IFN-stimulated gene 15 (ISG15) functions as

8/7/2019 IFN-stimulated gene 15 (ISG15) functions as

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IFN-stimulated gene 15 (ISG15) functions as a critical antiviral molecule

against influenza, herpes and Sindbis viruses

Deborah J. Lenschow et al (2007).-Washington University School of

Medicine

MUSYOKA THOMMAS MUTEMI

55610082

2010 JANUARY 06CHEMISTRTRY OF BIOFUNCTIONAL MOLECULES FOR INFECTIOUS

DISEASES 2

LECTURES: PROF.KAI and PROF KABASHIMA



HYPOTHESIS

� ISG 15 has an antiviral activity against RNA and DNA viruses in

vivo and mice lacking ISG15 have increased susceptibility to

virus infection.



OBJECTIVES

� Determination of in vivo ISG15 expression after virus

infection-comparison between wild type mice models

(ISG15+/+ ) and mutant forms (ISG15-/-)

�

Evaluate the susceptibility to different types of virusinfection-Between wild type mice models (ISG15+/+ )and

mutant forms (ISG15-/-)

� Determination if single point mutation on the C terminal motif

of ISG15 had any effect to its antiviral properties





Cont-Introduction

� ISG15 a ubiquitin homologue (15 Kda) is up regulated after viral infection

and conjugates to a wide array of host proteins constituting to the ISG15

pathway-but its activity is tightly regulated by specific signaling pathwaysthat have a role in innate immunity

� ISG15 intracellular conjugation (ISGlylation) to several host proteins

enhances cellular response to interferons-This is unlike ubiquitination

which targets proteins for degradation

�IFNs exert their function via a cognate cell surface receptors-Type 1 IFNshave a common receptor having 2 subunits-IFNAR-1 and 2 (IFN /)

pathogenesis studies with mice in which the receptor function has been

disrupted via target gene disruption shows their inability to establish an

antiviral state.



pres.doc-INF antiviral activity-schematic diagram presentation



Materials and Methods

� Mice models-obtained from the M.Aguet,Swiss Institute of experimentalcancer research

� Viruses-Influenza A virus (A/wsn/33),Influenza B virus (B/lee/40), HSV-

1,HV68,Sindbis viruses were used.

� Viral studies-depending on the virus type different standard protocols

were used as mentioned� Cytokine analysis (Quantification and qualification)-At the indicated time

periods serum cytokine levels were analyzed using cytometric bead array

mouse inflammation kit as directed by the manufacturer

� In vivo induction and analysis of ISG15-the different groups of mice

models were infected with the different types of viruses-lung sampleswere homogenized and mixed with SDS loading buffer and boiled for 30

min before western blot analysis-ISG15 expression was detected with anti-

ISG15 mAb-developed with goat anti-American hamster horseradish

peroxidase secondary Ab.For loading controls parallel probes were

probed with anti-b-actin mAb.



ISG 15 is induced in vivo after influenza infection

� Fig A and B-A westen blot

analysis of ISG 15 expression-Lung homogenates from ISG15+/+

or ISG15-/- that were mock

infected (M) or infected with

influenza virus A or B.

� Fig C and D-grow curve and

lethality curve-To determine the

susceptibility of the different

groups to virus infection-and also

to establish whether ISG15

accounted for all of the antiviral

effects- susceptibility of ISG15-/-

and IFN (IFN -/-) null mice

was compared

� Fig E and F -Lethality assessment

using different virus titers.



Characterization of influenza pathogenesis in ISG15+/+ or ISG15-/-

� Fig A- virus titer

determination by pfu assay

of lung homogenates

� Fig B- In vitro determination

of viral growth in MEFsisolated from ISG15+/+ (wild

type),ISG15-/- and STAT -/-

� Fig C and D- Serum cytokine

levels during course of

infection-3 days and 6 daysp.i respectively.



Determination of the broad spectrum activity of ISG 15 using - 1)HSV virus

� Fig A and B- Lethality

determination between

ISG15+/+ and ISG15-/-

after intracranial

infection of 20 p.f.u and2.0 p.f.u of HSV-1

� Fig C and D-evaluation

of a more physiological

route of infection-

corneal infection

� Fig E and F-

Determination whether

ISG15 is important for

resistance across

herpes virus



Demonstration that intact LRLRGG motif is required for ISG15 antiviral

activity

� Supp data- over expression of ISG15

by recombinant double sub genomic

Sindbis virus-results into protection

of IFN null mice from virus

infection. Fig A-Susceptibility

determination of ISG15+/+ or ISG15-

/- to Sindbis virus infection

� The use of a recombinant chimeric

virus-opportunity to define the aa

sequence within ISG15 required for

antiviral activity against Sindbis virus

� Fig B-Lethality evaluation.ISG15-/-

infected with dsTE12Q expressing

either WT ISG15-LRLRGG (filled

triangles)or mutant ISG15-LRLRAA

(filled circles)



Discussion

� IFNs are the most important early defense molecules against acute virus

infection

� This work defines ISG15 as an important host IFN-induced antiviral protein

that functions in vivo against several important human pathogens

� The loss of ISG15 results in increased susceptibility to both RNA and DNA

viruses

� The antiviral activity of ISG15 may be as a result of its cytokine activity or

its ability to conjugate to target proteins or b oth

� Intact LRLRGG motif is required for the antiviral capability of ISG15.

� In an attempt to evaluate ISG15 action mechanism,recombinant chimeric

Sindbis virus system was used to identify the aa required for antiviral

activity-mutation at the c terminal motif abrogated protection hence this

motif is critical for conjugation of ISG15 to target proteins

� Recent studies have identified >100 proteins targeted for

ISG15conjugation that encompass multiple cellular pathways



� Interestingly included in these target proteins are several known IFN

induced antiviral molecules such as PKR,Mx,RIGI and GBP-1

�

ISGylation of these antiviral molecules may regulate their activity duringviral infection

� Also ISG15 may conjugate to viral proteins within infected cells and alter

their function or localization



Recommendation

� Identification which proteins are altered by ISGylation during viral

infection, the fate of the proteins after conjugation to ISG15 and the role

of conjugation in its putative cytokine will be required to determine how

ISG15 functions in vivo as an antiviral molecule

�Definition of the molecular action mechanisms of ISG15 against specificviruses may lead to the development of potential therapies against these

important human pathogens



THANK YOU

OPEN DISCUSSION

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