry processes used by filoviruses is essential to gaining a complete understanding of the basis for the rapid and widespread tissue destruction and hemorrhagic disease associated with infection. Until recently, the specific details regarding processes of filovirus entry had remained elusive. Replicating filoviruses are restricted to maximum containment, biosafety level 4 (BSL4) research facilities, and studies of the molecular aspects of filovirus entry with wild-type filoviruses are difficult to perform (Peters et al., 1996). However, with the sequencing of filovirus genomes in recent years and the subsequent availability of molecular tools to study filovirus gene products outside BSL4 facilities, our understanding of the molecular aspects of filovirus entry and its impact on pathogenesis has advanced considerably. Currently, a preliminary conceptual framework has emerged, focusing on two main areas: (1) description of the filovirus envelope GP complex as a main viral determinant of both entry and subsequent pathogenic cellular responses; and (2) identification of host cell factors required for entry. As a result, a more thorough understanding of filovirus binding to host cell factors, initiation of infection, and the pathogenic consequences of those interactions should soon emerge as a basis for identifying more effective targets of drug treatment.
Chapter 5
Roles of Filoviral Matrix- and Glycoproteins in the Viral Life Cycle
Gabriele Neumann, Takeshi Noda, Ayato Takada, Luke D. Jasenosky, and Yoshihiro Kawaoka
The filovirus glycoprotein mediates virus entry into host cells and is therefore a determinant of host and tissue tropsism. Furthermore, its cytotoxicity to virus-infected cells may contribute to filovirus pathogenicity. The VP40 protein interacts with lipid bilayers and forms particles when expressed alone, indicating that it is equivalent to the matrix protein found in other RNA viruses. VP40 contains a short peptide motif similar to that identified in retroviral Gag protein, which is critical for particle assembly and budding. This motif has been shown to interact with cellular proteins of the ubiquitination and protein-sorting pathways; however, the role(s) of these interactions in viron formation and budding remains to be established.
Chapter 6
Virus Maturation
Larissa Kolesnikova and Stephan Becker
The assembly of filoviruses starts with the formation of nucleocapsids in the cytosol of the infected cells. Nucleocapsid formation is associated with the appearance of inclusions that are distributed around the nucleus. The inclusions contain high concentrations of nucleocapsid proteins and nucleocapsids. Mature nucleocapsids are found at later stages of infection outside the inclusions seemingly on their way to the plasma membrane. The transport of other viral components as the transmembrane glycoprotein GP and the matrix protein VP40 is closely associated with the host cell membranes. While the GP is transported to the plasma membrane via the classical secretory pathway, the major matrix protein VP40 is transported using the late retrograde endosomal pathway. This process is powered by the polymerization of actin. At the plasma membrane, nucleocapsids and a complex of matrix proteins, GP and lipids are assembled to viral particles. The release of infectious virions is accomplished by budding and depends on the interaction with cellular proteins.
Chapter 7
Filovirus Pathogenesis in Nonhuman Primates
Thomas W. Geisbert, Peter B. Jahrling, Tom Larsen, Kelly J. Davis, and Lisa E. Hensley
Infections with Ebola and Marburg viruses cause severe and fatal hemorrhagic disease in humans and nonhuman primates. While progress to define the mechanisms of filoviral pathogenesis has been made in the last decade, cultural mores, and a range of logistical problems, have hindered the systematic pathogenetic analysis of human filoviral infections. Nonhuman primate models of filoviral hemorrhagic fever (HF) have been developed, but with few exceptions, previous investigations examined animals naturally infected or killed when moribund, and shed little light on the pathogenesis of infection during the period before death. More recently, longitudinal analysis of pathogenetic events in nonhuman primate models of Ebola virus (EBOV) HF have revealed new and important findings. Specifically, tissue factor plays an important role in triggering the hemorrhagic complications that characterize EBOV infections, and dysregulation of protein C exacerbates disease. Moreover, replication of EBOV in endothelial cells was not consistently observed until the latter stages of disease, well after the onset of disseminated intravascular coagulation, suggesting that the characteristic coagulation abnormalities are not the direct result of filoviral-induced cytolysis of endothelial cells. Bystander lymphocyte apoptosis, previously described in end-stage tissues, occurred early in the disease course in intravascular as well as extravascular locations. Of note, apoptosis and loss of NK cells was a prominent finding suggesting the importance of innate immunity in determining the fate of the host. Accordingly, nonhuman primate models have been invaluable in identifying several new targets for chemotherapeutic interventions that may ameliorate the effects of filoviral HF.
Chapter 8
Ebola Virus Infection in the Guinea Pig
Elena Ryabchikova, Margarita Smolina, Antonina Grajdantseva, Jurii Rassadkin
Ebola virus causes severe disease with high mortality in humans and monkeys, and non-fatal illness in guinea pigs. Serial passages of Ebola virus in guinea pigs change the disease to highly fatal. A goal of the present work was to analyze the features of non-fatal Ebola infection in guinea pigs, and the changes in the pattern of the infection developing in the course of serial passages of the virus.
Examination of non-fatal infection in guinea pigs showed that Ebola virus causes focal inflammation in the liver. Replication of Ebola virus in non-fatally infected guinea pigs has been found only in the macrophages. Serial passages of Ebola virus resulted in two major events: widening of the set of cells supporting replication of Ebola virus, and abolition of inflammatory response to infected cells. These mechanisms may be responsible for the enhancement of pathological changes in visceral organs observed during the passages. Taken together, the data suggest that macrophages, the primary target of infection, play an important role in virus adaptation.
Chapter 9
Pathogenesis of Filovirus Infection in Mice
Mike Bray
Immunocompetent adult mice do not become ill when inoculated with filoviruses isolated from human cases. A variant of Ebola Zaire '76 virus that was adapted to mice through serial passage causes a rapidly lethal illness that resembles the human disease in terms of its target cells of infection, pattern of dissemination and organ injury, biochemical and hematological changes and pattern of cytokine responses, but differs in showing little evidence of coagulopathy. Mice are exquisitely sensitive to the virus injected intraperitoneally, but solidly resistant to the same agent injected subcutaneously. Both adaptive and innate immune responses play a role in resistance. The Type I interferon response is especially important in determining the outcome of infection. Knockout mice lacking an effective Type I response are susceptible to lethal infection by a number of non-adapted Ebola and Marburg viruses. The mouse-adapted virus appears to have been selected during serial passage for its ability to block the production of Type I interferon. Limited evidence suggests that the same change has rendered the virus somewhat attenuated for primates. Further research using mouse models of filovirus infection should focus on virus-cell interactions at the molecular level and on means of preventing viral suppression of innate immune responses.
Chapter 10
The Role of Endothelial Cells in Filovirus Hemorrhagic Fever
Hans-J. Schnittler, Ute Ströher, Tatiana Afanasieva, and Heinz Feldmann
Infection with Marburg and Ebola viruses, family Filoviridae, cause the most severe form of viral hemorrhagic fever (VHF). There is now considerable evidence that endothelial dysfunction largely contributes to the fatal outcome of filoviral HF. This review discusses the role of endothelial cells and addresses the potential pathomechanisms that may lead to structural and functional disturbances during filovirus infections. The virus-induced activation of cells of the mononuclear phagocytic system is discussed in regards to subsequent effects on endothelial cell functions such as disturbances of the barrier function and circulatory parameters finally leading to shock development. A working hypothesis is generated that will help to direct future research into the role of the endothelium in the pathogenesis of VHF.
Chapter 11
Modulation of Innate Immunity by Filoviruses
Christopher F. Basler and Peter Palese
Recent studies have begun to illuminate the interactions between filovirusesEbola and Marburg virusesand their hosts. Of particular interest is the interplay between theses viruses and the innate immune responses, since innate immunity constitutes the earliest host defense against infection. Filovirus infection can influence the host innate immune response in several ways. For example, in vivo filovirus infection induces the expression of proinflammatory cytokines. Often, however, this cytokine production does not clear the viral infection and may in fact contribute to pathology and death. At the same time, Ebola virus infection appears to block the production of type I interferons in at least some cell types and to block the antiviral effects of interferons. The viral proteins VP35 and VP24 have been implicated in these latter effects of viral infection. Finally, filovirus infection may inhibit the function of immunologically important cells, inhibit the activation of neutrophils, and destroy and/or inhibit the proliferation of monocytes/macrophages and lymphocytes.
Chapter 12
Cellular and Molecular Mechanisms of Ebola Pathogenicity and Approaches to Vaccine Development
Gary J. Nabel, Nancy J. Sullivan, and Zhi-yong Yang
Recent molecular analyses of Ebola viral gene products have suggested that the glycoprotein primarily mediates the cytotoxic effects of the Ebola virus. The glycoprotein is synthesized in a secreted (sGP) or full-length transmembrane (GP) form. GP binds preferentially to endothelial cells while sGP binds neutrophils and possibly some cells of the monocytic lineage. GP expression in cultured cells leads to cell rounding and detachment from the substrate. GP expression in vascular tissue explant cultures leads to the breakdown of the endothelial lining. These effects require a mucin-like domain of GP and appear to be mediated through the down-regulation of specific molecules on the cell surface. Critical mediators of cell adhesion to the matrix and immune signaling (e.g., integrins and MHC I) are among the affected cell-surface molecules. The effects of GP expression are consistent with, and may account for, the characteristic features of Ebola infection, including inflammatory dysregulation, immune suppression and loss of vascular integrity. Vaccines that employ GP as the immunogen have been shown to provide protective immunity in rodent and nonhuman primate models.
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