Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: inherent potential for biosynthetic versus live recombinant microbicides

Abstract

Background: Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of approximately 120-200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases) with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2.

Methods and results: Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6%) had potential to cleave the HSV-2 genome in > 100 but < 400 sites; 69(23.9%) in > 400 but < 700 sites; and the 9(3.1%) enzymes: BmyI, Bsp1286I, Bst2UI, BstNI, BstOI, EcoRII, HgaI, MvaI, and SduI cleaved in more than 700 sites. But for the 4: PacI, PmeI, SmiI, SwaI that had no sign of activity on HSV-2 genomic DNA, all 130(45%) other enzymes cleaved < 100 times. In silico palindromics has a PPV of 99.5% for in situ REase activity (2) Two models detailing how the REase EcoRII may be applied in developing interventions against HSV-2 are presented: a nanoparticle for microbicide development and a "recombinant lactobacillus" expressing cell wall anchored receptor (truncated nectin-1) for HSV-2 plus EcoRII.

Conclusion: Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.

Figure 1

This figure shows the chemical structures of nonoxynol-9 and C31G. A. Note the hydrophilic end with the hydroxyl ion at the extreme left; and the hydrophobic hydrocarbon-benzene complex. This property confers on this molecule the ability to complex with both hydrophilic (ionized) and hydrophobic molecules. The chemical formula and molecular mass of a single nonoxynol-9 molecule are respectively C₃₃H₆₀O₁₀ and 616.823 g/mol. B. C31G is a 1:1 mixed Micelle of Alkyl dimethyl amine oxide and Alkyl dimethylglycine (betaine).

Figure 2

This figure shows the deposited crystal structure of restriction endonuclease EcoRII mutant R88A in the European Molecular Biology Laboratory (EMBL) Protein database (entry 1nas6). A detailed structure of the N-domain, which contains the effector-binding cleft of EcoRII with putative DNA-binding residues H36, Y41, K92, R94, E96, K97 and R98, can be found from work by Zhou et al. [34].

Figure 3

The figure shows a simplified chemical structure of PGLA. X represents lactic acid while y represents glycolic acid. Notice the availability of the hydroxyl (-OH) and free hydrogen (+H) ions at lactic and glycolic extremities of the PLGA molecule respectively. This possibly accounts for diversity of PLGA solvent solubility. PLGA may thus effectively be used to complex both EcoRII and nonoxynol-9 by a two step emulsion of EcoRII first in PLGA; followed by a final emersion in nonoxynol-9.

Figure 4

This figure attempts to model the molecular binding of EcoRII to nonoxynol-9 or C31G (savvy) through the polyester PLGA. A. N-9 and EcoRII PLGA loaded nanoparticles: Note the orientation of the hydrogen and hydroxyl ions in the glycolic and lactic acids monomers of PLGA towards the hydroxyl and hydrogen ions in the N-9 and the REase nanoparticles model. The underlined dots signify that it is unknown which, covalent or hydrogen, bonds are involved. B. C31G and EcoRII PLGA loaded nanoparticles. Note that the chemical structure of Savvy is has been abbreviated to C31G, but is [C14H29 N(CH₃)₂O]_A^- + [C16H33 N (CH₃)₂CH₂COO]_B^-.