Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags

The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.The picornavirus genome is a single-stranded, positive-sense RNA strand that is translated into a single polyprotein of ∼250 kDa which is cleaved during translation to generate an N-terminal capsid protein precursor (P1) and a P2-3 precursor to the nonstructural proteins. Although genetic and some biochemical studies conducted over many years have implicated all the nonstructural proteins as having some role(s) in viral RNA synthesis, the precise biochemical functions of most of these proteins remain only vaguely defined.Picornaviral 2A protein sequences, defined by their position C-terminal to the capsid protein precursor in the viral polyprotein, are highly variable in size, sequence, and function among the different genera within the family Picornaviridae. In the enterovirus genus, the 2A protein is a small cysteine proteinase, homologous to trypsin-like small serine proteases, with a Cys residue substituting for the usual serine to form a catalytic triad in the active site of the protease. The 2A proteins of cardio- and aphthoviruses have no sequence similarity to the 2A proteins of enteroviruses or to any other known proteinases. While smaller in size, the aphthovirus 2A proteins are homologous to the C-terminal ends of 2A proteins from cardioviruses. Their coding sequence causes ribosomes to “skip” formation of a peptide bond at the junction of the 2A and downstream sequences, leading to production of two proteins from a single open reading frame (36). Other picornaviruses exhibit no identifiable activities in the 2A region.During translation of viral RNA, poliovirus (PV) 2A^pro catalyzes the cleavage of its own N terminus in cis, thereby releasing the capsid proteins in the P1 region from the nascent nonstructural proteins in the P2-3 region. A second 2A cleavage site in the polyprotein resides in the N-terminal portion of 3D. Cleavage of 3CD at this site generates two products, 3C′ and 3D′; however, removal of the 3CD scissile bond by mutagenesis had no effect on poliovirus replication in HeLa cells (19).In addition to cleavage of the viral polyprotein, 2A^pro catalyzes specific cleavages in a small number of host cell proteins whose activities affect virus replication in different ways. Cleavage of the translational initiation factor eIF4G prevents translation of capped mRNAs, generating a C-terminal cleavage product of eIF4G that stimulates utilization of the poliovirus internal ribosome entry site (IRES) (10, 16, 21). Poly(A) binding protein (PABP) is also cleaved by 2A^pro although this cleavage is preferentially targeted to PABP not associated with polysomes. 3C^pro is responsible for polysome-associated PABP cleavage, which appears to be responsible for synergistic inhibition of cap-dependent translation (9, 18, 35). 2A^pro was found to increase the stability of viral RNA and to stimulate and prolong translation in vitro, independent of the RNA stabilizing effect (17). All of these 2A-induced events serve to enhance viral protein synthesis. In addition, poliovirus infection or 2A expression in HeLa cells causes alterations in the nuclear pore structure resulting from cleavage of specific components of the nuclear pore complex (8, 15, 33). These alterations produce bidirectional increases in permeability of the nuclear envelope, which permit redistribution of cellular proteins that normally reside in the nucleus to the cytoplasm, where they are available for viral translation or other replication reactions.Several genetic studies have implicated a direct role for 2A in viral RNA replication. Deletion of a C-terminal negatively charged cluster of amino acids present in poliovirus 2A is lethal to viral RNA replication without significantly affecting 2A protease function, and growth could be rescued by providing wild-type 2A^pro in trans (20). Similarly, deletion of sequences coding for the first 45 amino acids of 2A greatly reduced replication of a subgenomic replicon in transfected cells; replication of this replicon was also rescued in trans when 2A was provided by a helper RNA (12). In another study using a dicistronic viral RNA with an encephalomyocarditis virus (EMCV) IRES inserted between the P1 and P2 coding regions so that the cis cleavage function of 2A was not required to generate viral proteins, deletion of 2A^pro coding sequences rendered the transcripts incapable of replication (27). Insertion of an IRES between the 2A and 2B genes generated a small-plaque virus, indicating that neither the intact P2 polypeptide nor the 2AB cleavage fragment of P2 is required for virus growth. Thus, although little insight has been provided into the possible function(s) of 2A in the mechanism of viral RNA replication, several studies have suggested that 2A contributes some essential role to this process.In an effort to develop new approaches to studying the potential role of polio 2A in viral RNA replication, we generated viable polioviruses that encode 2A proteins tagged with a fluorescent protein (FP), inserted at either of two different positions in 2A. We report here the characterization and properties of these viruses, and we describe for the first time the intracellular location and distribution of protein 2A during the course of virus infection in HeLa cells.