AAC Accepts, published online ahead of print on 10 January 2011 Antimicrob. Agents Chemother. doi:10.1128/AAC.01428-10 Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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IDENTIFICATION OF THE MINIMAL ACTIVE SEQUENCE OF AN ANTI-INFLUENZA
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PEPTIDE
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Jeremy C. Jones1, Erik W. Settles2, Curtis R. Brandt2,3, and Stacey Schultz-Cherry1*
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TN 38105, and Departments of 2Medical Microbiology and Immunology and
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53706.
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis,
Opthamology and Visual Sciences, University of Wisconsin-Madison, Madison, WI
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Running Title: Minimal sequence of an anti-influenza peptide
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Abstract Word Count: 67
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Text Word Count: 1,286
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*Corresponding author:
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Dr. Stacey Schultz-Cherry
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Department of Infectious Diseases
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St. Jude Children’s Research Hospital
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262 Danny Thomas Place, MS 320
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Memphis, TN 38105
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Phone: (901) 595-6629
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Fax: (901) 595-3099
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Email:
[email protected]
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ABSTRACT
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The antiviral peptide, Entry Blocker (EB), inhibits influenza virus replication by
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preventing attachment to cells.
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sequence that retained antiviral activity with IC50/EC50 values similar to the full-length EB
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peptide and several truncated variants that possessed up to 10-fold lower IC50 values.
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These data have implications for improving antiviral efficacy of EB-derived peptides
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while decreasing production costs and easing synthesis.
Here, we identified the minimal and optimal EB
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TEXT
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Influenza A viruses pose a considerable threat to human health (16, 18, 21, 22).
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Vaccination is a proven method to control infection, but viral antigenic drift necessitates
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a lengthy vaccine reformulation annually (7, 11, 19). Treatment of influenza by virus
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entry blockers (amantadine and rimantadine) or egress inhibitors (oseltamivir,
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zanamavir) limits transmission and disease severity (6, 8, 20, 23). However, increased
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resistance to these agents (2, 3, 9, 10, 12, 17, 24) supports the search for new antiviral
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therapies.
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We previously identified a 20-amino acid peptide derived from the fibroblast
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growth factor 4 signal sequence (Entry Blocker [EB]) that displayed broad-spectrum
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anti-influenza activity in vitro and in vivo (15).
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determine the minimal and optimal EB sequence required for antiviral activity. Thus, a
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library of peptides with serial deletions of a single residue from either the N- or C-
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terminus was synthesized (EZBiolab, Carmel, IN and St. Jude Children’s Research
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Hospital, Memphis, TN) and initially tested for inhibitory activity. All 32 synthesized
The goal of these studies was to
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peptides retained the N-terminal RRKK tetrapeptide to maintain solubility (Table 1).
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Full-length EB inhibits influenza virus by preventing attachment to host cells (15) as
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determined by blocking the viral-mediated hemagglution (HA) of chicken red blood cells
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(cRBCs), a commonly accepted indication of virus attachment (13, 14).
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screened the library of peptides for their ability to inhibit HA activity. Briefly, A/Puerto
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Rico/8/34 (PR/8, H1N1) virus was propagated in embryonated chicken eggs, sucrose-
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purified, and viral titer determined by HA activity. To screen the peptide library, the
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virus (64 HA units) was treated with 10 µM of each peptide for 1 hr at 37°C. Doubling
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dilutions of the virus/peptide mixture were incubated with cRBCs for 1 hr and the final
Thus, we
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dilution with agglutinated cRBCs recorded as the HA titer.
A significant decrease of
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this attachment-dependent activity was scored if the peptide inhibited ≥ 2 doubling
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dilutions below mock-treated virus. Our screen identified 11 “active” truncations of 13 –
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19 residues that maintained significant antiviral activity (≥ 89% reduction of HA activity
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compared to mock-treatment, Table 1). Up to 4 residues could be deleted from the C-
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terminus (A2-A5, Table 1), while up to 8 residues could be deleted from the N-terminus
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(B6-B12, Table 1), suggesting that sequence specific elements in the C-terminus of the
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peptide are less dispensable for antiviral activity. Peptides alone (10 µM) had no effect
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on cRBC agglutination.
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We determined the 50% inhibitory concentration (IC50) for several active peptides
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by treating PR/8 virus (64 HA units) with peptide concentrations ranging from 0 to 10 µM
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and subjecting samples to the HA assay as described. In keeping with our findings with
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EB (A2, Table 1), EBNP (Table 2) and Bultmann et al. (4), removing prolines (NP) had no
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effect on inhibitory activity. Thus, they were deleted from existing truncations to further
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reduce peptide size. Additionally, removal of the terminal proline has implications for
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simplified synthesis as it removes a secondary amine, which often interferes with
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coupling. Peptides B7NP-B10NP had IC50 values ranging from 0.3 to 3 µM (Table 2).
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However, peptides B11NP, B12NP and C1NP lost activity after proline removal. It is
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possible that these truncated peptides, unlike the full-length EB, require the proline for
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antiviral activity. Alternatively, the length of the peptides may also have a separate
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effect but since these effects are potentially co-variable with other changes this is
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difficult to determine specifically. Overall, our screen demonstrated that peptide B10NP
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(RRKKLAVLLALLA) was the minimal sequence that retained EB-like inhibitory activity.
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To optimize and potentially further reduce size, B10NP was modified to examine
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the importance of the RRKK solubility tetrapeptide and di-leucine repeats (Table 3).
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Modifying the RRKK tetrapeptide or either of the di-leucine repeats resulted in a loss of
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activity.
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activity. However, there was an increase in the IC50 value suggesting that the full RRKK
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tetrapeptide is optimal for antiviral activity. Modifying the charge of the tetrapeptide
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(B10ED, Table 3) also abolished activity. This may be due effects on peptide solubility.
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Substituting individual di-leucines (B108,9A and B1011,12A, Table 3) abolished anti-
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influenza activity analogous to our control peptide for EB (EBX, Table 2), which contains
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scrambled di-leucines and lacks inhibitory activity (1, 4, 15).
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obtained with vaccinia virus where the di-leucine repeats were important for antiviral
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activity (1). These studies suggest that the 13-amino acid B10NP (RRKKLAVLLALA) is
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the minimal peptide that maintains antiviral activity.
Peptide B10R (Table 3), lacking the N-terminal R, retained 75% of B10NP
Similar results were
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To determine the protective index of B10NP and B7NP as compared to EB,
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increasing concentrations of the peptides were pre-incubated with PR/8 virus (64 plaque
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forming units; PFU) and viral plaque formation measured in MDCK cells to determine
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the 50% effective concentration (EC50). In parallel, the same peptide concentrations
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were added to MDCK and A549 cells in serum-free and 5% serum containing media
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and cell death measured by MTS assay (Promega, Madison, WI) to determine the
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cytotoxic doses (CD50). All of the peptides had similar CD50 values of ~90 µM in MDCK
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cells and ~120 µM in A549 cells in the absence of serum and >150 µM in both cell types
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in the presence of 5% serum (Table 4). B10NP and EB had similar EC50 values (5 µM
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and 7 µM) and protective indices (18 and 12.2 respectively). In contrast, B7NP was
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significantly more active with an EC50 of 0.3 µM and a protective index of 351. These
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data demonstrate that while the 13 amino acid B10NP peptide has similar activity as EB,
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B7NP has significantly enhanced activity.
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Electron microscopic (EM) examination of PR/8 (512 HAU) virus pre-treated with
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mock (0 µM) or 10 µm peptides demonstrated that B7NP severely disrupted virions (Fig.
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1A) suggesting that at concentrations above the EC50 B7NP may be virucidal. Further,
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treatment of human RBCs with similar concentrations of B7NP induced lysis as
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measured by hemoglobin release (Fig. 1B). These properties were unique to the B7NP
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peptide.
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concentration tested (Fig. 1). In fact, the EM data suggests that the peptides may
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induce viral aggregation. This possibility is under investigation.
EB and B10NP did not disrupt the virion or lyse red blood cells at any
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In summary, these studies identify 2 important new derivatives of antiviral EB
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peptide; a minimal and optimal sequence, RRKKLAVLLALLA (B10NP) that confers
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similar anti-viral activity as EB, and a newly identified peptide, RRKKVALLAVLLALLA
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(B7NP) possessing significantly enhanced anti-viral and potentially virucidal activity. Like
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EB, B10NP and B7NP inhibit virus-cell attachment and reduce virus replication at low
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micromolar concentrations. Minimal toxicity and EC50 values near or considerably lower
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than EB produce attractive protective indices for B10NP and B7NP (Table 4). Of great
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interest, several of these EB peptides have broad spectrum activity against, not only
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influenza virus, but vaccinia virus (1) and HSV-1 (4), as well as other viruses (C. Brandt,
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unpublished data). The EB peptide blocks influenza attachment but with vaccinia and
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HSV-1 it prevents entry into the cell. For virucidal activity, EB is active against HSV-1
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and influenza but not vaccinia virus. These data suggest that EB interacts with specific
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but different proteins in each virus accounting for the different activities. Given the
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complexity of the HSV-1 and vaccinia virus envelopes we do not yet know the target in
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these viruses. Recently we showed that, at low concentrations (150.0
83.5 ± 22.0
85.5 ± 1.3
7.0 ± 2.1
Lowest IC50
>150.0
126.3 ± 23.0
105.3 ± 8.0
0.3 ± 0.2
Least Residues
>150.0
120.0 ± 28.0
90.0 ± 16.3
5.0 ± 4.0
NP
B7
NP
B10
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Serum Free a CD50 (µM)
b
Protective c Index
12.2 351 18
50% cytotoxic dose as determined by MTS viability assay b 0.01-10 µM of peptide was assayed for the ability to inhibit plaque formation (≈65 plaque forming units) of PR/8 virus in MDCK cells under serum free conditions. EC50 values were estimated from the dose response curves. C Protective index = CD50/EC50 in MDCKs under serum free conditions Results are indicative of at least 3 independent tests. a
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Figure 1. B7NP is virucidal at concentrations exceeding the IC50/EC50 A) Purified PR/8 (512
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HAU) virus was mock-treated (0 µM) or peptide-treated (10 µM EB, B7NP or 28 µM B10NP) for 1
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hr at 37°C. Samples were coated to grids, stained with phosphotungsic acid and visualized by
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electron microscopy. Scales bars: 1 µm (Mock, EB, B7NP), 2 µm (B10NP), B7NP inset scale
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bar:
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increasing concentrations of peptide (0.1–30 µM), or 1% SDS for 1 hr at 37ºC. Samples were
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subjected to centrifugation to pellet RBCs and the supernatants were read at A540 on a
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spectrophotometer to measure hemoglobin released. Values are normalized to 100% lysis (1%
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SDS), presented as the mean values ± standard error, and are representative of at least 3
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independent experiments. * P ≤ 0.001 as compared to mock treatment.
200 nm
B) A 2% solution of human RBCs was mock-treated (0 µM), treated with
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