Translation initiation and viral tricks

A variety of viral strategies are utilized for dominance of the host-cell protein synthetic machinery, optimization of viral mRNA translation and evasion of host-cell antiviral responses that act at the translational level. Many viruses exploit regulated steps in the initiation of cellular protein synthesis to their own advantage. They have developed some rather unconventional means for mRNA translation, which were probably adapted from specialized cellular mRNA translation systems. Regardless of the type of translational tricks exploited, viruses typically ensure efficient viral translation, often at the expense of host-cell protein synthesis.     

Adenovirus inhibition of cellular protein synthesis involves inactivation of cap-binding protein.

Adenovirus (Ad) infection results in a marked inhibition of cellular protein synthesis that initiates during the late phase of the viral infectious cycle. We show that the mechanism used for suppression of cellular protein synthesis during cell cycle progression is exploited by Ad to repress host and enhance late viral mRNA translation. Discrimination between cellular and late Ad mRNAs and inhibition of host protein synthesis are shown to involve viral-mediated underphosphorylation of cap-binding protein (CBP) and subsequent inactivation of CBP complex, a large enzymatic complex required for cap-dependent mRNA translation. Late Ad mRNAs, like those of poliovirus, possess the unique ability to translate independent of a normal cap recognition process and do not require the activity of CBP complex. Inhibition of cellular translation by these two viruses is quite similar, except that whereas CBP complex is proteolytically degraded by poliovirus, it is functionally inactivated by Ad.     

Capping strategies in RNA viruses

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.     

Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40.

Numerous viral and cellular RNAs are polycistronic, including several of the late mRNA species encoded by simian virus 40 (SV40). The functionally bicistronic major late 16S and functionally tricistronic major late 19S mRNA species of SV40 contain the leader-encoded open reading frames (ORFs) LP1, located upstream of the sequence encoding the virion protein VP1, and LP1*, located upstream of the sequence encoding the virion proteins VP2 and VP3. To determine how these leader ORFs affect synthesis of the virion proteins, monkey cells were transfected with viral mutants in which either the leader-encoded translation initiation signal was mutated or the length and overlap of the leader ORF relative to the ORFs encoding the virion proteins were altered. The levels of initiation at and leaky scanning past each initiation signal were determined directly by quantitative analysis of the viral proteins synthesized in cells transfected with these mutants. Novel findings from these experiments included the following. (i) At least one-third of ribosomes bypass the leader-encoded translation initiation signal, GCCAUGG, on the SV40 major late 16S mRNA. (ii) At least 20% of ribosomes bypass even the consensus translation initiation signal, ACCAUGG, when it is situated 10 bases from the 5' end on the major late 16S mRNA. (iii)O The presence of the leader ORF on the bicistronic 16S mRNA species reduces VP1 synthesis threefold relative to synthesis from a similar RNA that lacks it. (iv) At least half and possibly all VP1 synthesized from the bicistronic 16S mRNA species is made by a leaky scanning mechanism. (v) LP1 and VP1 are synthesized from the bicistronic 16S mRNA species at approximately equal molar ratios. (vi) Approximately half of the VP1 synthesized in SV40-infected cells is synthesized from the minor, monocistronic 16S mRNA even though it accounts for only 20% of the 16S mRNA present. (vii) The presence and site of termination of translation of the leader ORF on the late 19S mRNAs affect the relative as well as absolute rates of synthesis of VP2 and VP3.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.     

Infection of neuroblastoma cells by Semliki Forest virus. The interference of viral capsid protein with the binding of host messenger RNAs into initiation complexes is the cause of the shut-off of host protein synthesis

From ribosomal washes of neuroblastoma cells infected with Semliki Forest virus (SFV) a protein of Mr 33000 was purified, which comigrated with the viral capsid protein on sodium dodecyl sulfate/polyacrylamide gels and was recognized by antibodies against the capsid protein of SFV. This protein selectively inhibits the translation of host and early viral 42S mRNA in vitro, but has no effect on late viral 26S and encephalomyocarditis virus mRNA translation. Eukaryotic initiation factor 4B and cap-binding protein restore the translation of host and 42S mRNA to control levels. The capsid protein specifically prevents the binding of host mRNA into 80S initiation complexes, but has no effect on that of late viral mRNA. We propose that the capsid protein is the component responsible for the shut-off of host protein synthesis in SFV-infected cells and for the decreased translational activity of the crude ribosomal washes from these cells.     

5' sequence of vesicular stomatitis virus N-gene confers selective translation of mRNA.

The infection of cells by vesicular stomatitis virus results in the rapid inhibition of host-cell protein synthesis, but not of viral protein synthesis. To determine if this translational selectivity might be conferred by the viral mRNA, we constructed a plasmid (pUCLN beta-4) containing the 5' end of the viral nucleocapsid (N)-gene, including the ribosome binding site, fused in frame with the gene encoding beta-galactosidase, and compared it to a control plasmid (pMC1924) containing the cellular rabbit beta-globin gene 5' end fused with the beta-galactosidase encoding gene. Both plasmids contained identical promoter and 3' nontranslated regions and expressed similar levels of beta-galactosidase in the indicator cell line 293. In cells transfected with either plasmid, viral infection resulted in a approximately 70% decrease in protein synthesis by five hours. The level of beta-galactosidase from cells transfected with pMC1924 also decreased concomitantly with the decrease in total protein synthesis. However, the level of beta-galactosidase from cells transfected with pUCLN beta-4 was not affected by viral infection. Our data suggest that sequences in the 5' end of the viral mRNA allow for the selective translation of the viral message in the presence of an inhibited translational machinery.     

Poliovirus translation: a paradigm for a novel initiation mechanism

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.     

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.     

'Cap-tabolism'

distinctive feature of eukaryotic mRNA and small nuclear RNA (snRNA) that are transcribed by RNA polymerase II (Pol II) is the presence of a cap structure at their 5' end. This essential modification serves as an inviting 'landing pad' for factors that are involved in various cellular processes such as pre-mRNA splicing, nucleocytoplasmic RNA export and localization, and translation initiation. Because of the important functions mediated by the mRNA cap, this structure needs to be modified and/or degraded in a tightly controlled manner. Several cellular and viral systems implicated in cap metabolism have been uncovered recently; their analyses provide interesting new information on cell structure and function.     

Efficient translation of rotavirus mRNA requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor eIF4G and the mRNA 3' end

In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3'-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3' end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.     

Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation

Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Cα, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.     

Effect of high salt treatment on influenza B viral protein synthesis in MDCK cells

Based on the information that high salt inhibits the initiation of cellular mRNA translation which depends on the function of the 5'-terminal structure of mRNA, we compared the effect of high salt on translation of host cellular mRNAs and influenza viral mRNAs, both of which are of 5'-terminal structure. Brief exposure of influenza B virus-infected MDCK cells to high salt medium resulted in a dose-dependent inhibition of viral polypeptide synthesis as well as of cellular polypeptide synthesis, but it had less effect on synthesis of viral polypeptides, particularly nonstructural protein (NS). Under these conditions the Na+ content of the infected cells was significantly increased. A similar salt effect on in vitro translation of viral and cellular mRNAs extracted from infected cells was also observed. There was no significant difference in sensitivity to hypertonic block of in vivo translation of influenza viral mRNAs and vesicular stomatitis virus mRNAs, the latter of which possess a virus-directed structure at the 5'-terminus.     

Structural basis for competitive inhibition of eIF4G-Mnk1 interaction by the adenovirus 100-kilodalton protein

Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.     

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.