The cis -responsive element of Foot-and-mouth disease virus interacts with host cellular factor PCBP2 dependent on host specificity

I. Background: Foot-and-mouth disease virus (FMDV) is a highly contagious viral pathogen in cloven-hoofed animal including cattle and pig, yet progress in the molecular mechanisms of FMDV genome replication is notably lagging behind that for many RNA viruses. A positive single stranded RNA of FMDV encodes a single long open reading frame flanked by a long 5’-untranslated region (5’UTR) and a short 3’-UTR. The cis -responsive element (CRE) of 5’UTR is critical for FMDV genome replication. II. Methods and Results: Here, we described that poly(C)-binding protein 2 (PCBP2) is revealed as a CRE-binding cellular factor. The RNA immunoprecipitation experiment confirmed that the FMDV CRE interacts with PCBP2 protein. CRE derived from FMDV infection in pig bound stronger to PCBP2 protein of pig than cattle PCBP2, showing host specific RNA-protein interaction. In addition, PCBP2 interacts with FMDV 3B protein together with CRE. The interaction of PCBP and 3B protein with CRE also showed host-specific manners. III. Conclusions: These data suggest that cellular PCBP2 may serve as a host cellular factor of FMDV to facilitate viral replication through interaction with the viral genome and contribute to determine host susceptibility of FMDV variants. The inter-molecular interaction between cellular PCBP2 and FMDV 3B and CRE provides perspectives for antiviral strategy. 5’ ends of the FMDV genome and 3B protein and also formed an RNA-protein complex. This study indicates that PCBP2 association in the replication complex of FMDV is responsible for understanding of the finer replication initiation of FMDV. Further study on the identification of interaction details of PCBP2 and CRE or/and 3B might provide the molecular targets for development of vaccine and therapeutic interventions.


Introduction
The genomes of FMDV encode the viral proteins required for viral genome replication and contain cis-acting RNA sequences and structures at the end of 5' and 3' of the genome structure [1] Host intracellular factors that interact with the RNA elements are critical for successful maintenance of the virus life cycle and this functional interaction between host factors and viral RNA elements can be applied to manipulate the introduction of mutations into the viral genome for approach to rational attenuation [2].
The 5'-UTR plays a critical role in initiation of replication and translation of the FMDV genome [3]. FMDV RNA, which consists of a long 5'-UTR containing over 1,300 nucleotides (nt), shows extensive secondary structure with individual five regions. Cis-acting replication element (CRE), which is one of 5'-UTR, is a highly conserved stem-loop of 55 nt length. CRE is essential for FMDV genome RNA replication. Different picornavirus RNAs also have this element with a conserved "AAACA" motif in the loop region [4]. This element structure acts as the template for uridylylation of 3B (VPg) protein to produce 3BpU and /or 3BpUpU by the RNA polymerase 3D, providing a platform as a primer that initiates FMDV RNA synthesis [5].
The 3B uridylylation reaction requires that 3B binds to 3D and 3D RNA polymerase transfers UMP to 3B [6]. The interactions between 3B and 3D only reflect a partial aspect of the formation of the 3B uridylylation complex. The combination of protein-protein of 3B and 3D and protein-RNA interactions of 3B and CRE facilitate the formation of a functional 3B-3D replication complex. However, the direct interaction between 3B and 3D is not fully sufficient for the formation of the complete replication complex. The efficient formation of 3B-3D replication complex with CRE RNA substrate may require additional host cellular factors. To date, our understanding of host cell factors that are involved in the FMDV life cycle is limited. PCBP2 (hnRNP E2), a host cellular protein known to bind to the 5'UTR of the poliovirus genome, is involved in switching between viral translation and replication [7]. PCPB2 is a member of the cellular heterogeneous nuclear ribonucleoprotein (hnRNP) family, which is expressed in both the nucleus and cytoplasm [8]. hnRNPs are well known for their abilities to bind to cellular proteins and RNAs to facilitate many biological processes, such as mRNA stabilization, transcriptional regulation, translational control, and apoptotic program activation.
In this study, we used RNA-protein interaction assay to identify PCBP2 interacts with the 5 CRE RNA differentially dependent on host species of pig and cattle. Usage of a distinct host cell factor may provide additional targets for host susceptibility discrimination of FMDV. The CRE of FMDV functions as a platform to recruit viral and cellular proteins, which regulates viral RNA replication. Some of the host factors regulate FMDV RNA replication either by participating in the formation of RNA replication complex or binding to viral RNA. Here, we demonstrate that PCBP2 serves as a host cellular factor to facilitate the formation of FMDV RNA replication complex through interaction with CRE RNA and 3B protein.

Materials and Methods
Cell culture IBRS-2 (pig kidney epithelial cell) and MDBK (cattle kidney epithelial cell) were obtained from Korea Animal and Plant Quarantine Agency) and maintained in Dulbecco's modified  RT-PCR and FMDV RNA sequencing FMDV RNA was extracted using an automatic RNA extraction machine (MagNA Pure 96, Roche) according to the manufacture's instruction. The RNA was stored at -70℃ until use.
The cDNA was synthesized using the PrimeScript™ II 1st strand cDNA Synthesis Kit (TAKARA). In brief, a 10 µL reaction mixture was prepared containing 10 mM dNTP, 1 µL of oligo dT primer (50 µM), 3 µL RNase Free dH2O and 5 µL of viral RNA. The mixture was incubated for 5 min at 65°C then cooled immediately on ice. Next, the reaction was mixed with 10 µL of a second reaction mixture containing 5X PrimeScript II buffer, 0.5 µL of 40U/µL RNase inhibitor, 1 µL of enzyme, and RNase-free dH2O. The mixture was then incubated at 42°C for 45 min and then 70°C for 15 min. The entire genome was amplified using AccuPower® ProFi Taq PCR PreMix (BIONEER, Korea) according to the manufacturer's instructions with 9 overlapping pairs of FMDV-specific primers. RT-PCR products were analyzed by QIAxcel (Qiagen). Purified PCR products were either sequenced directly or after cloning into the pGEM-T easy vector (Promega, USA). DNA sequencing was performed using automatic DNA sequencer (ABI3730) using the BigDye Terminator v3.1 cycle sequencing kit (ABI, USA). Analysis of sequence identity and divergence was carried out using BioEdit software (version 7.2.5.). PCR product sequences were assembled with SeqMan Pro software (DNASTAR, Inc., Madison, WI, USA) using default parameters.

Preparation of CRE RNA of FMDV
The regions of interest, namely nucleotides 622-687 of the FMDV genome, were amplified using PCR with the FMDV cDNA clone. T7 promoter sequence was conjugated the 5'-end of viral RNA specific sequence for the forward primer. A control RNA was applied to PCR Laemmli sample buffer and then subjected to SDS-PAGE. 8

PCBP2 interacts with the CRE RNA of FMDV
The RNA structures present in CRE play a role in the FMDV life cycle and viral replication.
Mutational analysis of the RNA structures at the CRE has indicated that the stem-loop structures are essential for initiation of FMDV RNA replication. FMDV 3B protein binds to CRE at the priming stage of FMDV RNA replication. In addition to 3B protein, host cellular protein might involve the formation of CRE-3B complex and lead to efficient initiation of RNA replication. CRE RNA (O/JC/SKR/2014/P, which was derived from FMDV-infected pig in Korea at 2014) was prepared and coupled to Sepharose beads with CNBR activation (Fig. 1A).
We used VP3 gene of FMDV capsid protein as a control RNA or Sepharose beads only. The RNA-coupled beads were incubated with cytoplasmic lysates of IBRS-2 cells. Since PCBP2 was known to bind to the 5'UTR of the poliovirus, which is one of picornaviruses, genome, the interacting cellular proteins with CRE RNA were loaded in gels of SDS-PAGE and confirmed by immunoblotting with specific antibody to PCBP2 protein. As shown in Figure   1B, PCBP2 protein was identified in the CRE-bound fraction, not at the non-specific RNA (VP3) bound fraction and control IgG immunoblotting. This result indicates that PCBP2 is a host cellular protein interacting CRE RNA of FMDV.

Different PCBP2 derived from cattle and pig show differential binding to CRE dependent on host specificity
In other study, we identified different RNA secondary structures of CRE along with different host species of cattle and pig. We compared the amino acid variation of PCBP2 protein between cattle and pig. As shown in Fig. 2A, the amino acid composition of pig PCBP2 was different at one position with cattle one. Pig PCBP2 protein has an additional four amino acids (TLSQ) at position from 169 to 172 compared with cattle one (Fig. 2A).
To analyze the differential interaction of pig-derived PCBP2 and cattle one to the CRE RNA, we detected the direct interaction between two different PCBP2 proteins and FMDV CRE RNA. strongly interacted with cattle PCBP2 (Fig. 3C). These results indicate that the specificity of intermolecular interaction between CRE RNA and host cellular protein PCBP2 might contribute to host susceptibility of FMDV replication.

PCBP2 interacts with FMDV 3B protein together with CRE
The 5'UTR of FMDV functions as a platform to recruit viral and cellular proteins and not only directs IRES-dependent protein synthesis but also plays roles in CRE-dependent RNA  (Fig. 4B). These results indicate that formation of the functional replication complex of CRE RNA, 3B protein and PCBP2 is associated with host species susceptibility. We identified a potential factor PCBP2 that bind to the replication region, CRE, of the FMDV 5' UTR. PCBP2 previously was known to interact with the 5' UTR of the HCV genome [11]. Signals required for the replication and translation of positive-strand RNA viruses usually are located in the 5'-and 3'-terminal regions of the viral RNA. The sequences required for RNA replication and translation often overlap, and the regulatory mechanisms can be separated or shared. Some research indicates that the role of PCBP2 in HCV is to mediate IRES-dependent translation [12]. Also, a study showed that the HCV RNA level was reduced by PCBP2 short interfering RNA (siRNA) knockdown [13].

Discussion
PCBP2 also functioned in RNA replication and viral protein translation of poliovirus [14][15][16]. Furthermore, PCBP2 regulated the transition of poliovirus translation into viral genome replication [17]. Specific RNA-RNA interaction is necessary for flavivirus genome circularization [19] and picornaviruses require RNA-protein interaction [20], and cRNA-RNA sequences for dengue virus [21]. Circularization of FMDV Genome might coordinate viral translation and RNA synthesis and make a platform for the viral polymerase localization at the start site of RNA synthesis (allowing the viral and cellular proteins to interact with the terminal ends of the viral genome and, in turn, initiating translation or replication efficiently). In addition, the circularization may maintain the integrity of FMDV genome. In this study, PCBP2 was found to bind to both the 5' ends of the FMDV genome and 3B protein and also formed an RNAprotein complex.
This study indicates that PCBP2 association in the replication complex of FMDV is responsible for understanding of the finer replication initiation of FMDV. Further study on the identification of interaction details of PCBP2 and CRE or/and 3B might provide the molecular targets for development of vaccine and therapeutic interventions.