Effects of oligoribonucleotides-D-mannitol complexes on the - PowerPoint PPT Presentation

Effects of oligoribonucleotides-D-mannitol complexes on the hemagglutinin-glycan interactions Natalia Melnichuk 1, *, Zenoviy Tkachuk 1 , and Maryna Vivcharyk 1 1 Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine 150, Zabolotnogo Str., Kyiv - 143, Ukraine, 03680. * natalia.melnichuk8@gmail.com 1

Effects of oligoribonucleotides-D-mannitol complexes on the hemagglutinin-glycan interactions Our results showed that oligoribonucleotides-D mannitol complexes binds to HA of influenza virus and in this manner inhibit hemagglutinin- glycan interactions and reduces influenza A virus infectivity. Agglutination of the human red blood cells 0 (I) by influenza virus with and without RNA drugs * * Adapted to – Gopinath S.C.B,. Kumar P.K.R / Acta Biomaterialia (2013) 2

Abstract: The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. Oligoribonucleotides (ORNs) – have a wide range of biological activities and can be used in antiviral treatments since they play a key role in antiviral activity and can change a conformation of some proteins. However, the mechanism of ORNs-D-mannitol antiviral activity is still not clear. In this work was studied interactions between RNA drugs and НА , effect of RNA drugs on the НА -glycan interactions activity, the influenza A virus infectivity and viability cells infected with the influenza A virus in vitro . It was shown that decrease of HA activity and infectious titer of influenza A (A/FM/1/47(H1N1)) virus by a factor 4 and 2 lgTCID 50 respectively were observed after incubation of virus with ORNs-D-mannitol in comparison to the virus control. Reduction of the fluorescence intensity of HA of flu virus in the presence of the ORNs-D-mannitol was observed. This effect may indicate that interactions between HA and ORNs-D-mannitol are responsible for conformation changes of HA. Keywords: hemagglutinin; influenza A virus; RNA drugs 3

Introduction Single-stranded DNA (ssDNA) or RNA sequences can bind with high affinity and specificity to a wide range of target molecules, such as proteins, cell surface receptors, and even whole cells as well as other organic or inorganic molecules such as ATP, dyes, amino acids, drugs, or simple small. Theoretically, they can be used therapeutically in any disease for which extracellular blocking of protein – protein interactions is required. Yeast total RNA (ORNs) with the dominant fraction of 3-8 nucleotides has antiviral activity and can change a conformation of some proteins. The HA protein of influenza virus, a trimeric spike on the viral membrane, is responsible for hemagglutination and binding of the virus particles to the susceptible host cells. This binding mediates the subsequent entry of influenza viruses into host cells through membrane fusion, which is significant for initial viral infection. 4

Results and discussion RNA drugs effect on the infectivity of influenza A virus Effect of RNA drugs on the infectivity of influenza A virus was The infectious titer of virus, lgTCID 50 /0,1ml 9 7,8 studied on MDCK cells. The influenza A 7,1 8 virus incubated with ORNs (2,5 mg/ml) 5,8 7 and ORNs-D-mannitol (3,5 mg/ml) 30 6 min 20℃. 5 The infectious titer of influenza A virus 4 was determined by cytopathic effect 3 2 assay 48 hours after the influenza A 1 virus infection. 0 It was shown that the control influenza influenza virus influenza virus infectious titer of influenza A virus virus +ORNs +ORNs-D-mannitol decreased by 2 lgTCID 50 after incubation of virus with ORNs-D-mannitol at the Decrease of the influenza A virus infectious titer concentration 3,5 mg/ml in comparison after incubation with RNA drugs to the virus control. 5

Results and discussion RNA drugs effect on the viability of MDCK cells infected with the influenza A virus The influenza A virus was incubated 30 min 20℃ with ORNs and ORNs-D- mannitol at the concentration 2,5 mg/ml and 3,5 mg/ml respectively. Cell viability was determined by MTT-test 48 hours after the influenza A virus infection. By suppressing influenza A viral infection, oligoribonucleotides-D-mannitol complexes increase cell viability at the concentration 3,5 mg/ml. 120 87,2 100 97,48 100 Cell viability, % 80 64,9 60 40 20 0 control cells control influenza virus influenza virus+ORNs influenza virus+ORNs-D- mannitol Enhanced viability of MDCK cells, which have been infected by the influenza virus, thanks to RNA drugs 6

Results and discussion RNA drugs effect on HA-glycan interactions activity 300 HA titer of virus, HA units/0,1ml 256 Effect of RNA drugs on the 250 infectivity of influenza A virus was 200 studied on human red blood cells 0 (I). The influenza A virus was incubated with 150 128 ORNs (2,5 mg/ml) and ORNs-D-mannitol (3,5 mg/ml) 30 min 20℃. HA-glycan 100 interactions activity was determined 64 by agglutination assay. 50 Decrease of HA activity of influenza 0 A virus by a factor 4 was observed after control influenza influenza virus influenza virus virus +ORNs +ORNs-D-mannitol incubation of virus with ORNs-D- mannitol at the concentration 3,5 mg/ml Inhibition of agglutination in comparison to the virus control. of the human red blood cells 0 (I) by influenza A virus which were incubated with RNA drugs 7

RNA drugs effect Results and discussion on the fluorescence intensity of influenza A virus HA Fluorescence spectra HA under influence of the Fluorescence spectra HA ORNs-D-mannitol under influence of the ORNs 1600 Hemagglutinine+ORN:D-mannitol HA 4,3  1400 0,25  4,5  Hemagglutinine+ORN 1400 0,5  4,7  HA 4,3  0,74  5  0,25  4,5  1200 0,99  5,2  0,5  4,7  Fluorescence intensity, a.u. Fluorescence Intensity, a.u. 1200 0,74  5  1,23  5,4  0,99  5,2  1,48  5,6  1000 1,23  5,4  1,72  5.9  1000 1,48  5,6  1,96  6.1  1,72  5.9  2,2  6.3  1,96  6.1  800 2,44  6.5  2,2  6.3  800 2,67  6.7  2,44  6.5  2,67  6.7  2,9  7  600 2,9  7  3,2  7,2  600 3,2  7,2  3,38  7,4  3,38  7,4  3,6  7,6  3,6  7,6  400 400 3,8  4,8  3,8  4,8  4,1  4,1  200 200 0 0 300 320 340 360 380 400 420 440 300 320 340 360 380 400 420 440 Wavelength, nm Wavelength, nm Dependence of the fluorescence intensity Dependence of the fluorescence intensity of the HA on ORNs-D-mannitol concentration of the HA on ORNs concentration 1600 1400 1500 Intensity at 335 nm Intensity at 335nm ORN ORN:D-mannitol 1300 1400 1200 1300 1200 Kd=4,91E-6±9,19E-7 1100 Kd=4,41E-5±2,37E-5 0,0000000 0,0000015 0,0000030 0,0000045 0,0000060 0,0000075 0,0000000 0,0000015 0,0000030 0,0000045 0,0000060 0,0000075 Concentration (ORN:D-mannitol) ,M Concentration (ORN) , M 8 8

RNA drugs effect Results and discussion on the fluorescence intensity of influenza A virus HA The fluorescence melting curves were obtained from fluorescence spectra of protein-RNA solutions. For estimating the dissociation constant (K d ) next formula was used where ϴ is the fraction of Spectrofluorometer Jasco FP-8200 bound to total protein at the stoichiometric point and P 0 is the total protein concentration in the cuvette. It was observed that the fluorescence intensity of HA of flu virus reduced in the presence of the ORNs-D-mannitol. This effect may indicate that interactions between HA and ORNs-D-mannitol are responsible for conformation changes of HA. 9

Conclusions • Oligoribonucleotides-D-mannitol complexes reduce influenza A virus infectivity affecting the hemagglutinin-glycan interactions in vitro . • By suppressing influenza A viral infection, oligoribonucleotides-D- mannitol complexes increase cell viability in vitro . • Complexes of Oligoribonucleotides-D-mannitol interact with hemagglutinin of the influenza A virus, and in this manner inhibit HA- glycan interactions. 10