Viral IRES-mediated Translation Research Articles

HnRNP K Is a Novel Internal Ribosomal Entry Site-Transacting Factor That Negatively Regulates Foot-and-Mouth Disease Virus Translation and Replication and Is Antagonized by Viral 3C Protease.

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region. The regulation of internal initiation requires the interaction of IRES-transacting factors (ITAFs) with the IRES. In this study, we identified a novel ITAF, heterogeneous nuclear ribonucleoprotein K (hnRNP K), which negatively regulates foot-and-mouth disease virus (FMDV) translation and viral replication. Further investigation revealed that the KH2 and KH3 domains of hnRNP K directly bind to domains II, III, and IV of the FMDV IRES, resulting in the inhibition of IRES-mediated translation by interfering with the recognition of another positive ITAF, polypyrimidine tract-binding protein (PTB). Conversely, hnRNP K-mediated inhibition was antagonized by the viral 3C protease through the cleavage of hnRNP K at the Glu-364 residue during FMDV infection. Interestingly, the N-terminal cleavage product, hnRNP K1-364, retained partial inhibitory effects on IRES activity, whereas the C-terminal cleavage product, hnRNP K364-465, became a positive regulator of FMDV replication. Our findings expand the current understanding of virus-host interactions concerning viral recruitment and the modulation of ITAFs, providing new insights into translational control during viral infection.IMPORTANCE The translation of picornaviral genome RNA mediated by the internal ribosomal entry site (IRES) is a crucial step for virus infections. Virus-host interactions play a critical role in the regulation of IRES-dependent translation, but the regulatory mechanism remains largely unknown. In this study, we identified an ITAF, hnRNP K, that negatively regulates FMDV replication by inhibiting viral IRES-mediated translation. In addition, we describe a novel translational regulation mechanism involving the proteolytic cleavage of hnRNP K by FMDV protease 3C. The cleavage of hnRNP K yields two cleavage products with opposite functions: the cleavage product hnRNP K1-364 retains a partial inhibitory effect on IRES activity, and the cleavage product hnRNP K364-465 becomes a positive regulator of FMDV replication. Our findings shed light on the effect of a novel ITAF on the translational regulation of picornavirus and provide new insights into translational control during viral infection.

The Functional Role of the 3' Untranslated Region and Poly(A) Tail of Duck Hepatitis A Virus Type 1 in Viral Replication and Regulation of IRES-Mediated Translation.

The duck hepatitis A virus type 1 (DHAV-1) is a member of Picornaviridae family, the genome of the virus contains a 5′ untranslated region (5′ UTR), a large open reading frame that encodes a polyprotein precursor and a 3′ UTR followed by a poly(A) tail. The translation initiation of virus proteins depends on the internal ribosome-entry site (IRES) element within the 5′ UTR. So far, little information is known about the role of the 3′ UTR and poly(A) tail during the virus proliferation. In this study, the function of the 3′ UTR and poly(A) tail of DHAV-1 in viral replication and IRES-mediated translation was investigated. The results showed that both 3′ UTR and poly(A) tail are important for maintaining viral genome RNA stability and viral genome replication. During DHAV-1 proliferation, at least 20 adenines were required for the optimal genome replication and the virus replication could be severely impaired when the poly (A) tail was curtailed to 10 adenines. In addition to facilitating viral genome replication, the presence of 3′ UTR and poly(A) tail significantly enhance IRES-mediated translation efficiency. Furthermore, 3′ UTR or poly(A) tail could function as an individual element to enhance the DHAV-1 IRES-mediated translation, during which process, the 3′ UTR exerts a greater initiation efficiency than the poly(A)25 tail.

Promotion of Viral IRES-Mediated Translation Initiation under Mild Hypothermia.

Internal ribosome entry site (IRES)-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), human rhinovirus (HRV) or poliovirus (PV). Under mild hypothermic conditions (30 and 35°C), we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB) and upstream of N-Ras (unr), the IRES trans-acting factors (ITAFs) of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent.

Magnesium lithospermate B and rosmarinic acid, two compounds present in Salvia miltiorrhiza, have potent antiviral activity against enterovirus 71 infections

The aim of this study was to identify the active ingredients responsible for the anti-EV71 activity produced by Salvia miltiorrhiza extracts. A pGS-EV71 IRES-based bicistronic reporter assay platform was used for rapid analysis of compounds that could specifically inhibit EV71 viral IRES-mediated translation. The analysis identified 2 caffeic acid derivatives, magnesium lithospermate B (MLB) and rosmarinic acid (RA), which suppressed EV71 IRES-mediated translation at concentrations of 30μg/ml. We also found that MLB and RA inhibited EV71 infection when they were added to RD cells during the viral absorption stage. MLB had a low IC50 value of 0.09mM and a high TI value of 10.52. In contrast, RA had an IC50 value of 0.50mM with a TI value of 2.97. MLB and RA (100µg/ml) also reduced EV71 viral particle production and significantly decreased VP1 protein production. We propose that these two derivatives inhibit EV71 viral entry into cells and viral IRES activity, thereby reducing viral particle production and viral RNA expression and blocking viral VP1 protein translation. This study provides useful information for the development of anti-EV71 assays and reagents by demonstrating a convenient EV71 IRES-based bicistronic assay platform to screen for anti-EV71 IRES activity, and also reports 2 compounds, MLB and RA, which are responsible for the anti-EV71 activity of S. miltiorrhiza.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells.

Promotion of viral internal ribosomal entry site-mediated translation under amino acid starvation

Cap-dependent and internal ribosomal entry site (IRES)-mediated translation are regulated differently within cells. Viral IRES-mediated translation often remains active when cellular cap-dependent translation is severely impaired under cellular stresses induced by virus infection. To investigate how cellular stresses influence the efficiency of viral IRES-mediated translation, we used a bicistronic luciferase reporter construct harbouring IRES elements from the following viruses: encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV) or human rhinovirus (HRV). NIH3T3 cells transfected with these bicistronic reporter constructs were subjected to different cellular stresses. Increased translation initiation was only observed under amino acid starvation when EMCV or FMDV IRES elements were present. To identify cellular mechanisms that promoted viral IRES-mediated translation, we tested the involvement of eukaryotic initiation factor 4E-binding protein (4E-BP), general control non-depressed 2 (GCN2) and eukaryotic initiation factor 2B (eIF2B), as these are known to be modulated under amino acid starvation. Knockdown of 4E-BP1 impaired the promotion of EMCV and FMDV IRES-mediated translation under amino acid starvation, whereas GCN2 and eIF2B were not involved. To further investigate how 4E-BP1 regulates translation initiated by EMCV and FMDV IRES elements, we used a phosphoinositide kinase-3 inhibitor (LY294002), an mTOR inhibitor (Torin1) or leucine starvation to mimic 4E-BP1 dephosphorylation induced by amino acid starvation. 4E-BP1 dephosphorylation induced by the treatments was not sufficient to promote viral IRES-mediated translation. These results suggest that 4E-BP1 regulates EMCV and FMDV IRES-mediated translation under amino acid starvation, but not via its dephosphorylation.

A nucleo-cytoplasmic SR protein functions in viral IRES-mediated translation initiation.

A significant number of viral and cellular mRNAs utilize cap-independent translation, employing mechanisms distinct from those of canonical translation initiation. Cap-independent translation requires noncanonical, cellular RNA-binding proteins; however, the roles of such proteins in ribosome recruitment and translation initiation are not fully understood. This work demonstrates that a nucleo-cytoplasmic SR protein, SRp20, functions in internal ribosome entry site (IRES)-mediated translation of a viral RNA. We found that SRp20 interacts with the cellular RNA-binding protein, PCBP2, a protein that binds to IRES sequences within the genomic RNAs of certain picornaviruses and is required for viral translation. We utilized in vitro translation in HeLa cell extracts depleted of SRp20 to demonstrate that SRp20 is required for poliovirus translation initiation. Targeting SRp20 in HeLa cells with short interfering RNAs resulted in inhibition of SRp20 protein expression and a corresponding decrease in poliovirus translation. Our data have identified a previously unknown function of an SR protein (i.e., the stimulation of IRES-mediated translation), further documenting the multifunctional nature of this important class of cellular RNA-binding proteins.

Hepatitis C virus NS4A inhibits cap-dependent and the viral IRES-mediated translation through interacting with eukaryotic elongation factor 1A

SummaryThe genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1–20, and the central domain from residues 21–34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21–34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21–34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5 ′ untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5 ′ cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.

A peptide from autoantigen La blocks poliovirus and hepatitis C virus cap-independent translation and reveals a single tyrosine critical for La RNA binding and translation stimulation.

La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal "La motif" which is required for efficient interaction of La with IRNA and viral 5' untranslated region (5'-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5'-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5'-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.

Cell cycle regulation of hepatitis C and encephalomyocarditis virus internal ribosome entry site-mediated translation in human embryonic kidney 293 cells

We have established stably transformed human embryonic kidney cell lines (HEK293) containing bicistronic constructs to study regulation of viral internal ribosome entry site (IRES)-mediated translation in vivo. These cells produce Renilla luciferase (Rluc) in a cap-dependent manner, while Firefly luciferase (Luc) synthesis is mediated by IRES elements. Using these cell lines, we demonstrate here that IRES-mediated translation directed by both hepatitis C (HCV) and encephalomyocarditis (EMCV) virus varies with the cell cycle. Experiments involving arrest of the cell lines at different phases of the cell cycle, release of synchronized cells from cell cycle arrest, as well as direct sorting of the cells based on position in the cell cycle have shown that the activity of the HCV and EMCV IRES elements is lowest during the G2/M phase in HEK293 cells. These results suggest that cellular trans-acting factors either stimulate viral IRES-mediated translation during G1 and S phases or repress translation during the G2/M phase in HEK293 cells.

Nucleolin stimulates viral internal ribosome entry site-mediated translation

Previous results from our laboratory have identified a small (60 nt) RNA from the yeast S. cerevisiae that specifically inhibits internal ribosome entry site (IRES)-mediated translation programmed by poliovirus (PV) and hepatitis C virus (HCV) 5′-untranslated region (5′UTR). The yeast inhibitor RNA (called IRNA) was found to efficiently compete with viral 5′UTR for binding of several cellular polypeptides that presumably play important roles in IRES-mediated translation. One such IRNA (and 5′UTR)-binding protein has previously been identified as the La autoantigen. In this report, we have identified a 110-kDa IRNA-binding protein (which also interacts with viral 5′UTR) as nucleolin, a nucleolar RNA binding protein that was previously shown to translocate into the cytoplasm following infection of cells with poliovirus. We demonstrate that nucleolin (called C23) stimulates viral IRES-mediated translation both in vitro and in vivo. We also show that nucleolin mutants containing the carboxy-terminal RNA binding domains but lacking the amino terminal domain inhibit IRES-mediated translation in vitro. The translation inhibitory activity of these mutants correlates with their ability to bind the 5′UTR sequence. These results suggest a role of nucleolin/C23 in viral IRES-mediated translation.