Elsevier

Virology

Volume 446, Issues 1–2, November 2013, Pages 365-377
Virology

Protein–protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells

https://doi.org/10.1016/j.virol.2013.08.006Get rights and content
Under a Creative Commons license
open access

Highlights

  • We studied protein–protein interactions among WNV non-structural proteins, including self-interactions, and three-protein complexes.

  • NS2B likely plays key role in bridging proteins of the replication complex.

  • A model of transmembrane complex formation with a replication complex was suggested based upon these studies.

  • We further posit that the oligomeric state of the four TPs in the ER membrane may facilitate the formation of the virus vesicles.

Abstract

To study the membrane orientation of flavivirus non-structural proteins (NSPs) in the replication complex, the seven major West Nile (WN) NSPs were separately expressed in monkey cells, and their subcellular localization was investigated by imaging-based techniques. First, we observed by confocal microscopy that four small transmembrane proteins (TP) (NS2A, NS2B, NS4A, and NS4B) were located to the endoplasmic reticulum (ER), whereas the largest NSPs, NS1, NS3, and NS5 were not. We then analyzed the colocalization and the association of WN NSPs using the methods of confocal microscopy, fluorescence resonance energy transfer (FRET), and biologic fluorescence complementation (BiFC). Through these combined imaging techniques, protein–protein interactions (PPI) among WNNSPs were detected. Our data demonstrate that there are interactions between NS2A and NS4A, and interactions of NS2B with three other TPs (NS2A, NS4A, and NS4B) as well as the expected interaction with NS3. PPI between NS2A and NS4B or between NS4A and NS4B were not detected. By the criteria of these techniques, NS5 interacted only with NS3, and NS1 was not shown to be in close proximity with other NSPs. In addition, homo-oligomerization of some NSPs was observed and three-way interactions between NS2A, NS4A, and NA4B with NS2B–NS3 were also observed, respectively. Our results suggest that the four TPs are required for formation of transmembrane complex. NS2B protein seems to play a key role in bringing the TPs together on the ER membrane and in bridging the TPs with non-membrane-associated proteins (NS3 and NS5).

Keywords

NS protein interactions
Transmembrane complex
Flavivirus replication complex

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