The Repurposing of Cellular Proteins during Enterovirus A71 Infection

Figure 1. Reprogramming of the cellular environment upon EV-A71 infection. Following viral infection, several host RNA binding proteins, collectively referred to as ITAFs, change their sub-cellular localization. (a) These cellular ITAFs are primarily localized in the nucleus or cytoplasm while some of them can be shuttled between nucleus and cytoplasm based on their cellular functions. Regardless, upon EV-A71 infection, all ITAFs will be relocalized into the cytoplasm to regulate IRES-mediated translation. (b) The 5′-UTR of EV-A71 genome consists of the cloverleaf structure (SLI) and the IRES (SLII-VI). Cellular ITAFs bind to specific region(s) within IRES to either positively or negatively regulate IRES-mediated translation. Positive and negative ITAFs are indicated in green and red, respectively. The ITAFs, of which the effect on translation is not discovered yet, are indicated in grey. Some ITAFs can also bind the SLI region to regulate viral replication.

Figure 2. The EV-A71 5′-UTR contains phylogenetically conserved stem-loops (SLs) that adopt well-defined 3D structures. Depicted is a predicted secondary structure of the 5′-UTR of EV-A71, and 3D models of each individual stem-loop. The four nucleotides: adenosine (red), uridine (green), guanosine (blue), cytidine (yellow) are color coded in the regions that are predicted to be single stranded and most likely to interact with listed ITAFs. The base-paired nucleotides are shown in grey. 3D structures of SL I and II are published PDB structures while SLs III–VI were produced using the FAFFAR module of the ROSETTA software suit. The lowest energy structure for each stem-loop has been selected from a pool of 10,000 structures. The structures of SLI (8DP3) and SLII (5V17) were obtained from the PDB. Note, the structure of SLI of EV-A71 has not been published yet. Shown here is the SLI of the coxsackievirus B3 (CVB3) that was selected due to the high secondary structural homology between SLI regions of EV-A71 and CVB3.

Figure 3. Cellular ITAFs bind with specificity to short and degenerate sequence motifs. Depicted are published PDB structures of cellular ITAFs bound to RNA. These structures have been experimentally determined by both X-ray and nuclear magnetic resonance (NMR) spectroscopy. RNA has been color coded: adenosine (red), uridine (green), guanosine (blue), cytidine (yellow). ITAFs that bind to double-stranded RNA have the RNA depicted as a cartoon structure. The identity and PDB ids of the depicted ITAFs are as follows: (a) Argonaute 2 (5KI6), (b) hnRNP F (2KFY), (c) Far -Upstream Binding Protein 2 (4B8T), (d) hnRNP A1 (4YOE), (e) Polypyrimidine tract-binding protein 1 (2N3O), (f) Human Antigen R (6G2K), (g) DEAD-Box Protein 3 (6O5F), (h) Staufen homolog 1 (6HTU). Note, the crystal structure of the hnRNP A1-RNA complex (4YOE) was solved with a 5′-AGU-3′ sequence bound to only its RRM1 domain, confirming that a single-strand AG dinucleotide is the minimal specificity motif.

Figure 4. EV-A71 infection perturbs normal RNA metabolism, which leads to a shift in cellular homeostasis. Shown in steps 1–7 are some of the main cellular mechanisms that occur in the host cell. The mechanisms that are illustrated as faded are the ones that are affected by EV-A71 infection and are inhibited specifically due to the relocalization of cellular proteins to be used as ITAFs in EV-A71 IRES-mediated (cap-independent) translation. A few of the ITAFs, hnRNPA1, FBP1, FBP2, GADD34, and HuR, are used to represent the whole repertoire of ITAFs to help explain different viral mechanisms adapted by EV-A71 to facilitate its IRES-mediated translation. Positive ITAFs are colored in green while negative ITAFs are colored in red. These ITAFs are shown at the cellular functions that they regulate prior to EV-A71 infection. Note, a cleaved product of eIF4G is still involved in translation initiation, even though it is shown as faded due to cleavage by viral protease 3Cpro.