Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Abstract

The human rhinovirus (HRV) 3C and 2A proteases (3C pro and 2A pro , 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, 3C pro 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 2A pro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3C pro , 3D, and mutant derivatives thereof, we show that 3C pro 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 3C pro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2A pro fusion proteins, we demonstrate formally that 2A pro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3C pro 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 2A pro activity and not 3C pro activity, which is observed only later in infection. The results thus define the temporal activities of 2A pro and 3CD/3C pro activities in HRV serotype16 infection. IMPORTANCEThe human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.

Verdinexor (KPT-335), a Selective Inhibitor of Nuclear Export, Reduces Respiratory Syncytial Virus Replication In Vitro

“…CLSM and image analysis. Fixed or live (transfected)-cell samples were imaged as described previously (15,47). Digitized fluorescent cell images were collected using a Nikon Ti Eclipse CLSM with a Nikon 60ϫ/1.40 numeric aperture oil immersion lens (Plan Apo VC OFN25 DIC N2; optical section of 0.5 m) and the NIS Elements AR software; data from four individual scans were averaged to obtain the final images.…”

“…Digitized fluorescent cell images were collected using a Nikon Ti Eclipse CLSM with a Nikon 60ϫ/1.40 numeric aperture oil immersion lens (Plan Apo VC OFN25 DIC N2; optical section of 0.5 m) and the NIS Elements AR software; data from four individual scans were averaged to obtain the final images. Digital images were analyzed as described previously using ImageJ software to determine the fluorescence intensity above background (Fb) in the nucleus (Fn) compared to that in the cytoplasm (c) and to determine the nuclear-to-cytoplasmic fluorescence ratio (Fn/c) (47).…”

Innate Immune Evasion by Human Respiratory RNA Viruses

“…These activities are thought to influence host immune response signaling for which cytosolnucleus communication and trafficking is essential. It recently became clear that rhinovirus 2A protease activity also plays a role in targeting rhinovirus 3C protein to the nucleus [113,114], however, it is not clear what 3C protease is doing there exactly [114,115].…”

The Nonstructural Proteins 3 and 5 from Flavivirus Modulate Nuclear-Cytoplasmic Transport and Innate Immune Response Targeting Nuclear Proteins

“…Interestingly, the viral protease is located early after infection (8 hrs for DENV and 12 hrs for ZIKV) in the perinuclear region as well as in the nucleus of the cells and at 24 hpi the protein is present in the cytoplasm in the perinuclear region. Degradation of nucleoporins by the activity of viral proteases located in the perinuclear region, such as the 2A protease from poliovirus, or in the nucleus such as the 3CD protease-polymerase from rhinovirus 35,36 have been reported, suggesting that NS3 located in this two particular locations could reach and cut its target NUPs.…”

“…It has been widely reported that degradation of nucleoporins occurs by the activity of viral proteases located in the perinuclear and the nuclear regions, such as the 2A and 3CD from rhinovirus 35,36 . Nup98 is degraded early after DENV and ZIKV infection and it is located in both the nuclear and the cytoplasmic regions of the NPC; thus, it is possible that its degradation may be mediated by the protease present in the perinuclear region or in the nucleus of the infected cells.…”