Poliovirus 2A(Pro) increases viral mRNA and polysome stability coordinately in time with cleavage of eIF4G

Poliovirus (PV) 2A protease (2A(Pro)) cleaves eukaryotic initiation factors 4GI and 4GII (eIF4GI and eIF4GII) within virus-infected cells, effectively halting cap-dependent mRNA translation. PV mRNA, which does not possess a 5' cap, is translated via cap-independent mechanisms within viral protease-modified messenger ribonucleoprotein (mRNP) complexes. In this study, we determined that 2A(Pro) activity was required for viral polysome formation and stability. 2A(Pro) cleaved eIF4GI and eIF4GII as PV polysomes assembled. A 2A(Cys109Ser) (2A(Pro) with a Cys109Ser mutation) protease active site mutation that prevented cleavage of eIF4G coordinately inhibited the de novo formation of viral polysomes, the stability of viral polysomes, and the stability of PV mRNA within polysomes. 2A(Cys109Ser)-associated defects in PV mRNA and polysome stability correlated with defects in PV mRNA translation. 3C(Pro) activity was not required for viral polysome formation or stability. 2A(Pro)-mediated cleavage of eIF4G along with poly(rC) binding protein binding to the 5' terminus of uncapped PV mRNA appear to be concerted mechanisms that allow PV mRNA to form mRNP complexes that evade cellular mRNA degradation machinery.     

Poly(rC) binding proteins mediate poliovirus mRNA stability

The 5'-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5'-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5' cloverleaf RNA and rendered poliovirus mRNA unstable. A 5'-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5'-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5' cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5' cloverleaf RNA.     

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.     

Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis

Chimeric poliovirus RNAs, possessing the 5' nontranslated region (NTR) of hepatitis C virus in place of the 5' NTR of poliovirus, were used to examine the role of the poliovirus 5' NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3' NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5'-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5'-terminal cloverleaf and 3' NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.     

Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.     

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.     

Genetic analysis of a poliovirus/hepatitis C virus (HCV) chimera: interaction between the poliovirus cloverleaf and a sequence in the HCV 5' nontranslated region results in a replication phenotype

Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5'-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5' nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.     

Rotavirus Nonstructural Protein NSP3 is not required for viral protein synthesis

Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5' end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3' end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3' ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.     

Eucaryotic initiation factor 4B of wheat germ binds to the translation initiation region of a messenger ribonucleic acid

Purified preparations of eucaryotic initiation factor 4B (eIF4B) from wheat germ bind the monocistronic, uncapped, mRNA satellite tobacco necrosis virus RNA (STNV RNA) in nitrocellulose-mediated binding assays. This reaction is mRNA specific and yields dissociation constants (Kd) in the 10(-7)-10(-8) M range, depending upon the particular enzyme preparation tested. Purified wheat germ eIF4A, in the presence or absence of ATP, does not bind STNV RNA efficiently, but added eIF4A and ATP do enhance the efficiency of the eIF4B-dependent binding of STNV RNA. Wheat germ eIF4B binds the oligonucleotide containing the 5'-terminal 52 nucleotides of STNV RNA (designated 1-52) with the same affinity as intact STNV RNA. This binding affinity is less with the 1-44 oligonucleotide of STNV RNA and does not occur with the 1-33 oligonucleotide of STNV RNA that contains the 5'-terminal untranslated region and the initiator AUG codon at positions 30-32 of this mRNA. Wheat germ eIF4B therefore binds the translation initiation region of STNV RNA, and this binding requires up to 20 nucleotides on the 3' side of the initiator AUG codon of this mRNA. Wheat germ eIF4B also efficiently binds an oligonucleotide containing nucleotides from positions 13-52 in from the 5' terminus of STNV RNA, thereby establishing that the postulated 5'-terminal stem and loop secondary structure of STNV RNA [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] is not functional or essential for this specific binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translation Driven by Picornavirus IRES Is Hampered from Sindbis Virus Replicons: Rescue by Poliovirus 2A Protease

Alphavirus replicons are very useful for analyzing different aspects of viral molecular biology. They are also useful tools in the development of new vaccines and highly efficient expression of heterologous genes. We have investigated the translatability of Sindbis virus (SV) subgenomic mRNA bearing different 5′-untranslated regions, including several viral internal ribosome entry sites (IRESs) from picornaviruses, hepatitis C virus, and cricket paralysis virus. Our findings indicate that all these IRES-containing mRNAs are initially translated in culture cells transfected with the corresponding SV replicon but their translation is inhibited in the late phase of SV replication. Notably, co-expression of different poliovirus (PV) non-structural genes reveals that the protease 2A (2A^pro) is able to increase translation of subgenomic mRNAs containing the PV or encephalomyocarditis virus IRESs but not of those of hepatitis C virus or cricket paralysis virus. A PV 2A^pro variant deficient in eukaryotic initiation factor (eIF) 4GI cleavage or PV protease 3C, neither of which cleaves eIF4GI, does not increase picornavirus IRES-driven translation, whereas L protease from foot-and-mouth disease virus also rescues translation. These findings suggest that the replicative foci of SV-infected cells where translation takes place are deficient in components necessary to translate IRES-containing mRNAs. In the case of picornavirus IRESs, cleavage of eIF4GI accomplished by PV 2A^pro or foot-and-mouth disease virus protease L rescues this inhibition. eIF4GI co-localizes with ribosomes both in cells electroporated with SV replicons bearing the picornavirus IRES and in cells co-electroporated with replicons that express PV 2A^pro. These findings support the idea that eIF4GI cleavage is necessary to rescue the translation driven by picornavirus IRESs in baby hamster kidney cells that express SV replicons.     

Poliovirus-encoded 2C polypeptide specifically binds to the 3'-terminal sequences of viral negative-strand RNA.

The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5' untranslated region of the positive strand and its complementary 3'-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3'-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3'-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3'-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F.

Most eukaryotic mRNAs contain a 5'cap structure and a 3'poly(A) sequence that synergistically increase the efficiency of translation. Rotavirus mRNAs are capped, but lack poly(A) sequences. During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3' end. We looked for cellular proteins that could interact with NSP3A, using the two-hybrid system in yeast. Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI). The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co-immunoprecipitation and in vitro with NSP3A produced in Escherichia coli. In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected. PABP is removed from the eIF4F complex after incubation in vitro with the C-terminal part of NSP3A, but not with its N-terminal part produced in E.coli. These results show that a physical link between the 5' and the 3' ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation. These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut-off of cellular protein synthesis.     

Efficient cleavage of ribosome-associated poly(A)-binding protein by enterovirus 3C protease

Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2A(pro)) or 3C protease (3C(pro)). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3C(pro) is more efficient in cleaving PABP in ribosome-enriched fractions than 2A(pro) in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3C(pro)-mediated cleavage and inhibits 2A(pro)-mediated cleavage. These results suggest that 3C(pro) plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases.     

5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.     

NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI from Degradation by the Proteasome

The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.In eukaryotes, eukaryotic translation initiation factor 4G (eIF4G) plays a central role in the recruitment of ribosomes to the mRNA 5′ end and is therefore critical for the regulation of protein synthesis (14). Two homologues of eIF4G, eIF4GI and eIF4GII, have been cloned (15). Although they differ in various respects, both homologues clearly function in translation initiation. The most thoroughly studied of these is eIF4GI, which serves as a scaffolding protein for the assembly of eIF4F, a protein complex composed of eIF4E (the mRNA cap-binding factor) and eIF4A (an ATP-dependent RNA helicase). Thus, via its association with the mRNA cap-binding protein eIF4E and with another translation initiation factor (eIF3) which is bound to the 40S ribosomal subunit, eIF4GI creates a physical link between the mRNA cap structure and the ribosome, thus facilitating cap-dependent translation initiation (25). eIF4GI functions also in cap-independent, internal ribosome entry site (IRES)-mediated translation initiation. For instance, upon picornavirus infection, eIF4G is rapidly attacked by viral proteases. The resulting eIF4GI cleavage products serve to reprogram the cell''s translational machinery, as the N-terminal cleavage product inhibits cap-dependent translation of host cell mRNAs by sequestering eIF4E while the C-terminal cleavage product stimulates IRES-mediated translation of viral mRNAs (23). Similarly, apoptotic caspases cleave eIF4G into an N-terminal fragment that blocks cap-dependent translation and a C-terminal fragment that is utilized for IRES-mediated translation of mRNAs encoding proapoptotic proteins (22).The regulation of eIF4GI cleavage by viral proteases or apoptotic caspases has been extensively studied. Little is known, however, about the regulation of eIF4GI steady-state levels. Yet the eIF4GI amount that exists at a given moment results from the sum of the effects of de novo synthesis and ongoing degradation. Many cellular proteins are physiologically degraded by the proteasome. This has been shown to be true for eIF4GI, as the factor can be degraded by the proteasome in vitro (5) and in living cells (6). However, how eIF4GI targeting for or protection from destruction by the proteasome is regulated remains unknown.There are two major routes to degradation by the proteasome. In the more conventional route, polyubiquitinated proteins are targeted to the 26S proteasome. Alternatively, a few proteins can be degraded by the 20S proteasome (and sometimes by the 26S proteasome) in a ubiquitin-independent manner (16). Interestingly, it has been shown recently that a few of these proteins (1, 2, 13) can be protected from degradation by the 20S proteasome by binding to the NAD(P)H quinone-oxydoreductase 1 (NQO1). It has been proposed that NQO1 may interact with the 20S proteasome and may consequently block access of target proteins to the 20S degradation core. Because eIF4GI can be degraded in vitro by the 20S proteasome (5) and since it appears that proteasomes can degrade eIF4GI in living cells independently of ubiquitination (6), we asked whether NQO1 could protect eIF4GI from degradation by the proteasome.     

Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay.

Polysome and messenger ribonucleoprotein (mRNP) preparations from various mammalian cells contain tightly bound nuclease activity that causes degradation of the mRNA in the preparations. This activity was found to cosediment with all polysome size classes as well as with free mRNPs and to remain associated with the mRNPs released from polysomes by treatment with EDTA. No association with ribosomal subunits was evident. The rates of mRNA degradation were not affected by serial dilution, an indication that enzyme and substrate are tightly associated. beta-Globin mRNA in purified reticulocyte polysomes was cleaved at AU sequences in the 3'-terminal region. Cleavages at the same sites occurred when deproteinized reticulocyte RNA was incubated with mouse sarcoma 180 (S-180) polysomes. The S-180 preparations caused additional cleavages, primarily at UG sequences. A P40 mRNA in S-180 polysomes was cleaved primarily in the 3' noncoding region, but the cleavages in a P21 mRNA were seen in the 5' noncoding region only. Actin mRNA was cleaved in an internal region, yielding large relatively stable 3'- and 5'-terminal fragments. These data suggest the occurrence of highly specific interactions between one or more mRNA-bound nucleases and individual mRNA species.     

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.     

Influenza Virus mRNA Translation Revisited: Is the eIF4E Cap-Binding Factor Required for Viral mRNA Translation?

Influenza virus mRNAs bear a short capped oligonucleotide sequence at their 5' ends derived from the host cell pre-mRNAs by a "cap-snatching" mechanism, followed immediately by a common viral sequence. At their 3' ends, they contain a poly(A) tail. Although cellular and viral mRNAs are structurally similar, influenza virus promotes the selective translation of its mRNAs despite the inhibition of host cell protein synthesis. The viral polymerase performs the cap snatching and binds selectively to the 5' common viral sequence. As viral mRNAs are recognized by their own cap-binding complex, we tested whether viral mRNA translation occurs without the contribution of the eIF4E protein, the cellular factor required for cap-dependent translation. Here, we show that influenza virus infection proceeds normally in different situations of functional impairment of the eIF4E factor. In addition, influenza virus polymerase binds to translation preinitiation complexes, and furthermore, under conditions of decreased eIF4GI association to cap structures, an increase in eIF4GI binding to these structures was found upon influenza virus infection. This is the first report providing evidence that influenza virus mRNA translation proceeds independently of a fully active translation initiation factor (eIF4E). The data reported are in agreement with a role of viral polymerase as a substitute for the eIF4E factor for viral mRNA translation.