Abstract
Studies presented herein establish the antiviral activity of non-toxic derivatives of pure animal neurotoxins against HSV-1 infections in vivo and in vitro. Subsequent investigations delineated the molecular forms of the peptides responsible for the antiviral effect. Those antiviral forms of the peptide were also discovered to inhibit several protein kinases as well as HSV-1 protein synthesis, reactions that may reflect on the antiviral mechanism. Peptide-induced kinase inhibitions and the antiviral effect in vitro were both dependent on peptide concentration.The neurotoxoid protected suckling mice from experimental HSV-1-induced encephalitis by inhibiting virus replication in brain tissue. Protection appeared dose-dependent in this animal model. Cutaneous skin lesions in hairless mice were dramatically reduced by replicate peptide injections initiated after experimental infection.The neurotoxoid reduced the TCID(,50) of HSV-1 in BHK cells. This response was dose-related and was observed on adequate contact of the cells with peptide either before or after infection. Evidence also suggested the peptide undergoes turnover in the cells.Pretreatment of the cells with toxoid prior to challenge with HSV-1 resulted in a reduced uptake of fluorescein-conjugated HSV-1 antibodies. Likewise, the peptide inhibited ('3)H-leucine uptake into TCA-insoluble proteins of uninfected BHK cells, again a concentration dependent reaction.Both the antiviral and kinase-inhibitory effects are those of the peptide monomer, not of dimers and polymers. Some polymers can be dissociated to the active, monomeric form in dilute urea solutions. The oxidized toxoid monomer inhibited the cAMP independent protein kinase of myelin and the cAMP dependent heart kinase and its C-subunit. While the precursor peptide, the toxin, had no effect on the heart enzyme and its C-subunit, it did inhibit the myelin kinase in the presence of urea. The kinase inhibition reactions were non-competitive based on kinetic analyses and on the fact that neither peptide was phosphorylated by the enzymes.While a direct relationship between kinase inhibition and the antiviral response is not proven by these studies, the correlations between these responses to different forms of the peptide are suggestive. The relationships between protein phosphorylations, protein synthesis and subsequent virus replications should guide further studies into the peptide mechanisms.