Viral entry inhibitors targeted to the membrane site of action

Abstract

The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The "class I" fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutinin-neuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses-human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases-the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.

FIG. 1.

Inhibition of HPIV3, HeV, and NiV infection by cholesterol-tagged HPIV3 HRC peptides. (A) CV1 cell monolayers were infected with wild-type HPIV3 at a multiplicity of infection (MOI) of 6.7 × 10⁻⁴ in the presence of increasing concentrations of HPIV3 HRC peptides that either were left untagged or were tagged with cholesterol at the C or the N terminus. After a 90-min incubation, cells were overlaid with methylcellulose, and plaques were stained and counted after 18 h. The percentage of inhibition of viral entry (compared to results for control cells infected in the absence of inhibitors) is shown as a function of the (log-scale) concentration of the HPIV3 HRC peptide. Data points are means (± standard deviations) for triplicate samples. These data are representative of results from three to five experiments. (B and C) 293T cell monolayers were infected with HeV (B) or NiV (C) pseudotyped virus at an MOI of 0.25 in the presence of increasing concentrations of the HPIV3 HRC peptide, either left untagged or tagged at the N or the C terminus. After 24 h, cells were collected and fixed for FACS analysis. The percentage of inhibition of viral entry (compared to results for control cells infected in the absence of inhibitors) is shown as a function of the (log-scale) concentration of the HPIV3 HRC peptide. The data are representative of results from three to five experiments. (D and E) Vero cell monolayers were infected with 50 to 80 PFU of HeV (D) or NiV (E) in the presence of HPIV3 peptides, tagged at either the N or the C terminus, at the indicated concentrations (x axis). After a 30-min incubation, cells were overlaid with agarose to prevent cell-to-cell spread of virus, and plaques were stained and counted after 3 days of incubation. The percentage of inhibition of viral entry is shown as a function of the (log-scale) concentration of the HPIV3 HRC peptide. The data are representative of results from three to five experiments.

FIG. 2.

Cholesterol-tagged peptides insert into the target cell membrane and create a protective shield. (A) CV1 cell monolayers were incubated with increasing concentrations of HPIV3 HRC peptides that were either left untagged or tagged with cholesterol at the N or the C terminus. After 60 min, the cells were washed and infected with wild-type HPIV3 at an MOI of 5 × 10⁻⁴. Ninety minutes later, cells were overlaid with methylcellulose, and plaques were stained and counted after 24 h. The percentage of inhibition of viral entry (compared to results for control cells infected in the absence of inhibitors) is shown as a function of the (log-scale) concentration of the HPIV3 HRC peptide. (B) CV1 cell monolayers were incubated with increasing concentrations of HPIV3 HRC peptides that were either left untagged or tagged with cholesterol at the N or the C terminus. After 60 min, the cells were washed and stained with anti-HRC antibodies. Data points are means (± standard deviations) for triplicate samples. (C) CV1 cell monolayers were incubated either with 1 μM HRC peptides tagged with cholesterol at the N or the C terminus or with 10 μM untagged peptides. After 60 min, cells were washed with cholesterol depletion medium (or with plain medium); then they were infected with wild-type HPIV3 at an MOI of 5 × 10⁻⁴. Ninety minutes later, cells were overlaid with methylcellulose, and plaques were stained and counted at 18 h. The percentage of inhibition of viral entry for each peptide is shown for cells washed with cholesterol depletion medium or with plain medium. (D) CV1 cells were incubated with inhibitory concentrations of tagged or untagged HPIV3 peptides. After 60 min, cells were washed with cholesterol depletion medium or plain medium and were then stained with anti-HRC antibodies and an HRP-conjugated protein G secondary antibody.

FIG. 3.

Preaddition of C-terminally tagged peptides curtails multicyle replication of Hendra virus at picomolar concentrations. Vero cell monolayers were pretreated with ½ log dilutions of the N-terminally or C-terminally tagged HPIV HRC peptide at the indicated concentrations (x axis) for 60 min in EMEM-10. Cells were then washed with PBS to remove unbound peptides prior to infection with 0.05 MOI of HeV for 30 min. The medium containing the virus was then removed; fresh medium was added; and the cells were incubated at 37°C for 18 h. Monolayers were then fixed and immunolabeled as described previously (2). The percentage of inhibition of viral entry is shown as a function of the (log-scale) concentration of the HPIV3 HRC peptide. The data are averages of results from four experiments, and values are expressed as means ± standard errors (SE).

FIG. 4.

C-terminally cholesterol-tagged peptides inhibit SV5 (HPIV5). CV1 cell monolayers were infected with wild-type SV5 at a multiplicity of infection (MOI) of 3 × 10⁻⁴ in the presence of increasing concentrations of HPIV3 HRC peptides that were either left untagged or tagged with cholesterol at the C terminus. After 180 min, cells were overlaid with methylcellulose, and plaques were stained and counted at 36 h. The percentage of inhibition of viral entry (compared to results for control cells infected in the absence of inhibitors) is shown as a function of the (log-scale) concentration of the HPIV3 HRC peptide. Data points are means (± standard deviations) for triplicate samples. The data are representative of results from three to five experiments.

FIG. 5.

(A) The sequence spanning residues 449 to 484 of the F protein of HPIV3 is shown in the standard N-to-C and in the opposite C-to-N direction, to highlight the possible conserved interactions with the HRN peptide. (B) Alignment of the sequence spanning residues 449 to 484 of the F protein of HPIV3 with the corresponding region of HPIV5. Red, identical amino acids; green, conservative substitutions.