Inhibition of herpes simplex virus DNA synthesis by pokeweed antiviral protein

Abstract

Pokeweed antiviral protein at a concentration of 3 FM inhibited both the synthesis and release of infectious herpes simplex virus type 1 in cell culture by 90 and 99%, respectively. Addition of pokeweed antiviral protein to Vero cell monolayers before virus infection was 10 to 15% more effective in reducing virus yields than was the simultaneous addition of the antiviral protein with virus inoculum. Viral DNA synthesis was inhibited by 90% in cells which had been exposed to the antiviral protein, whereas cellular DNA synthesis was unaffected. No significant inhibition in the synthesis of the majority of viral infected-cell polypeptides was observed early postinfection (7 h), with the exception of infected cell polypeptides 4 and 41, whose syntheses were reduced by 38 and 25%, respectively. At 9 to 21 h postinfection, however, the synthesis of individual infected cell polypeptides was reduced by 48 to >99%.Pokeweed (Phytolacca americana) antiviral protein (PAP) is a basic protein with a molecular weight of 29,000 (29K) (9). PAP has been shown to inhibit the transmission of several plant viruses (20,22) as well as the multiplication of poliovirus (21), influenza virus (20) and herpes simplex virus (HSV) (1) in cell culture. PAP has also been reported to inhibit eucaryotic protein synthesis in cell-free systems derived from rabbit reticulocytes (13) and in poliovirus-infected HeLa cells (21). This inhibition is due to the enzymatic inactivation of ribosomes, which in turn primarily inhibits the action of elongation factor EF-2 (6, 13). In contrast, PAP causes little cytotoxicity to uninfected cells (11, 21), suggesting that PAP is unable to enter intact, uninfected cells. It has been postulated, therefore, that the antiviral activity of PAP is dependent upon. a virus-cell interaction which facilitates the entry of PAP into the host cell, followed by the inactivation of the ribosomes of the host cell by PAP (21).The results presented in this paper demonstrate that the antiviral activity of PAP against herpes simplex virus is due to a selective inhibition of viral DNA synthesis rather than an enzymatic inactivation of the ribosomes of the host cell. We also present evidence which indicates that (i) PAP inhibits not only the synthesis of infectious virus but also release from the infected cell and (ii) a PAP-cell interaction is an initial step in the antiviral mechanism of PAP, followed by a virus-cell interaction.

Polynucleotide: adenosine glycosidase activity of saporin-L1: effect on DNA, RNA and poly(A)

“…All of the biological properties of RIPs were attributed to the inactivation of ribosomes. However, some observations could not be equated with this view : (i) an effect of RIPs on viral DNA synthesis independent of the action of ribosomes was postulated by Teltow et al [8], who observed that pokeweed anti-viral protein (PAP) inhibits viral DNA synthesis to a much greater extent than cell protein synthesis in cells infected with the herpes simplex virus ; (ii) histological aspects of lesions induced by RIPs [9] are quite different from those obtained with other known protein synthesis inhibitors, and (iii) some aspects of the antiviral activity of RIPs could not be attributed to this property [10][11][12]. Furthermore, preliminary results showed that some saporins released adenine from all RNAs tested, and also from poly(A) and DNA, but did not affect ribomononucleotides, thus being actually polynucleotide :adenosine glycosidases [13].…”

A New Class of DNA Glycosylase/Apurinic/Apyrimidinic Lyases That Act on Specific Adenines in Single-stranded DNA

“…RIP Activity on pUC18 DNA-The amounts of protein and DNA are indicated in the figure legends. Reactions were carried out in 10 mM HEPES, pH 7.5, 0.1 mM ZnSO 4 , and the products were resolved by gel electrophoresis in 16 mM HEPES-KOH, 16 mM sodium acetate, 0.8 mM EDTA (18). For the inhibition study with NaBH 4 , a stock solution (1 M) was freshly prepared immediately prior to use.…”

“…RIP Activity on ODNs-1 g of gelonin or PAP or 2 g of ricin, in 10 mM HEPES, pH 7, 0.1 mM ZnSO 4 , was incubated at 37°C for 1 h with the appropriate oligonucleotide in a reaction volume of 10 l at protein/ DNA molar ratios of 2:1. To assay for DNA glycosylase activity in the absence of spontaneous cleavage, a post-treatment with 10 mM spermidine for 30 min at 37°C was performed.…”

“…20. The oligonucleotides were added after a preincubation for 5 min at 37°C of the proteins in the reaction buffer used above but supplemented with 10 mM NaCl or NaBH 4 . After 5 min at 37°C, the samples were post-treated with spermidine as above and subjected to SDS-PAGE on a polyacrylamide 4 -20% gradient gel after addition of loading buffer (0.1 M sodium phosphate, pH 6.0, 4% SDS, 10% glycerol, 2% ␤-mercaptoethanol, 0.01% bromphenol blue) and boiling.…”

“…The glycosylase/AP lyase-DNA covalent intermediate can be trapped by reduction of the imino intermediate with NaBH 4 (22) and visualized by autoradiography after SDS-PAGE when a 5Ј 32 P end-labeled ODN is used as substrate (20,22,(25)(26)(27)(28).…”