Entry mechanisms and pathologic effects mediated by the envelope glycoproteins of the human immunodeficiency virus type 1, Marburg, and Ebola viruses.

摘要

Cellular entry by enveloped viruses is a central event in pathogenesis and depends critically on the binding of the viral envelope glycoprotein (GP) to a cell surface receptor(s) and/or coreceptor(s). The objective of this thesis project was to characterize the molecular methods by which the envelope GP of the human immunodeficiency virus type 1 (HIV-1), Marburg (MBG), and Ebola (EBO) viruses bind to host cell factors to initiate infection and to determine the pathogenic consequences of those interactions. For HIV-1, the hypothesis was investigated that HIV dementia (HIVD) results largely from alterations in the gp120 envelope protein, influencing receptor/coreceptor use. Through functional characterization of primary gp120 V3 sequences, the CCR5 chemokine receptor was identified as a primary coreceptor for brain-derived viruses regardless of HIVD diagnosis, indicating that alteration of coreceptor specificity is not necessary for HIVD development. In contrast, an alternate mechanism of coreceptor use was characterized that allows for HIV-1 entry into CD4-negative human fetal astrocytes in a coreceptor-dependent manner only when CD4 is provided in trans by donor cells. Such a process may be active in facilitating neurodegeneration and inciting dementia. For the filoviruses MBG and EBO, GP-mediated entry and pathogenesis were characterized by using pseudotype viruses carrying an HIV-1 backbone and packaged by MBG or EBO GP. While these pseudotype viruses infected a comparable wide range of cell types, it was discovered that the MBG and EBO GP utilize distinct pathways to enter cells and incite cellular dysregulation. To identify cellular factors that facilitate virus uptake, a genetic complementation strategy combining selectable filovirus pseudotype viruses with a retroviral expression library was implemented to isolate cells exhibiting reconstitution of permissivity for filovirus entry. Library cDNA inserts encoding for the folate receptor-alpha (FR-alpha) protein were recovered from reconstituted cells. Utilizing further genetic and biochemical strategies, FR-alpha was identified as a cellular cofactor that mediates entry by both MBG and EBO viruses. Taken together, these discoveries should expand our understanding of the pathogenic life cycles of HIV-1, MBG, and EBO viruses and may facilitate the development of more effective treatments of these devastating and lethal infections.