Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.

Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.

Publication date: Jan 23, 2024

Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al. , Cell Rep. 33, 108234 (2020); L. Miorin et al. , Proc. Natl. Acad. Sci. U. S.A. 117, 28344-28354 (2020); and M. Frieman et al. , J. Virol. 81, 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.

Concepts Keywords
Antiviral nuclear pore
Coronavirus nuclear transport
Nuclear ORF6
Quantitative SARS-CoV-2

Semantics

Type Source Name
disease MESH severe acute respiratory syndrome
disease IDO host
disease IDO cell

Original Article

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