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

Effects of oligoribonucleotides-D-mannitol complexes

• n the hemagglutinin-glycan interactions

Natalia Melnichuk1,*, Zenoviy Tkachuk1, and Maryna Vivcharyk1

1Institute of Molecular Biology and Genetics, National Academy of Sciences of

Ukraine 150, Zabolotnogo Str., Kyiv - 143, Ukraine, 03680.

*natalia.melnichuk8@gmail.com

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Effects of oligoribonucleotides-D-mannitol complexes on the hemagglutinin-glycan interactions

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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)

Our results showed that

• ligoribonucleotides-D mannitol

complexes binds to HA of influenza virus and in this manner inhibit hemagglutinin- glycan interactions and reduces influenza A virus infectivity.

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 lgTCID50 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

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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,

• r simple small. Theoretically, they can be used

therapeutically in any disease for which extracellular blocking

• f

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

• f some proteins.

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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.

Results and discussion

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7,8 7,1 5,8 1 2 3 4 5 6 7 8 9

control influenza virus influenza virus +ORNs influenza virus +ORNs-D-mannitol

The infectious titer of virus, lgTCID50/0,1ml

Decrease of the influenza A virus infectious titer after incubation with RNA drugs

RNA drugs effect on the infectivity of influenza A virus

Effect of RNA drugs on the infectivity of influenza A virus was studied on MDCK cells. The influenza A virus incubated with ORNs (2,5 mg/ml) and ORNs-D-mannitol (3,5 mg/ml) 30 min 20℃. The infectious titer of influenza A virus was determined by cytopathic effect assay 48 hours after the influenza A virus infection. It was shown that the infectious titer of influenza A virus decreased by 2 lgTCID50 after incubation

• f virus with ORNs-D-mannitol at the

concentration 3,5 mg/ml in comparison to the virus control.

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RNA drugs effect on the viability of MDCK cells infected with the influenza A virus

Enhanced viability of MDCK cells, which have been infected by the influenza virus, thanks to RNA drugs

100 64,9 87,2 97,48 20 40 60 80 100 120

control cells control influenza virus influenza virus+ORNs influenza virus+ORNs-D- mannitol

Cell viability, %

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.

Results and discussion

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RNA drugs effect

• n HA-glycan interactions activity

256 128 64 50 100 150 200 250 300 control influenza virus influenza virus +ORNs influenza virus +ORNs-D-mannitol

HA titer of virus, HA units/0,1ml

Inhibition of agglutination

• f the human red blood cells 0 (I) by influenza A

virus which were incubated with RNA drugs

Effect of RNA drugs on the infectivity of influenza A virus was studied on human red blood cells 0 (I). The influenza A virus was incubated with ORNs (2,5 mg/ml) and ORNs-D-mannitol (3,5 mg/ml) 30 min 20℃. HA-glycan interactions activity was determined by agglutination assay. Decrease of HA activity of influenza A virus by a factor 4 was observed after incubation

• f

virus with ORNs-D- mannitol at the concentration 3,5 mg/ml in comparison to the virus control.

Results and discussion

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Results and discussion

300 320 340 360 380 400 420 440 200 400 600 800 1000 1200 1400

Fluorescence intensity, a.u. Wavelength, nm

Hemagglutinine+ORN HA 4,3 0,25 4,5 0,5 4,7 0,74 5 0,99 5,2 1,23 5,4 1,48 5,6 1,72 5.9 1,96 6.1 2,2 6.3 2,44 6.5 2,67 6.7 2,9 7 3,2 7,2 3,38 7,4 3,6 7,6 3,8 4,8 4,1

300 320 340 360 380 400 420 440 200 400 600 800 1000 1200 1400 1600

Hemagglutinine+ORN:D-mannitol HA 4,3 0,25 4,5 0,5 4,7 0,74 5 0,99 5,2 1,23 5,4 1,48 5,6 1,72 5.9 1,96 6.1 2,2 6.3 2,44 6.5 2,67 6.7 2,9 7 3,2 7,2 3,38 7,4 3,6 7,6 3,8 4,8 4,1

Fluorescence Intensity, a.u. Wavelength, nm

0,0000000 0,0000015 0,0000030 0,0000045 0,0000060 0,0000075 1100 1200 1300 1400

Kd=4,41E-5±2,37E-5 Intensity at 335nm

Concentration(ORN), M ORN

0,0000000 0,0000015 0,0000030 0,0000045 0,0000060 0,0000075 1200 1300 1400 1500 1600

Kd=4,91E-6±9,19E-7 Intensity at 335 nm Concentration(ORN:D-mannitol),M

ORN:D-mannitol

RNA drugs effect

• n the fluorescence intensity of influenza A virus HA

Fluorescence spectra HA under influence of the ORNs Fluorescence spectra HA under influence of the ORNs-D-mannitol Dependence of the fluorescence intensity

• f the HA on ORNs concentration

Dependence of the fluorescence intensity

• f the HA on ORNs-D-mannitol concentration

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Results and discussion

The fluorescence melting curves were

• btained from fluorescence spectra of protein-RNA

solutions. For estimating the dissociation constant (Kd) next formula was used where ϴ is the fraction of bound to total protein at the stoichiometric point and P0 is the total protein concentration in the cuvette.

Spectrofluorometer Jasco FP-8200

RNA drugs effect

• n the fluorescence intensity of influenza A virus HA

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.

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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
• f

Oligoribonucleotides-D-mannitol interact with hemagglutinin of the influenza A virus, and in this manner inhibit HA- glycan interactions.