Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May 18;10(5):e0126239.
doi: 10.1371/journal.pone.0126239. eCollection 2015.

Novel heparan sulfate-binding peptides for blocking herpesvirus entry

Pranay Dogra  1 , Emily B Martin  2 , Angela Williams  2 , Raphael L Richardson  3 , James S Foster  2 , Nicole Hackenback  2 , Stephen J Kennel  4 , Tim E Sparer  1 , Jonathan S Wall  4
Affiliations

Novel heparan sulfate-binding peptides for blocking herpesvirus entry

Pranay Dogra et al. PLoS One. .

Abstract

Human cytomegalovirus (HCMV) infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV) infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs), serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Heparin-reactive peptides reduce MCMV infection in vitro.
(A) Peptides (500 μg/ml) with different net charges and lengths were incubated with cells 30 min prior to addition of MCMV (~100 pfu/well). Bars represent the average of the percent reduction in infection compared to PBS-treated control from three independent experiments with at least three replicates in each + SD. (B) p5+14 and CGGY-p5G (control peptide) were serially diluted and assayed in a plaque reduction assay as described in materials and methods.
Fig 2
Fig 2. p5+14 binding to cells is charge dependent.
(A) Predicted α-helix structure of peptide p5+14 based on ITASSER modeling. (B) Biotinylated peptide p5+14 (left panel) or CGGY-p5G (right panel) was added to MEF 10.1 cells followed by addition of Alexa Fluor 594-conjugated streptavidin (red). Nuclei are stained blue with Hoechst and F-actin stained green with Alexa Fluor 488-conjugated phalloidin.
Fig 3
Fig 3. Soluble heparin interferes with peptide inhibition of virus infection.
The effect of heparin on the activity of peptide and MCMV viral infectivity in vitro when (A) pre-incubated with the peptide (100 μM), (B) incubated with the cells before adding virus, and (C) pre-incubated with virus alone in a plaque reduction assay. Bars represent the average of the percent reduction in infection compared to PBS-treated control from three independent experiments with at least three replicates in each + SD. Statistical significance is indicated as: * = p<0.05, ** = p<0.01, *** = p<0.001.
Fig 4
Fig 4. Peptide interacts with cell surface heparan sulfate but not chondroitin sulfate to mediate anti-viral activities.
(A) MEF 10.1 cells were treated with heparinase I, II, III or chondroitinase ABC and the amount of bound peptide was assessed as described in materials and methods. (B) Cells were treated with heparinase I (1U/ml) or (C) chondroitinase ABC (1U/ml) and peptide p5+14 was added. The amount of plaque reduction of MCMV infection in each treatment was measured in a plaque reduction assay. Bars represent the average of the percent reduction in infection compared to PBS-treated control from three independent experiments with at least three replicates in each + SD. Statistical significance is indicated as: * = p<0.05, ** = p<0.01, *** = p<0.001, NS = non-significant difference in the reduction of infection.
Fig 5
Fig 5. Peptide p5+14 blocks adsorption of MCMV.
(A) MCMV was preincubated with p5+14 peptide (100 μM) before diluting the virus/peptide to an ineffective peptide concentration (1 μM) and assayed in the plaque reduction assay as described in materials and methods. As a control, virus and peptide (100 μM) were added to cells simultaneously. (B) Cells were incubated with p5+14 peptide either prior to virus adsorption, during virus adsorption, after virus adsorption (at 4°C) but prior to fusion, or after fusion (at 37°C). Plaque reduction was measured in a plaque reduction assay. Bars represent the average of the percent reduction in infection compared to PBS-treated controls from three independent experiments with at least three replicates in each + SD. Statistical significance is indicated as: * = p<0.05, ** = p<0.01, *** = p<0.001, NS = non-significant difference in the reduction of infection compared to PBS treated control wells. (C) Adsorption of HCMV TB40/E-pp150-GFP (MOI 10) fusion protein expressing HCMV was measured via flowcytometry in the presence of p5+14 (green), control peptide CGGY-p5G (orange) and PBS (blue). Red line represents uninfected cells. Inset is a scatter plot of the mean fluorescence intensity (MFI) for GFP with the line representing average of 3 replicates +/- SD for the different treatments.
Fig 6
Fig 6. Comparison of the efficacy of p5+14 and peptide G2 to reduce MCMV infection in vitro.
Peptides G2 and p5+14 were added at different concentrations (100, 10, 1 μM) in a plaque reduction assay as described in materials and methods. Bars represent the average of the percent reduction in infection compared to PBS-treated control from three independent experiments with at least three replicates in each + SD. Statistical significance is indicated as: * = p<0.05, ** = p<0.01, *** = p<0.001, NS = non-significant difference in the reduction of infection compared to PBS-treated control wells.
Fig 7
Fig 7. Peptide p5+14 inhibits HCMV and HSV infections in vitro.
Peptide p5+14 (100 μM) was added in a plaque reduction assay using HCMV (TB40/E) on different cell types (HFF, HAEC, and ARPE-19) and HSV-1 or HSV-2 on VERO cells. Bars represent the average of the percent reduction in infection compared to PBS-treated control from three independent experiments with at least three replicates in each + SD. Statistical significance is indicated as: * = p<0.05, ** = p<0.01, *** = p<0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mocarski ES, Shenk T, Griffith P, Pass RF. Cytomegaloviruses In: Knipe DM, Howley PM, editors. Fields Virology (Sixth Edition), Sixth ed. Philadelphia: Lippincott Williams & Wilkins; 2013. p. 1960–2014.
    1. Crough T, Khanna R. Immunobiology of human cytomegalovirus: from bench to bedside. Clinical microbiology reviews. 2009;22(1):76–98, Epub 2009/01/13. 10.1128/cmr.00034-08 - DOI - PMC - PubMed
    1. Gerna G, Baldanti F, Revello MG. Pathogenesis of human cytomegalovirus infection and cellular targets. Human immunology. 2004;65(5):381–6. 10.1016/j.humimm.2004.02.009 - DOI - PubMed
    1. Schleiss MR. Cytomegalovirus in the neonate: immune correlates of infection and protection. Clinical & developmental immunology. 2013;2013:501801 Epub 2013/09/12. 10.1155/2013/501801 - DOI - PMC - PubMed
    1. Griffiths P, Plotkin S, Mocarski E, Pass R, Schleiss M, Krause P, et al. Desirability and feasibility of a vaccine against cytomegalovirus. Vaccine. 2013;31 Suppl 2:B197–203. Epub 2013/04/26. 10.1016/j.vaccine.2012.10.074 . - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

Funding came from Physicians Medical, Education and Research Foundation (PMERF) at the University of Tennessee Medical Center, Knoxville and a Scholarly and Research Incentive Fund (SARIF) award from the Office of Research Engagement at the University of Tennessee, Knoxville.

LinkOut - more resources