Rhinovirus 16 2A protease affects nuclear localization of 3CD during infection

Erin WALKER, Sarah CROFT, Ke-jun Wei, David Jans, Alex J. Fulcher, Reena GHILDYAL

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Abstract

The human rhinovirus (HRV) 3C and 2A proteases (3Cpro and 2Apro, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3Cpro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2Apro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3Cpro, 3D, and mutant derivatives thereof, we show that 3Cpro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3Cpro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2Apro fusion proteins, we demonstrate formally that 2Apro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cpro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2Apro activity and not 3Cpro activity, which is observed only later in infection. The results thus define the temporal activities of 2Apro and 3CD/3Cpro activities in HRV serotype16 infection.
Original languageEnglish
Pages (from-to)11032-11042
Number of pages11
JournalJournal of Virology
Volume90
Issue number24
DOIs
Publication statusPublished - Dec 2016

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Human rhinovirus
Enterovirus
Rhinovirus
proteinases
Infection
Cytoplasm
infection
cytoplasm
Polyproteins
Virus Replication
virus replication
Green Fluorescent Proteins
green fluorescent protein
Human Activities
Proteins
proteins
Picornavirus picornain 2A
mutants

Cite this

WALKER, Erin ; CROFT, Sarah ; Wei, Ke-jun ; Jans, David ; Fulcher, Alex J. ; GHILDYAL, Reena. / Rhinovirus 16 2A protease affects nuclear localization of 3CD during infection. In: Journal of Virology. 2016 ; Vol. 90, No. 24. pp. 11032-11042.
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Rhinovirus 16 2A protease affects nuclear localization of 3CD during infection. / WALKER, Erin; CROFT, Sarah; Wei, Ke-jun; Jans, David; Fulcher, Alex J.; GHILDYAL, Reena.

In: Journal of Virology, Vol. 90, No. 24, 12.2016, p. 11032-11042.

Research output: Contribution to journalArticle

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T1 - Rhinovirus 16 2A protease affects nuclear localization of 3CD during infection

AU - WALKER, Erin

AU - CROFT, Sarah

AU - Wei, Ke-jun

AU - Jans, David

AU - Fulcher, Alex J.

AU - GHILDYAL, Reena

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Y1 - 2016/12

N2 - The human rhinovirus (HRV) 3C and 2A proteases (3Cpro and 2Apro, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3Cpro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2Apro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3Cpro, 3D, and mutant derivatives thereof, we show that 3Cpro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3Cpro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2Apro fusion proteins, we demonstrate formally that 2Apro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cpro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2Apro activity and not 3Cpro activity, which is observed only later in infection. The results thus define the temporal activities of 2Apro and 3CD/3Cpro activities in HRV serotype16 infection.

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