Anti-heparan Sulfate Peptides That Block Herpes Simplex Virus Infection in Vivo

Abstract

Heparan sulfate (HS) and its highly modified form, 3-O-sulfated heparan sulfate (3-OS HS), contribute strongly to herpes simplex virus type-1 (HSV-1) infection in vitro. Here we report results from a random M13-phage display library screening to isolate 12-mer peptides that bind specifically to HS, 3-OS HS, and block HSV-1 entry. The screening identified representative candidates from two-different groups of anti-HS peptides with high positive charge densities. Group 1, represented by G1 peptide (LRSRTKIIRIRH), belongs to a class with alternating charges (XRXRXKXXRXRX), and group 2, represented by G2 peptide (MPRRRRIRRRQK), shows repetitive charges (XXR-RRRXRRRXK). Viral entry and glycoprotein D binding assays together with fluorescent microscopy data indicated that both G1 and G2 were potent in blocking HSV-1 entry into primary cultures of human corneal fibroblasts and CHO-K1 cells transiently expressing different glycoprotein D receptors. Interestingly, G2 peptide isolated against 3-OS HS displayed wider ability to inhibit entry of clinically relevant strains of HSV-1 and some divergent members of herpesvirus family including cytomegalovirus and human herpesvirus-8. To identify functional residues within G1 and G2, we performed point mutations and alanine-scanning mutagenesis. Several arginine and a lysine residues were needed for anti-HSV-1 activity, suggesting the importance of the positively charged residues in virus-cell binding and virus-induced membrane fusion. In vivo administration of G1 or G2 peptide as a prophylactic eye drop completely blocked HSV-1 spread in the mouse cornea as evident by immunohistochemistry. This result also highlights an in vivo significance of HS and 3-OS HS during ocular herpes infection.

Zebrafish: Modeling for Herpes Simplex Virus Infections

“…33 The differences also point to the possibility that the levels of HSV infectivity during zebrafish development from early stages of embryos to adult form may vary and a lot of it can be monitored using time-lapse confocal imaging. Since our group has already identified and characterized HS recognizing anti-HS and anti-3-OS HS peptides, 7,8 it will be interesting to test the effects of the peptides in blocking HSV infection and associated inflammation in the zebrafish model. Similarly, testing HSV infectivity in receptor-specific knockouts will also be very useful to understand their in vivo significance.…”

“…21 Ideally, zebrafish in a 96-well format can be treated with antibodies 50 or compounds targeting specific cells/tissues or modified antiviral peptides with an objective to have higher efficacy. For instance, peptide generated against human 3-OS HS 7,8 can be tested against the zebrafish 3-OS HS model since virus is able to infect zebrafish. Likewise, elucidating the antiviral activity of new drugs or peptides can be done and activation or blockage of specific pathways may be identified.…”

“…5,6 In addition, phage display screening generated peptides against HS and 3-OS HS significantly impaired HSV infection in both cell culture and in vivo mouse corneal model. 7,8 Recently, our group provided the first evidence of zebrafish encoded 3-OST modified HS receptor mimicking human 3-OST generated HS in allowing HSV-1 entry and spread. [9][10][11] Our results raised the possibility that zebrafish can provide a reasonable ground to study the role of 3-OST generated receptors in HSV-1 infection by making 3-OST isoform-specific knockouts.…”

“…4 Using primary human cell culture model and a mouse model we have demonstrated the in vivo significance of HS and 3-OS HS in HSV-1 infection. [5][6][7][8] Quite interestingly, zebrafish Danio rerio widely expresses 3-OSTs, which are required for embryonic development and also for generating HSV receptors. [9][10][11][12][13][14] Supporting the idea that zebrafish 3-OSTs can generate the receptors, we have found evidence that similar to the human 3-OST-3 isoform, zebrafish encoded 3-OST-3 also allows HSV-1 entry.…”

“…Further, an advantage of using a zebrafish model is to test peptides generated against specific 3-OST isoforms. 7,8 The kinetics of inhibition can be determined in short duration of time together with toxicological evaluation, which again is not possible with present murine and rabbit models against HSV infection. The information generated by the zebrafish experiments can identify useful inhibitors to prevent HSV-1-induced pathological damages especially during ocular corneal infection or neuronal damages.…”