Viral Proteinase Requirements for the Nucleocytoplasmic Relocalization of Cellular Splicing Factor SRp20 during Picornavirus Infections

Infection of mammalian cells by picornaviruses results in the nucleocytoplasmic redistribution of certain host cell proteins. These viruses interfere with import-export pathways, allowing for the cytoplasmic accumulation of nuclear proteins that are then available to function in viral processes. We recently described the cytoplasmic relocalization of cellular splicing factor SRp20 during poliovirus infection. SRp20 is an important internal ribosome entry site (IRES) trans-acting factor (ITAF) for poliovirus IRES-mediated translation; however, it is not known whether other picornaviruses utilize SRp20 as an ITAF and direct its cytoplasmic relocalization. Also, the mechanism by which poliovirus directs the accumulation of SRp20 in the cytoplasm of the infected cell is currently unknown. Work described in this report demonstrated that infection by another picornavirus (coxsackievirus B3) causes SRp20 to relocalize from the nucleus to the cytoplasm of HeLa cells, similar to poliovirus infection; however, SRp20 is relocalized to a somewhat lesser extent in the cytoplasm of HeLa cells during infection by yet another picornavirus (human rhinovirus 16). We show that expression of poliovirus 2A proteinase is sufficient to cause the nucleocytoplasmic redistribution of SRp20. Following expression of poliovirus 2A proteinase in HeLa cells, we detect cleavage of specific nuclear pore proteins known to be cleaved during poliovirus infection. We also find that expression of human rhinovirus 16 2A proteinase alone can cause efficient cytoplasmic relocalization of SRp20, despite the lower levels of SRp20 relocalization observed during rhinovirus infection compared to poliovirus. Taken together, these results further define the mechanism of SRp20 cellular redistribution during picornavirus infections, and they provide additional insight into some of the differences observed between human rhinovirus and other enterovirus infections.     

Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation

Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRES-dependent translation, indicating a novel function for the cytoplasmic form of this protein.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5'-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.     

A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis

The upstream of N-Ras (Unr) protein is involved in translational regulation of specific genes. For example, the Unr protein contributes to translation mediated by several viral and cellular internal ribosome entry sites (IRESs), including the PITSLRE IRES, which is activated at mitosis. Previously, we have shown that translation of the Unr mRNA itself can be initiated through an IRES. Here, we show that UNR mRNA translation and UNR IRES activity are significantly increased during mitosis. Functional analysis identified hnRNP C1/C2 proteins as UNR IRES stimulatory factors, whereas both polypyrimidine tract-binding protein (PTB) and Unr were found to function as inhibitors of UNR IRES-mediated translation. The increased UNR IRES activity during mitosis results from enhanced binding of the stimulatory hnRNP C1/C2 proteins and concomitant dissociation of PTB and Unr from the UNR IRES RNA. Our data suggest the existence of an IRES-dependent cascade in mitosis comprising hnRNP C1/C2 proteins that stimulate Unr expression, and Unr, in turn, contributes to PITSLRE IRES activity. The observation that RNA interference-mediated knockdown of hnRNP C1/C2 and Unr, respectively, abrogates and retards mitosis points out that regulation of IRES-mediated translation by hnRNP C1/C2 and Unr might be important in mitosis.     

Heterogeneous nuclear ribonucleoprotein C modulates translation of c-myc mRNA in a cell cycle phase-dependent manner

The c-myc proto-oncogene plays a key role in the proliferation, differentiation, apoptosis, and regulation of the cell cycle. Recently, it was demonstrated that the 5' nontranslated region (5' NTR) of human c-myc mRNA contains an internal ribosomal entry site (IRES). In this study, we investigated cellular proteins interacting with the IRES element of c-myc mRNA. Heterogeneous nuclear ribonucleoprotein C (hnRNP C) was identified as a cellular protein that interacts specifically with a heptameric U sequence in the c-myc IRES located between two alternative translation initiation codons CUG and AUG. Moreover, the addition of hnRNP C1 in an in vitro translation system enhanced translation of c-myc mRNA. Interestingly, hnRNP C was partially relocalized from the nucleus, where most of the hnRNP C resides at interphase, to the cytoplasm at the G(2)/M phase of the cell cycle. Coincidently, translation mediated through the c-myc IRES was increased at the G(2)/M phase when cap-dependent translation was partially inhibited. On the other hand, a mutant c-myc mRNA lacking the hnRNP C-binding site, showed a decreased level of translation at the G(2)/M phase compared to that of the wild-type message. Taken together, these findings suggest that hnRNP C, via IRES binding, modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.     

Heterogeneous Nuclear Ribonucleoprotein L Interacts with the 3′ Border of the Internal Ribosomal Entry Site of Hepatitis C Virus

Translation initiation of hepatitis C virus (HCV) RNA occurs by internal entry of a ribosome into the 5′ nontranslated region in a cap-independent manner. The HCV RNA sequence from about nucleotide 40 up to the N terminus of the coding sequence of the core protein is required for efficient internal initiation of translation, though the precise border of the HCV internal ribosomal entry site (IRES) has yet to be determined. Several cellular proteins have been proposed to direct HCV IRES-dependent translation by binding to the HCV IRES. Here we report on a novel cellular protein that specifically interacts with the 3′ border of the HCV IRES in the core-coding sequence. This protein with an apparent molecular mass of 68 kDa turned out to be heterogeneous nuclear ribonucleoprotein L (hnRNP L). The binding of hnRNP L to the HCV IRES correlates with the translational efficiencies of corresponding mRNAs. This finding suggests that hnRNP L may play an important role in the translation of HCV mRNA through the IRES element.     

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import: IMPLICATIONS FOR HIV-1 GENE EXPRESSION*

Human immunodeficiency virus type 1 (HIV-1) co-opts host proteins and cellular machineries to its advantage at every step of the replication cycle. Here we show that HIV-1 enhances heterogeneous nuclear ribonucleoprotein (hnRNP) A1 expression and promotes the relocalization of hnRNP A1 to the cytoplasm. The latter was dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of the FG-repeat nuclear pore glycoprotein p62. hnRNP A1 and vRNA remain colocalized in the cytoplasm supporting a post-nuclear function during the late stages of HIV-1 replication. Consistently, we show that hnRNP A1 acts as an internal ribosomal entry site trans-acting factor up-regulating internal ribosome entry site-mediated translation initiation of the HIV-1 vRNA. The up-regulation and cytoplasmic retention of hnRNP A1 by HIV-1 would ensure abundant expression of viral structural proteins in cells infected with HIV-1.     

Cell-type-specific repression of internal ribosome entry site activity by double-stranded RNA-binding protein 76

Translation of picornavirus plus-strand RNA genomes occurs via internal ribosomal entry at highly structured 5' untranslated regions. In addition to canonical translation factors, translation rate is likely influenced by supplementary host and viral trans-acting factors. We previously reported that insertion of a heterologous human rhinovirus type 2 internal ribosomal entry site (IRES) into the poliovirus (PV) genome, generating the chimeric virus PV-RIPO, selectively abrogates viral translation and propagation in neurons, which eliminate poliovirus's signature neuropathogenicity. While severely deficient in cells of neuronal lineage, the rhinovirus IRES promotes efficient propagation of PV-RIPO in cancer cells. Tumor-specific IRES function can be therapeutically exploited to direct viral cytotoxicity to cancer cells. Neuron-glioma heterokaryon analysis implicates neuronal trans-dominant inhibition in this effect, suggesting that host trans-acting factors repress IRES function in a cell-type-specific manner. We identified a set of proteins from neuronal cells with affinity for the rhinovirus IRES, including double-stranded RNA-binding protein 76 (DRBP76). DRBP76 associates with the IRES in neuronal but not in malignant glioma cells. Moreover, DRBP76 depletion in neuronal cells enhances rhinovirus IRES-driven translation and virus propagation. Our observations suggest that cell-type-specific association of DRBP76 with the rhinovirus IRES represses PV-RIPO translation and propagation in neuronal cells.     

Differentiation-Induced Internal Translation of c-sis mRNA: Analysis of the cis Elements and Their Differentiation-Linked Binding to the hnRNP C Protein

In previous reports we showed that the long 5' untranslated region (5' UTR) of c-sis, the gene encoding the B chain of platelet-derived growth factor, has translational modulating activity due to its differentiation-activated internal ribosomal entry site (D-IRES). Here we show that the 5' UTR contains three regions with a computer-predicted Y-shaped structure upstream of an AUG codon, each of which can confer some degree of internal translation by itself. In nondifferentiated cells, the entire 5' UTR is required for maximal basal IRES activity. The elements required for the differentiation-sensing ability (i.e., D-IRES) were mapped to a 630-nucleotide fragment within the central portion of the 5' UTR. Even though the region responsible for IRES activation is smaller, the full-length 5' UTR is capable of mediating the maximal translation efficiency in differentiated cells, since only the entire 5' UTR is able to confer the maximal basal IRES activity. Interestingly, a 43-kDa protein, identified as hnRNP C, binds in a differentiation-induced manner to the differentiation-sensing region. Using UV cross-linking experiments, we show that while hnRNP C is mainly a nuclear protein, its binding activity to the D-IRES is mostly nuclear in nondifferentiated cells, whereas in differentiated cells such binding activity is associated with the ribosomal fraction. Since the c-sis 5' UTR is a translational modulator in response to cellular changes, it seems that the large number of cross-talking structural entities and the interactions with regulated trans-acting factors are important for the strength of modulation in response to cellular changes. These characteristics may constitute the major difference between strong IRESs, such as those seen in some viruses, and IRESs that serve as translational modulators in response to developmental signals, such as that of c-sis.     

RNA-binding protein hnRNP D modulates internal ribosome entry site-dependent translation of hepatitis C virus RNA

Hepatitis C virus (HCV) is one of the major causative agents of virus-related hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. Translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of the genome. Here, we report that a cellular protein, hnRNP D, interacts with the 5′ border of HCV IRES (stem-loop II) and promotes translation of HCV mRNA. Overexpression of hnRNP D in mammalian cells enhances HCV IRES-dependent translation, whereas knockdown of hnRNP D with small interfering RNAs (siRNAs) inhibits translation. In addition, sequestration of hnRNP D with an interacting DNA oligomer inhibits the translation of HCV mRNA in an in vitro system. Ribosome profiling experiments reveal that HCV RNA is redistributed from heavy to light polysome fractions upon suppression of the hnRNP D level using specific siRNA. These results collectively suggest that hnRNP D plays an important role in the translation of HCV mRNA through interactions with the IRES. Moreover, knockdown of hnRNP D with siRNA significantly hampers infection by HCV. A potential role of hnRNP D in HCV proliferation is discussed.     

Apigenin Inhibits Enterovirus-71 Infection by Disrupting Viral RNA Association with trans-Acting Factors

Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC₅₀ value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC₅₀ was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5′-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.     

Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2-dependent RNA granules

Central nervous system myelination requires the synthesis of large amounts of myelin basic protein (MBP) at the axon-glia contact site. MBP messenger RNA (mRNA) is transported in RNA granules to oligodendroglial processes in a translationally silenced state. This process is regulated by the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binding to the cis-acting A2 response element (A2RE). Release of this repression of MBP mRNA translation is thus essential for myelination. Mice deficient in the Src family tyrosine kinase Fyn are hypomyelinated and contain reduced levels of MBP. Here, we identify hnRNP A2 as a target of activated Fyn in oligodendrocytes. We show that active Fyn phosphorylates hnRNP A2 and stimulates translation of an MBP A2RE-containing reporter construct. Neuronal adhesion molecule L1 binding to oligodendrocytes results in Fyn activation, which leads to an increase in hnRNP A2 phosphorylation. These results suggest that Fyn kinase activation results in the localized translation of MBP mRNA at sites of axon-glia contact and myelin deposition.     

Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.     

IRES-mediated pathways to polysomes: nuclear versus cytoplasmic routes

Eukaryotic mRNA initiates translation by cap-dependent scanning, ribosome shunting and cap-independent internal ribosome entry. Internal ribosome entry was first discovered for cytoplasmic RNA viruses but has also been identified for DNA viruses and cellular mRNAs. An internal ribosome entry site (IRES) directs internal binding of ribosomes and nucleates the formation of a translation initiation complex. Current research is aimed at identifying interactions between IRES elements and RNA-binding proteins known as ITAFs (IRES trans-acting factors). Here we compare IRES elements from cytoplasmic RNA viruses with those of cellular mRNAs and DNA viruses with nuclear mRNA synthesis, and suggest that ITAF composition and IRES function directly reflect the site of synthesis of mRNA and the history of its pathway to polysomes.     

Polypyrimidine tract-binding protein inhibits translation of bip mRNA

Translation initiation of human Bip mRNA is directed by an internal ribosomal entry site (IRES) located in the 5' non-translated region. No trans-acting factor possibly involved in this process has as of yet been identified. For the encephalomyocarditis virus and other picornaviruses, polypyrimidine tract-binding protein (PTB) has been found to enhance the translation through IRES elements, probably by interaction with the IRES structure. Here, we report that PTB specifically binds to the central region (nt 50-117) of the Bip 5' non-translated region. Addition of purified PTB to rabbit reticulocyte lysate and overexpression of PTB in Cos-7 cells selectively inhibited Bip IRES-dependent translation. On the other hand, depletion of endogenous PTB or addition of an RNA interacting with PTB enhanced the translational initiation directed by Bip IRES. These suggest that PTB can either enhance or inhibit IRES-dependent translation depending on mRNAs.