Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNA miR-122.

microRNAs (miRNAs) are small RNAs that in general down-regulate the intracellular abundance and translation of target mRNAs. We noted that sequestration of liver-specific miR-122 by modified antisense oligonucleotides resulted in a dramatic loss of hepatitis C virus (HCV) RNA in cultured human liver cells. A binding site for miR-122 was predicted to reside close to the 5' end of the viral genome, and its functionality was tested by mutational analyses of the miRNA-binding site in viral RNA, resulting in reduced intracellular viral RNA abundance. Importantly, ectopic expression of miR-122 molecules that contained compensatory mutations restored viral RNA abundance, revealing a genetic interaction between miR-122 and the viral RNA genome. Studies with replication-defective viral RNAs demonstrated that miR-122 affected mRNA abundance by positively modulating RNA replication. In contrast, interaction of miR-122 with the 3'-noncoding region (3'NCR) of the cellular mRNA encoding the cationic amino acid transporter CAT-1 resulted in the down-regulation of CAT-1 protein abundance. These findings provide evidence that a specific miRNA can regulate distinct target mRNAs in both a positive and negative fashion. The positive role of miR-122 in viral replication suggests that this miRNA could be targeted for antiviral therapy.

Takeover of host ribosomes by divergent IRES elements.

The ribosome is the macromolecular machinery in the host cell for which all viruses have to compete. Early in infection, the viral mRNAs have to compete with the host for both the ribosomes and for the limited pool of eukaryotic initiation factors that are needed to facilitate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition, certain viruses have evolved to recruit ribosomes to IRESs (internal ribosome entry sites), highly specialized RNA elements that are located at the 5'-end of the viral genomes. Here, we discuss how divergent IRES elements can recruit ribosomes and start protein synthesis with only a minimal set of eukaryotic translation initiation factors, and how this mode of translation initiation aids viral gene amplification during early onset of innate immune responses.

Translation inhibition during the induction of apoptosis: RNA or protein degradation?

The induction of apoptosis leads to a substantial inhibition of protein synthesis. During this process changes to the translation-initiation factors, the ribosome and the cellular level of mRNA have been documented. However, it is by no means clear which of these events are necessary to achieve translational shutdown. In this article, we discuss modifications to the translational apparatus that occur during apoptosis and examine the potential contributions that they make to the inhibition of protein synthesis. Moreover, we present evidence that suggests that a global increase in the rate of mRNA degradation occurs before the caspase-dependent cleavage of initiation factors. Increased mRNA decay is temporally correlated with the shutdown of translation and therefore plays a major role in the inhibition of protein synthesis in apoptotic cells.

Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element.

Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes. Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.

Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition.

Infection of eukaryotic cells with lytic RNA viruses results in extensive interactions of viral gene products with macromolecular pathways of the host, ultimately leading to death of the infected cells. We show here that infection of cells with poliovirus results in the cytoplasmic accumulation of a variety of shuttling and non-shuttling nuclear proteins that use multiple nuclear import pathways. In vitro nuclear import assays using semi-permeabilized infected cells confirmed that nuclear import was blocked and demonstrated that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the nuclear pore complex (NPC) was prevented. Analysis of components of the NPC revealed that two proteins, Nup153 and p62, were proteolyzed during poliovirus infection. These results suggest that the cytoplasmic relocalization of numerous cellular proteins is caused by the inhibition of multiple nuclear import pathways via alterations in NPC composition in poliovirus-infected cells. Blocking of nuclear import points to a novel strategy by which cytoplasmic RNA viruses can evade host immune defenses, by preventing signal transduction to the nucleus.

Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source.

The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.

Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function.

Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5' untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES-40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES-40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.

Regulation of host cell translation by viruses and effects on cell function.

Viruses have evolved a remarkable variety of strategies to modulate the host cell translation apparatus with the aim of optimizing viral mRNA translation and replication. Recent studies have revealed that modulation of both host and viral mRNA translation can be accomplished by selective alteration of translation factors in virus-infected cells. These findings provide new insights into the functioning of the translational apparatus in both uninfected and infected cells.

Initiation of protein synthesis from the A site of the ribosome.

Positioning of the translation initiation complex on mRNAs requires interaction between the anticodon of initiator Met-tRNA, associated with eIF2-GTP and 40S ribosomal subunit, and the cognate start codon of the mRNA. We show that an internal ribosome entry site located in the genome of cricket paralysis virus can form 80S ribosomes without initiator Met-tRNA, eIF2, or GTP hydrolysis, with a CCU triplet in the ribosomal P site and a GCU triplet in the A site. P-site mutagenesis revealed that the P site was not decoded, and protein sequence analysis showed that translation initiates at the triplet in the A site. Translational initiation from the A site of the ribosome suggests that the repertoire of translated open reading frames in eukaryotic mRNAs may be greater than anticipated.

Distinct mRNAs that encode La autoantigen are differentially expressed and contain internal ribosome entry sites.

Analysis by reverse transcription-polymerase chain reaction has suggested the existence of at least two La autoantigen-encoding mRNAs that contain different 5' noncoding regions (NCRs) linked to the same La coding region (Troster, H., Metzger, T. E., Semsei, I., Schwemmle, M., Winterpacht, A., Zabel, B., and Bachmann, M. (1994) J. Exp. Med. 180, 2059-2067). La-encoding transcripts La1 and La1' contain 115- and 483-nucleotide 5' NCRs, respectively. To determine whether the various La transcripts are functional mRNAs, the expression and polysomal association of natural La1 and La1' RNAs were examined. Although La1 transcripts were ubiquitously expressed in human tissues, La1' transcripts were predominantly expressed in peripheral blood leukocytes, especially in B, T, and natural killer cells. Both La1 and La1' transcripts associated with polysomes in natural killer cells, suggesting that these transcripts were functional mRNAs. Upon activation of B cells with the mitogens phorbol 12-myristate 13-acetate and ionomycin, the amount of La1' mRNA, but not La1, declined. In contrast, after chemical activation of T cells, the amount of La 1 mRNA, but not La1', declined. The mechanism by which the La1 and La1' 5' NCRs initiate translation initiation was tested in cultured human HeLa cells and in two different in vitro translation systems. It was found that both 5' NCRs can mediate translation initiation by internal initiation. These findings indicate that the constitutive expression of La1 mRNA and the tissue-specific expression of La1' mRNA can both allow La protein synthesis under conditions when cap-dependent translation is compromised, such as inflammation, apoptosis, or certain viral infections.

Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites.

Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5' end of the viral RNA and is more active than the IRES located at the 5' end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5' end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray.

Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5' end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5' noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

Functional coupling between replication and packaging of poliovirus replicon RNA.

Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687-1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726-1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.

Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells.

The poliovirus 3' noncoding region (3'NCR) is involved in the efficient synthesis of viral negative-stranded RNA molecules. A strong interaction between a 105-kDa host protein and the wild-type 3'NCR, but not with a replication-defective mutant 3'NCR, was detected. This 105-kDa protein was identified as nucleolin which predominantly resides in the nucleolus and has been proposed to function in the folding of rRNA precursor molecules. A functional role for nucleolin in viral genome amplification was examined in a cell-free extract which has been shown to support the assembly of infectious virus from virion RNA. At early times of viral gene expression, extracts depleted of nucleolin produced less infectious virus than extracts depleted of fibrillarin, another resident of the nucleolus, indicating a functional role of nucleolin in the early stages of the viral life cycle in this in vitro system. Immunofluorescence analysis of uninfected and infected cells showed a nucleocytoplasmic relocalization of nucleolin, but not of fibrillarin, in poliovirus-infected cells. Relocalization of nucleolin was not simply a consequence of virally induced inhibition of translation or transcription, because inhibitors of translation or transcription did not induce nucleolar-cytoplasmic relocalization of nucleolin. These findings suggest a novel virus-induced mechanism by which certain nucleolar proteins are selectively redistributed in infected cells.

In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules.

Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues. The methylated guanosine residue closely resembles the 5' terminal cap structure present on all eukaryotic mRNA molecules. The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from Saccharomyces cerevisiae. These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.