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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 complex In: Positive-Strand Rna Viuses
N.B. When citing this work, cite the original published paper. Permanent link to this version:
Pham Tue Hung Tran1, Naveed Asghar1, Roger Karlsson2, Ander Karlsson2, Magnus Johansson1, Wessam Melik1 1School of Medical Sciences, Örebro University, Örebro, Sweden
2Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; Nanoxis Consulting AB, Gothenburg, Sweden
Contact person: hung.tran@oru.se
However, it is little-known whether host proteins can also participate in the formation and maintenance of these compartments. Recently, it has been shown that the host protein RTN3.1A is important for rearrangement of ER membrane, and stabilization of RC (2).
Experiments
Screening of host proteins interacting with Kunjin, Langat, Zika
replication complex
Infect A549 cells with KUNV, LGTV, ZIKV TMT-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 function in 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
LGTV
KUNV
Aims
Enrichment of RCs by Co-immunoprecipitation
In this project, we attempted to identify host proteins that interact with RC and function in the formation of replication compartments during Flavivirus infection.
Because the MS data from using the ER membrane of Flavivirus-infected cells may contain false positive results, we attempted to enrich the ER fractions having RC by CoIP with anti NS1 Flavivirus antibodies. We then will identify the proteins by a quantitative MS technique, TMT-MS.
The candidate proteins will be characterized by using molecular techniques such as gene knock down, overexpression, and microscopy techniques.
Total ER LGTV KUNV ZIKV Total ER Non infection A D C B
Conclusions and future perspectives
This work was supported by grants to Magnus Johansson by the Knowledge Foundation.
Acknowledgements
Introduction
Figure 1: A549 cells were infected with 0.01 MOI of ZIKV (A), LGTV (B), KUNV (C) or not infected (D). After 4-6 days, cells were fixed and
immuno-stained with anti
dsRNA antibody
indicating virus
replication and the formation of RC. Cells then were harvested and lysated to purify the ER membranes.
Scale bar is 10 µm.
Figure 2: Western Blot analysis of cell lysates and purified ERs. 8ug of
proteins were loaded each well.
Membranes were labeled with anti-NS1 antibodies for TBEV and KUNV infected samples, anti-NS5 antibody for ZIKV infected samples. There were clear enrichments of viral proteins in the purified ERs. These ER samples were used for MS analysis.
Figure 3: The purified ER samples were analyzed by MS to identify host proteins expressed during Flavivirus infections. Compared to non-infected samples, we identified 168, 636, and 447 host proteins expressed in LGTV, KUNV, ZIKV infected ER, respectively. There were 73 host proteins expressed in both infected samples. However, we cannot exclude the false positive results from this analysis.
50kDA- 80kDA-
115kDA-NS1 NS5 Input IP Input IP Input IP
LGTV KUNV ZIKV
Bead GFP-taged bead
Figure 4: Western Blot analysis of the ER inputs and the elutions after IP using beads tagged with anti-NS1 antibodies. 500 ng of proteins were loaded each well. There were significant enrichments of viral proteins following Co-IP. These samples will be used for TMT-MS analysis.
(1) Apte-Sengupta S, Sirohi D, Kuhn RJ. Coupling of replication and assembly in flaviviruses. Current opinion in virology. 2014;9:134-42.
(2) Aktepe TE, Liebscher S, Prier JE, Simmons CP, Mackenzie JM. The Host Protein Reticulon 3.1A Is Utilized 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 Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts. Current protocols in cell biology. 2015;68:3.27.1-33.
During infection and eclipse time, Flaviviruses induce invagination of the endoplasmic reticulum (ER) membrane to form compartments, anchoring their viral replication complexe (RC). The rearrangements of ER membrane require modifications in ER membrane lipid constituents or binding of proteins to bend the ER membrane. It was suggested that transgenetically expressing Flaviviral proteins NS1, NS2A, NS4A, NS4B could remodel the ER membrane (Reviewed in
