Studies of Ebola Virus Glycoprotein-Mediated Entry and Fusion by Using Pseudotyped Human Immunodeficiency Virus Type 1 Virions: Involvement of Cytoskeletal Proteins and Enhancement by Tumor Necrosis Factor Alpha

Abstract

The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates. To date, no effective therapies have been identified. To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope. Fusion was detected by cleavage of the fluorogenic substrate CCF2 by ␤-lactamase-Vpr incorporated into virions and released as a result of virion fusion. Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1. These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion. Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol). Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion. Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs. In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion. We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells. Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis. Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.

Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion

“…The markedly different actin requirements for HIV-1 fusion at the cell surface (FFWO) and for virus entry/fusion through a conventional pathway (13,48,62) are in line with the notion that these processes occur at distinct cellular sites. Our findings thus appear to support the model that HIV-1 normally enters permissive cells via endocytosis (13), whereas a small fraction of virions trapped between adjacent cells can undergo fusion with the PM.…”

“…Unlike the adherent NP2-derived (this study) and HeLa-derived cells (13,48,62), in which the HIV-1 fusion appears to occur in an actin-independent manner, disruption of actin filaments significantly reduced fusion with CEM.CCR5 cells. Our finding that actin inhibitors also reduce the efficiency of virus uptake by CEM.CCR5 cells is consistent with published data on the actin dependence of HIV-1 uptake by CD4 ϩ T cells (83).…”

“…ϩ T Cells Are Partially Dependent on Intact Actin-Whereas HIV-1 fusion with HeLaderived cells is not affected by actin disruption (13,48,62), the actin cytoskeleton has been implicated in infection of CD4 ϩ T cells (53,63). We therefore examined the actin dependence of HIV-1 fusion with the lymphoid cell line, CEM.NKR-CCR5-Luc, engineered to express CCR5 and containing a Tat-inducible luciferase expression cassette (31).…”

Hepatitis C Virus Glycoproteins Mediate Low pH-dependent Membrane Fusion with Liposomes

“…Particles produced without any envelope expression construct, and consequently not infectious, were denoted "no env pp." To incorporate specifically ␤-lactamase (BlaM) into HIVbased particles, a chimeric protein encoding BlaM linked to the N terminus of the HIV-1 viral protein R (Vpr) was co-expressed with the other HIV viral vectors (44,45). Cell supernatants containing pseudoparticles were harvested 36 h after transfection and concentrated 100-fold by centrifugation through 2 ml of a 25% sucrose cushion at 100,000 ϫ g in a Beckman SW 41 rotor for 120 min at 4°C, followed by resuspension in PBS.…”

“…To compare the amounts of viruses, the blots were probed with antibodies against the capsid proteins of HIV and MLV as follows: the mouse anti-p24 of HIV (antibody AG3.0, obtained through the AIDS Research and Reference Reagent Program, division of AIDS, NIAID, NIH, from Dr. Jonathan Allan) and the anti-p30 of MLV (goat antiserum raised against the Rauscher-MuLV p30; ViroMed). Monoclonal anti-␤-lactamase antibody was from Chemicon International (AB3738), and the rabbit polyclonal anti-Vpr antibody was a kind gift from Dr. Warner Greene (45).…”