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 06
CHEMISTRTRY 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 virus
infection-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
 INTRODUCTION

• Antiviral actions of interferons- IFN are molecules of a multigene family

of a vast group of glycoproteins collectively known as the cytokines-
Named so after their ability to interfere with viral replication (Blatt et al
1996, Diaz et al 1996)
• 1st IFN antiviral ability discovered in 1957 by Issacs and Lindemann on
their viral interference study-reported influenza infected chicken cells
secreted a factor that mediated the transfer of a virus resistant state
active against both homologous and heterogous viruses (Samuel CE 2001-
Clinical microbiology)
• This seminal observation along that of Nagano and Kujima 1958 set the
stage for subsequent studies that have led to the elucidation of the IFN
system in exquisite detail
• Type 1 IFNs play a critical role in the innate immune response to viral
infection-induction of numerous ISGs including important antiviral
molecules such as PKR,RNAse L,Mx- upto date 3 kinds of human IFNs have
been approved by FDA for clinical use (http://www.fda.gov/cber/products
 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 pathways
that 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 IFNs
have a common receptor having 2 subunits-IFNAR-1 and 2 (IFN α/β) –
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 experimental

cancer 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 samples
were 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 IFNαβ (IFN αβ-/-) 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 MEFs
isolated from ISG15+/+ (wild
type),ISG15-/- and STAT -/-
• Fig C and D- Serum cytokine
levels during course of
infection-3 days and 6 days
p.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 and
2.0 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 IFNαβ 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 during
viral 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 specific
viruses may lead to the development of potential therapies against these
important human pathogens
 THANK YOU

OPEN DISCUSSION