The Nonstructural Protein 2C of a Picorna-Like Virus Displays Nucleic Acid Helix Destabilizing Activity That Can Be Functionally Separated from Its ATPase Activity

Abstract

b Picorna-like viruses in the Picornavirales order are a large group of positive-strand RNA viruses that include numerous important pathogens for plants, insects, and humans. In these viruses, nonstructural protein 2C is one of the most conserved proteins and contains ATPase activity and putative RNA helicase activity. Here we expressed 2C protein of Ectropis obliqua picorna-like virus (EoV; genus Iflavirus, family Iflaviridae, order Picornavirales) in a eukaryotic expression system and determined that EoV 2C displays ATP-independent nucleic acid helix destabilizing and strand annealing acceleration activity in a concentration-dependent manner, indicating that this picornaviral 2C is more like an RNA chaperone than like the previously predicted RNA helicase. Our further characterization of EoV 2C revealed that divalent metal ions, such as Mg 2؉ and Zn 2؉ , inhibit 2C-mediated helix destabilization to different extents. Moreover, we determined that EoV 2C also contains ATPase activity like that of other picornaviral 2C proteins and further assessed the functional relevance between its RNA chaperone-like and ATPase activities using mutational analysis as well as their responses to Mg 2؉ . Our data show that, when one of the two 2C activities was dramatically inhibited or almost abolished, the other activity could remain intact, showing that the RNA chaperone-like and ATPase activities of EoV 2C can be functionally separated. This report reveals that a picorna-like virus 2C protein displays RNA helix destabilizing and strand annealing acceleration activity, which may be critical for picornaviral replication and pathogenesis, and should foster our understanding of picorna-like viruses and viral RNA chaperones.

The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB

“…2E), suggesting that EV71 3AB unwinds RNA-RNA helix more efficiently. Altogether, our data show that EV71 3AB has a nucleic acid helix-destabilizing activity for both RNA and DNA helices, and this feature is similar with the nonstructural protein 2C from Ectropis obliqua picorna-like virus (EoV), which is also able to destabilize both RNA and DNA helices (Cheng et al, 2013).…”

“…Thus, RNA chaperone activity is generally believed to be required for efficient replication and translation of vRNAs (Ivanyi-Nagy et al, 2005;Mir and Panganiban, 2006a;Zúñiga et al, 2009). So far, the list of virus-encoded RNA chaperones keeps growing and includes retroviruses nucleocapsid (NC) protein, HIV-1 Vif and Tat, coronavirus nucleocapsid (N) protein, hantavirus nucleocapsid (N) protein, flavivirus core protein, hepatitis D virus small delta antigen (SdAg), cypovirus capsid protein VP5, EoV nonstructural protein 2C, and poliovirus 3AB (Baric et al, 1988;Batisse et al, 2012;Cheng et al, 2013;DeStefano and Titilope, 2006;Huang et al, 2003;Ivanyi-Nagy et al, 2008;Kuciak et al, 2008;Mir and Panganiban, 2006b;Rein et al, 1998;Yang et al, 2014). Our current study adds EV71 3AB as a new member of this list.…”

“…Previous studies have shown that a variety of RNA chaperones are also able to destabilize/unwind DNA-DNA or DNA-RNA duplexes (Cheng et al, 2013), thus being named nucleic acid chaperones (DeStefano and Titilope, 2006). To further examine whether the helix-destabilizing activity of EV71 3AB is specific for RNA helix, we constructed two different nucleic acid helix substrates, Rn/D ( Fig.…”

“…To further characterize and verify the RNA chaperone activity of EV71 3AB, a classic assay for measuring helix destabilization and annealing stimulation of RNA chaperones (Cheng et al, 2013;DeStefano and Titilope, 2006;Gangaramani et al, 2010;Heath et al, 2003;Yang et al, 2014) was adapted for EV71 3AB. As illustrated in Fig.…”

Challenges associated with research on RNA viruses of insects

“…Since then, advances in the knowledge of these groups including the unique intergenic IRES of dicistrovirids [7,19,56], the characteristics of proteins 2C and 3C of iflaviruses [57,58], the specific host responses to viral infection [5,59,60] and other details of pathogenesis [3] will facilitate the generation of infectious clones for use in basic research and applied biotechnology (Figure 1). …”