Poliovirus 2A&amp;lt;sup&amp;gt;pro&amp;lt;/sup&amp;gt; induces the nucleic translocation of poliovirus 3CD and 3C&amp;prime; proteins

Tian, Wenwu; Cui, Zongqiang; Zhang, Zhiping; Wei, Hongping; Zhang, Xian‐En

doi:10.1093/abbs/gmq112

Cited by 12 publications

(10 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The presence of 3CD in the nuclei of infected cells has been noted for a number of picornaviruses (6,27,28), but the mechanism by which this large protein enters the nucleus has been unclear. Together with other recent studies (29)(30)(31), the results here suggest that a unique mechanism regulates nuclear localization of picornavirus 3CD/3D, with protease activity being key. When put into the context of the current knowledge of picornaviral protease cleavage of NPC proteins, our data strongly suggest that cleavage of distinct Nups by 2A pro and 3C pro in a temporally regulated fashion is required to effect the disruption of nucleocytoplasmic trafficking, in part to enable nuclear localization of the large 3CD protein, which cannot otherwise easily access the host nucleus.…”

Section: Discussionsupporting

confidence: 84%

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

Walker

Jensen

Croft

et al. 2016

J Virol

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 84%

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

Walker

Jensen

Croft

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…In the end of our studies, we also addressed the usefulness of the protease antibodies in confocal microscopy and performed a study examining the subcellular localization of the proteases during acute CVB3 infection of HeLa cells. It has been previously described that the poliovirus 3C pro precursor 3CD, when expressed as a fusion protein, translocates into the nucleus after poliovirus infection or transfection with 2A pro , in a 2A pro dependent manner (Sharma et al, 2004;Tian et al, 2011). Similarly, HRV16…”

Section: Discussionmentioning

confidence: 99%

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Laitinen

Svedin

Kapell

et al. 2018

Journal of Virological Methods

View full text Add to dashboard Cite

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2A and 3C), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2A cause direct damage to the infected heart and tools to investigate 2A and 3C expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2A and 3C; Two monoclonal 2A antibodies and one 3C antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3C antibody detects all of the EV species B (EV-B) viruses tested and that the 2A antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2A and 3C, and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.

show abstract

“…However, these proteins localized to the cytoplasm of uninfected cells [51]. Furthermore, 2A pro of PV was demonstrated to be responsible for the redistribution of 3CD to the nucleus [52]. Subsequently, the HRV 3C protease was demonstrated to have a nuclear-targeting ability by causing the degradation of nuclear pore components [53].…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Sun

Wang

Wen

et al. 2019

Virol J

View full text Add to dashboard Cite

Background The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. Methods In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. Results Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and V max and K m values were determined to be 16.52 nmol/min and 50.78 μM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. Conclusions This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the V max and K m values were 16.52 nmol/min and 50.78 μM, respectively, using the Lineweaver–Burk plot.

show abstract

Poliovirus 2A<sup>pro</sup> induces the nucleic translocation of poliovirus 3CD and 3C′ proteins

Cited by 12 publications

References 31 publications

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

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

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Contact Info

Product

Resources

About