Restriction of Influenza virus multiplication induced by PK peptide treatment

Restriction of Influenza virus multiplication induced by PK peptide treatment G. Conti, P. Portincasa, S. Conti, V. Magliani, L. Polonelli Dept. of Pathology and Lab. Medicine, Microbiology Section, Univ. Parma Gramsci 14, 43100 Parma, Italy Confluent monolayers of LLC-MK2 cells were infected with Ulster 73 virus at a m.o.i. of 20 p.f.u. in pre-warmed phosphate buffer saline ( P.B.S.) pH 7.4. After a 40 minute adsorption period , LLC-MK2 cells were treated with different amounts of PK which were left for the duration of the whole experiment. PK diluent was added to the maintenance medium in infected untreated cells from the beginning of infection as a control. After 24 hours, the supernatant of infected and treated cell monolayers was collected, centrifuged at 2,000 x g to remove cell debris and assayed by HA titration. The compound did not interfere with virus-specific receptors located on red blood cells. Virus production was also determined in a different set of experiments in which a molecule called SK was used as negative internal control. Virus titers were determined in triplicate samples either by measuring the hemagglutination activity ( HA ) in the culture medium of infected and treated LLC-MK2 cells . In the first set of experiments the effect of PK on Ulster 73 multiplication was determined in the permissive LLC-MK2 host cells by HA assay . Cellular monolayers were infected with Ulster 73 virus at the multiplicity of infection of 20 pfu / cell . The aim was to define the concentration of the drug completely inhibiting viral growth : 10, 20, 40, 60, 70, 80 g / ml were the amounts of PK added to the maintenance medium from the beginning of the infectious cycle . PK was able to suppress virus replication in a dose-dependent fashion : 10, 20, 40 and 60 μg/ml. did not interfere with virus growth, whereas at 70 g /ml there was an interfering activity and at 80 g /ml virus production was completely blocked . Such inhibiting effect was absolutely absent in infected cells treated with SK. Similar results of inhibition were obtained using other cellular types such as MDCK and AGMK 37RC cellular monolayers treated with PK. In a different set of experiments the multiplication of Ulster 73 strain was assessed in LLC-MK2 cells treated with 80 g / ml of PK and, respectively, different amounts of compounds added at the same time at the beginning of the infectious cycle; the haemagglutination assay test, HA, was used to measure total infectious virus production at various times after the beginning of treatment until 24 hours p.i. In particular, PK and Laminarin were preincubated for 10 minutes ( 80 µl/ml of PK and 64 μl/ml of Laminarin ) and then added, at the beginning of the infectious cycle, to the maintenance medium of LLC-MK2 cells infected by influenza virus. In an other experiment PK and Laminarin were added together ( without preincubation ) to the maintenance medium of infected LLC-MK2 cells at the 0 time( i.e: at the end of the absorption period ) and then left for the whole experiment. The same experiments were done with SK. The results were consistent with production of virus particles in condition of PK as well as SK treatments suggesting an interfering action performed by Laminarin. In fact, PK alone inhibited virus production whereas SK alone had no effect. These results were obtained by HA assayes at 24 hours p.i. In order to verify the proper biological role of virus–coded haemagglutinin ,HA, polypeptide a set of experiments involving the haemadsorption assays on monolayers of both untreated or PK-treated LLC-MK2 cells infected by Ulster 73 virus was performed . This was necessary because the haemadsorption of red blood cells correlates not only with the integrity of the glycosylated haemagglutinin molecule but also with its correct insertion into the cellular membrane of the infected cells. The values of haemadsorption were expressed as optical density measurements at 420 nm.obtained after a very long period , 24 hours from the beginning of the infectious cycle , in treated as well as PK–untreated monolayers of infected LLC-MK2 cells. Significant differences between untreated versus treated - infected cells are clearly observed: in fact , the haemadsorption values obtained indicate a great reduction of virus-coded haemagglutinin molecules on the plasma membrane of Ulster 73-infected cells treated with 80μg./ml.of PK . For comparison , the results referring to LLC-MK2 cells other than MDCK (Madin Darby Canine Kidney) and AGMK-37RC (African Green Monkey Kidney) cellular types all permissive to the infection with Ulster 73 virus , treated with the same inhibiting concentration of PK, showed such the inhibiting effect in which there was a great reduction of viral haemagglutinin molecules on the cytoplasmic membrane of infected cells. All these experiments were performed with type A influenza virus Ulster /73, H7N1, which is an avian strain. PK, at 80 μg/ ml, was also effective against type A, NWS/33,H1N1, a neurovirulent human strain . Since after 24 hours from the beginning of the infectious cycle influenza virus seems to escape from the inhibiting action performed by PK, a set of experiments was performed in which different amounts of PK ( 10,20,40,60,70,80µg/ml ) were added to the maintenance medium at intervals of 12 hours starting from 24 hrs. post infection ( p.i.) until 72hrs p. i. In these conditions the inhibiting action of PK, added at 80 μg/ml, was restored for whole experiment and the values of HA were negative. On the contrary, no inhibiting effects were shown by SK treatment. 14C-mannose pulse-labelling experiments of LLC-MK2 cells infected by Ulster 73 virus in presence of PK from the beginning of the infection, showed a marked reduction of glycosylation of haemagglutinin HA, suggesting a possible interfering mechanism performed by PK The restriction on viral multiplication, the evidence that lowering the multiplicity of infection PK acts at a lower concentration, the interfering action on HA glycosylation, seem results of interest also taking into consideration that PK peptide has an high sequence omology with the antibody recognizing the fusion peptide of virus-coded HA.