Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

By Sue Abbasi and Amy Turner

Emergence of Marburg

August of 1967In Marburg, Germany, 3 laboratory workers became ill with a hemorrhagic disease after processing organs from African Green Monkeys. Total of 32 cases of whom 7 died.

Ebola Virus

1976A related virus emerged. It received its name from the Ebola river.In Zaire (now Congo) and Sudan2 distinct Ebola virus, Sudan ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV)In 1994, the third Ebola virus species originates. In Cote d’Ivore ebolavirus (CIEBOV).1994-2005

SymptomsIt causes severe viral hemorrhagic fever.The symptoms are internal bleeding, liver damage and dysfunction, disseminated intravascular coagulation, and shock.

Filovirus Structure.Marburg and Ebola viruses have enveloped virions 80 nm in diameter and of varying lengths.They have filamentous particles between 800 and 1200 nm long.The virions contain a negative-sense, single stranded RNA genome 19 kb long, wrapped in a helical nucleocapsid. .

The Structure of Marburg and Ebola

These viruses contain 7 genes.The gene coding for the nucleocapsid protein (NP) is located closest to the 3’ end of the genomeThe gene coding for the RNA polymerase (L) is located closet to the 5’ end.

NP-VP35-VP30-VP24-membrane protein. It is a interferon signaling antagonist.L Protein-VP40- Associates with membranes and resembles matrix protein. Also directs budding of virus envelopes at the plasma membrane to form virions.GP-

The Study This study shows that MARV and EBOV inhibit IFN-α/β signaling but utilize different pathways.Differences-Transcriptions strategiesThe structure of their replication promotersThe use of mRNA editing to express the surface glycoprotein by EBOV

IFN α/β and yIFN alpha and IFN gamma- antiviral cytokinesIFNα/β - Trigger innate antiviral defense mechanism and promote adaptive immunity.IFNy-modulate adaptive immune response.Both signaling results in tyrosine phosphorylation and activation of Janus kinases JAK1 and Tyk2. -pSTAT2 and pSTAT1 heterodimerize and associate with interferon regulatory factor (IRF9) to form a complex that is translocated to the nucleus to activate genes involved in antiviral response.IFNy signaling activates JAK1 and 2, resulting in tyrosine phosphorylation of STAT1. Homodimerization and translocation to the nucleus. Gene expression is induced.

MARV VIRUS BLOCKS THROUGH The JAK/STAT PATHWAYMARV blocks the phosphorylation of Janus Kinases and their target STAT proteins in response to type I and type II interferon and interleukin 6.

THE EBOV VIRUS BLOCKS IFN SIGNALINGEBOV inhibits IFN signaling through its EBOV VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. MARV inhibits IFN α/β- induced tyrosine phosphorylation of STAT1

and 2 by MARV VP40.MARV also inhibits IFNγ-induced STAT phosphorylation and tyrosine phosphorylation of upstream JAK by VP40.

Figure 1 MARV Infection prevents IFN-mediated phosphorylation and Nuclear translocation of STAT proteinsA. Huh-7 cells were infected with

MARV or ZEBOV . Then at 24 hours, cells were treated with IFN-α or IFN-γ. Western Blotting using antibodies against STAT 1 or STAT 2.

Goal; To demonstrate that tyrosine phosphorylation of STAT1 and STAT2 is reduced in MARV but not in ZEBOV.Results; MARV virus interferes with an early step of JAK/STAT

FIGURE 1CGoal; Immunofluorescence is used to determine the cellular localization of STAT.Results; EBOV and MARV both inhibit nuclear translocation of STAT1.

MARV Infection prevents IFN-mediated phosphorylation and Nuclear translocation of STAT proteinsI;;;;;

FIGURE 2 IFNα-induced tyrosine phosphorylation of Janus kinases is inhibited in MARV- but not in EBOV-infected

cells

.

• Huh -7 cells were used the same way then treated with IFN-α. Western Blotting was used.

• Goal; To see the effect of JAK 1 and TYK2.

• Results; Inhibition of JAK STAT signaling pathway by MARV takes place upstream of p-JAK.

FIGURE 2B Huh-7 cells were treated at different times with IFN-α to determine the activation of STAT 1 and TYK2.

GOAL; To discover at which time point does the MARV replication cycle occur. Result; It occurs at 4hThe binding of MARV to its receptor does not trigger the IFN antagonist function

Figure CMARV-infected and IFN-treated cells were treated with different PTPs; SO, PTP1B and DMSO. PTP1B- dephosphorylates Tyk2 and Jak2, SO-It inhibits multiple phosphatasesGoal; To determine whether MARV inhibits JAK1 phosphorylation through protein tyrosine phosphatases.Results; MARV inhibits p-Tyk2 therefore, inhibitory effects do not depend on active cellular PTPs.

Figure 3Vero cells were transfected with plasmids then the cells were mock treated or with IFNB and cells were infected with New Castle Disease Virus-NDV-GFP. This is IFN sensitive. GFP (green fluorescent protein) is used to determine where the gene is expressed.Control-pCAGGSNipah Virus W Protein (known inhibitors of IFN signaling)ZEBOV VP24 (known inhibitors of IFN signaling)ZEBOV VP40MARV VP24MARV VP30MARV VP35MARV VP40MARV NP

Figure 3. MARV VP40 acts as an IFN antagonistGoal; To identify the viral protein mediating the antagonistic effects observed in MARV infected cells. EBOV and MARV proteins where assessed for their capacity to counteract the antiviral effects of IFN B

Results; The only MARV protein tested that permitted the NDV-GFP replication in IFNB treated cells was MARV VP40

Figure 3BHuh-7 cells were transfected with different plasmids of viral proteins from rabies virus, ZEBOV, and MARV. After 24 hrs, they were stimulated with IFN alpha and Beta and stained with anti-STAT2 antibodies.Rabies virus phosphoprotein blocks the nuclear translocation of STAT2 into the nucleus.+ControlEmpty Vector is the –controlGoals; To confirm that MARV VP40 antagonizes IFN signaling through intracellular distribution of STAT2 in cells.Results; MARV VP40 and ZEBOV VP24 led to inhibition of STAT2 accumulation in the nucleus. MARV VP40 is the viral protein interfering with IFN signaling.

Figure 4 STAT1 or STAT2 fused to a C-terminal GFP in Huh-7 cells of different plasmids and treated IFNα/β . Cells were lysed and assayed by western blot for tyrosine phosphorylated STAT1 (p-STAT1-GFP), STAT2 (p-STAT2-GFP) and overexpressed proteins.Langat virus NS5 protein has demonstrated in the past that inhibits STAT1 and 2. It served as a control.

MARV VP40 inhibits IFN-induced STAT and Jak phosphorylation• Goals; Assess the impact of MARV VP40 on IFN induced signaling in the absence of

other viral proteins.• Results; MARV VP40 inhibited p-STAT1 and p-STAT2.

Figure 4BResults; MARV VP40 led to a reduction in IFNy induced STAT1 tyrosine Phosphorylation.

Figure 4C

• 293T cells were transfected with different plasmids and then treated with IFNα/β or IFNγ.

• Western blot analysis• Goal; What is the impact of MARV VP40

on the phosphorylation of Janus Kinases.

• Results; MARV VP40 inhibits both kinases. This shows that MARV VP40 uses a different mechanism than EBOV to block IFN signaling.

MARV VP40 inhibits ISRE- and GAS-induced gene expression

What is the functional significance of the observed inhibition?

Methods•Used a reporter gene assay to determine the impact of MARV VP40 on IFNβ and IFNγ-induced transcription•CAT gene •293 T cells used for transfection•MARV VP24 and ZEBOV VP24 were used as controls•ISG54 promoter (IFNα/β) contains an interferon stimulated response element (ISRE)•GAS element (three gamma activated sequence elements) activated by IFNγ

MARV VP40 inhibits ISRE- and GAS-induced gene expression

Assess the impact of MARV VP40 upon IFNγ-induced production of IP-10

IP-10 is an immune cell chemoattractant protein secreted by several cell types in response to IFNγ

MethodsHUVECs were transfected with expression plasmids, treated with IFNγ and cell supernatants were tested for the presence of IP-10 by ELISA

How specific is this effect?

MethodSimilar assay performed testing the impact of viral protein expression on TNFα-induced secretion of IL-8 which is mediated by the NF-κB signaling pathway

Conclusion

MARV VP40 is specific for Jak-STAT signaling and does not have an effect on the induction of NF-κB-mediated signaling

IL-6-induced activation of STAT1 and STAT3

Analyzing the effect that MARV VP40 has on IL-6-induced phosphorylation of STAT1 and STAT3

Why IL-6?Because in Jak1- cells, p-STAT1 and p-STAT3 as induced by IL-6 were greatly absent or reduced (respectively)

IL-6 signaling pathway is a non-IFN, Jak-STAT signaling pathway

Does MARV VP40 inhibit phosphorylation of over-expressed Jak1?

Over-expression of Janus kinases leads to their tyrosine phosphorylation and to the phosphorylation of STAT proteins

First: determine the phosphorylation state of HA-Jak1 and STAT2-GFP in transfected cells

Over-expression of HA-Jak1 led to tyrosine phosphorylation of endogenous STAT1 and STAT3 (Lane 2)

This phosphorylation was inhibited only by MARV VP40

Is the same result seen with over-expression of HA-Tyk2?

No inhibition of tyrosine phosphorylation of over-expressed HA-Tyk2 or subsequent STAT2-GFP phosphorylation by ANY of the filoviral proteins

Is the late domain of MARV VP40 critical for inhibition of signaling?

MARV VP40’s late domain (PPPY) Positioned residues 16-19Mediates VP40 interaction with the cellular protein Tsg101Contributes to its budding function

MethodsPPPY motif was mutated to AAAA (M40-AAAA)

Looked ForBudding (VLPs)Ability to suppress IFNα/β-induced signaling via STAT1 phosphorylationTranscription of the ISG54 promoter