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Postprint

This is the accepted version of a paper presented at Positive-Strand RNA Viuses,KILLARNEY, Co.Kerry, Ireland, June 9-13,2019.

Citation for the original published paper:

Tran, P T., Asghar, N., Karlsson, R., Karlsson, A., Johansson, M. et al. (2019)Screening of host proteins interacting with Kunjin, Langat, Zika replication complexIn: Positive-Strand Rna Viuses

N.B. When citing this work, cite the original published paper.

Permanent link to this version:http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-76400

Pham Tue Hung Tran1, Naveed Asghar1, Roger Karlsson2, Ander Karlsson2, Magnus Johansson1, Wessam Melik1

1School of Medical Sciences, Örebro University, Örebro, Sweden2Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; Nanoxis Consulting AB, Gothenburg, SwedenContact person: hung.tran@oru.se

However, it is little-known whether host proteins can also participate in the formationand maintenance of these compartments. Recently, it has been shown that the hostprotein RTN3.1A is important for rearrangement of ER membrane, and stabilization ofRC (2).

Experiments

Screening of host proteins interacting with Kunjin, Langat, Zikareplication complex

Infect A549 cellswith KUNV, LGTV,

ZIKVTMT-MS

Mass Spectrometry (MS)

Enrichment of RC by Co-IP

Extract ERs by ultracentrifugation

(3)

ER purification by ultracentrifugation

References

In this project, we aimed to identify of host proteins that interact with RC and functionin the formation of replication compartments.

Identification of host protein expression in infected cells

Virus replication in infected cells

50kDA- NS1

Total ER Total ER

NS1

80kDA-115kDA- NS5

ZIKV

LGTVKUNV

Aims

Enrichment of RCs by Co-immunoprecipitation

In this project, we attempted to identify host proteins that interact with RC and functionin the formation of replication compartments during Flavivirus infection.

Because the MS data from using the ER membrane of Flavivirus-infected cells maycontain false positive results, we attempted to enrich the ER fractions having RC by CoIPwith anti NS1 Flavivirus antibodies. We then will identify the proteins by a quantitativeMS technique, TMT-MS.

The candidate proteins will be characterized by using molecular techniques such as geneknock down, overexpression, and microscopy techniques.

Total ER LGTV KUNV ZIKV

Total ER

Non infection

A

DC

B

Conclusions and future perspectives

This work was supported by grants to Magnus Johansson by the Knowledge Foundation.Acknowledgements

Introduction

Figure 1: A549 cellswere infected with0.01 MOI of ZIKV (A),LGTV (B), KUNV (C) ornot infected (D). After4-6 days, cells werefixed and immuno-stained with antidsRNA antibodyindicating virusreplication and theformation of RC. Cellsthen were harvestedand lysated to purifythe ER membranes.Scale bar is 10 µm.

Figure 2: Western Blot analysis of celllysates and purified ERs. 8ug ofproteins were loaded each well.Membranes were labeled with anti-NS1 antibodies for TBEV and KUNVinfected samples, anti-NS5 antibodyfor ZIKV infected samples. There wereclear enrichments of viral proteins inthe purified ERs. These ER sampleswere used for MS analysis.

Figure 3: The purified ER sampleswere analyzed by MS to identify hostproteins expressed during Flavivirusinfections. Compared to non-infectedsamples, we identified 168, 636, and447 host proteins expressed in LGTV,KUNV, ZIKV infected ER, respectively.There were 73 host proteinsexpressed in both infected samples.However, we cannot exclude the falsepositive results from this analysis.

50kDA-

80kDA-115kDA-

NS1

NS5

Input IP Input IP Input IP LGTV KUNV ZIKV Bead GFP-

tagedbead

Figure 4: Western Blot analysis of the ER inputs and the elutions after IP using beadstagged with anti-NS1 antibodies. 500 ng of proteins were loaded each well. Therewere significant enrichments of viral proteins following Co-IP. These samples will beused for TMT-MS analysis.

(1) Apte-Sengupta S, Sirohi D, Kuhn RJ. Coupling of replication and assembly in flaviviruses. Currentopinion in virology. 2014;9:134-42.(2) Aktepe TE, Liebscher S, Prier JE, Simmons CP, Mackenzie JM. The Host Protein Reticulon 3.1A IsUtilized by Flaviviruses to Facilitate Membrane Remodelling. Cell reports. 2017;21(6):1639-54.(3) Williamson CD, Wong DS, Bozidis P, Zhang A, Colberg-Poley AM. Isolation of EndoplasmicReticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent ResistantMembrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected PrimaryFibroblasts. Current protocols in cell biology. 2015;68:3.27.1-33.

During infection and eclipse time, Flaviviruses induce invagination ofthe endoplasmic reticulum (ER) membrane to form compartments,anchoring their viral replication complexe (RC). The rearrangementsof ER membrane require modifications in ER membrane lipidconstituents or binding of proteins to bend the ER membrane. It wassuggested that transgenetically expressing Flaviviral proteins NS1,NS2A, NS4A, NS4B could remodel the ER membrane (Reviewed in(1)). Lesca,2018