Potent Inhibition of Late Stages of Hepadnavirus Replication by a Modified Cell Penetrating Peptide

Abstract

Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)8 having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)8 peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)8 caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)8 may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication

“…Because the human HBV has an extremely narrow host range, we used DHBV-infected PDH culture and Pekin ducks as model systems. The DHBV represents a reference model validated by us and others for the evaluation of novel anti-HBV approaches in preclinical studies 19, 20, 21, 22, 23, 24, 25, 26, 27…”

“…Hepatocytes were seeded on collagen-coated 12-well plates (Biocoat, Becton Dickinson, Heidelberg, Germany) at a density of 1 × 10 6 cells per well, and the growth medium was supplemented with 5% fetal calf serum and changed daily. Antisense PNAs (2 μM final) conjugated to CPPs or CPPs alone (2 μM final) were added to the culture medium daily starting from 2 hr post-infection, as previously described 21 . The PDHs were treated during 6 days and lysed on day 7, followed by viral replication analysis.…”

The Antimicrobial and Antiviral Applications of Cell-Penetrating Peptides

“…While trying to improve the effi ciency of their CPP-PNA conjugate [ 110 ] by adding a fatty acid domain, they created a CPP, which was an effi cient antiviral agent even without the conjugated PNA. The infected cells treated with the peptide also demonstrated the tendency to release naked viral nucleocapsids that are extremely immunogenic, which could be used for vaccine-like immune system stimulation in the infected organism [ 111 ].…”

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

“…In this regard, increasing evidences indicate that CatLip, resulting from chemical conjugation of CPPs to a lipid domain (such as fatty acids), have improved cellular uptake and endosomal escape [ 30 , 31 ]. Our recent discovery of anti-DHBV activity exhibited by oligoarginine, encouraged us to evaluate different CatLip for their ability to inhibit viral replication in vitro , in primary duck hepatocytes cells (PDHs) and in stably DHBV-transfected chicken hepatoma cells (LMH-D2) [ 32 ].…”

“…Thus, such PNA-CatLip conjugates appear as pertinent anti-HBV agents. Interestingly, we have recently showed that CatLip peptides alone, in the absence of their PNA cargo, were able to inhibit viral replication [ 32 ].…”