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. 2013 Sep 12;7(9):e2430.
doi: 10.1371/journal.pntd.0002430. eCollection 2013.

A Fusion-Inhibiting Peptide Against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

Free PMC article

A Fusion-Inhibiting Peptide Against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

Jeffrey W Koehler et al. PLoS Negl Trop Dis. .
Free PMC article


For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Stem-based peptides inhibit both RVFV and VSV.
Peptides were screened for inhibition of the pseudotyped reporter viruses RVF-VSV-luc (A and C) and VSV-luc (B and D). Virus was incubated with peptide RVFV-6, -7, -8, -9, or -10 (A, B) or serial dilutions of RVFV-6, RVFV-10, or the scrambled peptides RVFV-6sc or RVFV-10sc (C, D) prior to infecting a monolayer of Vero E6 cells. Luciferase activity (RLU) was measured approximately 18 h later. Percent inhibition was calculated based on the virus-only controls. Error shown is the standard deviation of the mean. Data are representative of at least 2 experiments.
Figure 2
Figure 2. Further characterization of the RVFV stem peptides show broad, non-toxic viral inhibition.
Serial dilutions of peptide were incubated with RVFV-ZH501 (A), EboZ-eGFP (B), or ANDV (C), before infecting a monolayer of Vero E6 cells. Percent inhibition was determined for each virus using virus-only controls. (D) MTT toxicity assay results after overnight incubation of Vero E6 cells with RVFV-6 or RVFV-6sc peptides. Absorbance was measured approximately 18 h after adding the diluted peptide in triplicate. The dashed line in (D) represents the average signal generated by the mock treated control cells. Error shown is the standard deviation of the mean. Data are representative of at least 2 experiments.
Figure 3
Figure 3. RVFV-6 does not prevent virus from binding to cells.
RVFV-MP12 (A) or EboZ-eGFP (B) was incubated with 50 µM RVFV-6 prior to the addition to a confluent monolayer of Vero E6 cells. After a 1 h adsorption, cells were rinsed with PBS, and RNA was harvested using TRIzol. Real time RT-PCR was conducted in triplicate to quantify the relative amount of viral RNA bound to cells, and results are combined from duplicate experiments. NTC is the no template control, PC (positive control) is RNA purified from either RVFV-MP12 or EboZ-eGFP. Untreated virus was mock treated without peptide. Error shown is the standard deviation of the mean.
Figure 4
Figure 4. RVFV-6 binds to cells independent of RVFV GnGc expression.
Vero E6 cells were transfected with a plasmid expressing RVFV GnGc. Forty-eight h later, either the biotin-labeled RVFV-6 peptide or the biotin-labeled RVFV-6sc peptide was added to the cells followed by washing with PBS. Cells were fixed, and peptide binding was identified using an anti-biotin antibody conjugated to Texas Red. Nuclei were stained with DAPI (blue).
Figure 5
Figure 5. Activation of the viral fusion process is required for RVFV-6 binding to RVFV Gc.
Biotin-conjugated RVFV-6, RVFV-6sc, or no peptide was pre-bound to avidin beads before the addition of RVFV-MP12. Beads were washed to remove unbound virus and treated as indicated with 1) lysis buffer and wash, 2) pH 5.2 treatment followed by lysis buffer and wash, or 3) no pH 5.2 treatment and no lysis buffer. Protein bound to the avidin beads were resolved by SDS-PAGE and probed with the anti-RVFV Gc antibody 4D4. Data represent at least 3 separate experiments.
Figure 6
Figure 6. RVFV-6 inhibits both RVFV and VSV cell:cell fusion.
Vero E6 cells were transfected with an expression plasmid expressing either the RVFV GnGc or VSV G. Twenty-four h later, cells were harvested and seeded into chamber slides. Eighteen h later, cells were incubated for 1 h with 50 µM RVFV-6 peptide followed by a pH 5.2 treatment. Medium was added to raise the pH, and slides were incubated for 5 h prior to methanol fixing and Giemsa staining. Fusion events (indicated with arrows) are shown in (A) and were quantified by number of syncytia per field of view at 100× in (B). Statistical significance was assessed by a paired, two-tailed t test. * (p = 0.001); ** (p<0.0001). Results are representative of at least 3 experiments.
Figure 7
Figure 7. Molecular hypothesis of RVFV-6 mechanism of action.
Panel A shows the initial stages of the membrane fusion process in bunyaviruses (adapted from [8]). Receptor-binding triggers uptake of virions by endocytosis. Acidification of the endocytic vesicle likely initiates Gn/Gc dissociation. Conformational rearrangements of domains I and II in Gc lead to trimer formation and insertion of the fusion peptide into the endosomal vesicle membrane. Trimer formation exposes the stem binding sites (shown in black) with affinity for the Gc stem and RVFV-6. The main molecular elements involved are: (I) glycoprotein Gc having four main components including [a] domains DI, DII, and DIII shown in green, orange/yellow, and blue, respectively; [b] the fusion loop shown in red; [c] the stem shown as small red cylinders; and [d] the transmembrane domains shown as a magenta cylinder; (II) the glycoprotein Gn, depicted as the receptor-binding protein of bunyaviruses, colored pink; and (III) host-cell receptors, shown in light-blue. Panel B: Zippering action: After acidification of the endocytic vesicle, the stem regions relocate, moving towards the host membrane through a zippering reaction. Docking of the stems into the stem binding sites is an essential step of membrane fusion that leads to opening of a pore or channel. RVFV-6 blocking: RVFV-6 molecules, shown as green cylinders, outcompete the stem fragment and lead to inhibition of fusion by blocking the movement of the native stem. Panel C: Molecular model of the RVFV Gc trimer complex. A molecular surface is used to highlight the three domains of the protein (colored as indicated in A). The stem fragments are shown as a red α-helices docked into the stem binding sites (black surface areas), and stem residues are highlighted using a stick representation.

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Support for this research was provided by the Defense Threat Reduction Agency (DTRA, under project number 4.10062_09_RD_B and the United States Department of Defense (DoD) High-Performance Computing Modernization Program ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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