Initiation of translation is an evolutionarily conserved process in yeast and humans. Efficient initiation of translation of cellular mRNAs is dependent upon the presence of a 7-methylguanosine cap structure at the 5′ end of the RNA, a relatively unstructured 5′ untranslated region, and a poly(A) tail at the 3′ end. The 7-methylguanosine cap structure is recognized by eIF4E, the cap binding protein. This interaction facilitates the recruitment of the small ribosomal subunit and all interacting complexes to the 5′ end of the mRNA. The ribosome then scans to the initiating AUG codon. Hepatitis C virus mRNA lacks a 5′-cap structure, and the 5′ untranslated region is highly structured and contains multiple AUG codons. These characteristics suggest that translation of the viral mRNA does not occur by a 5′-end-dependent mechanism. Instead, translation of the viral mRNA occurs by internal entry of the ribosome within the 5′UTR (67
). The 5′UTR of HCV mediates translation of the downstream open reading of a bicistronic transcript in poliovirus-infected cells, further demonstrating that translation does not occur by a 5′-end-dependent mechanism (70
The identification of cell proteins required for internal ribosome binding provides insight into the mechanism of IRES-dependent initiation. In vitro binding and UV cross-linking assays have been used to identify cell proteins that may be needed for efficient internal initiation mediated by the HCV IRES, including La protein (3
), polypyrimidine tract-binding protein (2
), poly(rC)-binding protein 2 (64
), and Nsap1 (35
). The yeast S. cerevisiae
has been used as a model organism to understand 5′-end-dependent initiation (23
). The study of IRES-mediated translation in this organism should also provide insight into the mechanism of internal ribosome binding. Internal initiation mediated by the HCV IRES in S. cerevisiae
was demonstrated by the production of β-galactosidase from a bicistronic mRNA in which the IRES was inserted between the yeast ADE3
and bacterial lacZ
genes. These findings establish a functional assay for identifying proteins required for IRES-mediated initiation.
Mutations known to abrogate HCV IRES-mediated internal initiation in mammalian cells also prevented β-galactosidase synthesis in yeast, providing genetic evidence for internal initiation. Translation of fragmented, uncapped RNAs derived from the bicistronic RNA could account for the production of β-galactosidase. Results of Northern analysis demonstrate the presence of full-length bicistronic mRNA in yeast. Even though smaller RNAs were detected in strains harboring the bicistronic plasmid, they were also present in cells containing the plasmid vector (Fig. ). If low levels of fragmented RNAs are present, these RNAs would lack a 5′-cap structure, and perhaps a poly(A) tail, and would be poorly translated in wild-type yeast (8
). Uncapped cellular mRNAs are found only in mitochondria in yeast and are translated in a prokaryotic-like manner with formyl-met
as the initiating codon (18
). The uncapped, nonpolyadenylated RNAs of the yeast L-A virus and satellite M virus are translated efficiently in the cytoplasm only when host cell transcripts are uncapped by the major coat protein of the virus, a trick to defeat the mRNA surveillance systems of the cell (45
). These considerations, together with the observation that initiation via the HCV IRES in yeast is efficient (Fig. ), make it highly unlikely that β-galactosidase activity is a consequence of translation of fragmented RNAs.
Activation of the nonsense-mediated decay pathway could lead to the production of fragmented RNAs in the cytoplasm of yeast cells. Bicistronic mRNAs contain two open reading frames and might trigger the nonsense-mediated decay pathway. If β-galactosidase is produced by translation of aberrant RNAs generated by nonsense-mediated decay, then β-galactosidase activity should be abolished in strains lacking the UPF1
gene. Both 5′-dependent and internal initiation was reduced 1.5-fold in a UPF1
null strain compared to an isogenic wild-type yeast strain (Fig. ). The reduction in 5′-dependent initiation is consistent with a suggested role for Upf1p during translation initiation (21
). The 1.5-fold decrease in HCV IRES-mediated initiation suggests that Upf1p is also required during internal ribosome binding.
RNA splicing of the bicistronic transcript could lead to the production of RNAs that are translated by 5′-end-dependent initiation. This possibility seems highly unlikely. Less than 1% of the transcripts synthesized in S. cerevisiae
are spliced (43
). The requirements for RNA splicing in S. cerevisiae
are much more stringent than in mammalian cells. All introns within pre-mRNA transcripts of yeast have UACUAAC sequences 20 to 55 nucleotides from their 3′ ends (39
), and an almost invariant GUAUGUU at their 5′ ends (50
). A minimum of 40 nucleotides separate the 5′ splice site and branch point sequence (36
), while a stretch of Un
followed by a conserved (U/C)AG is found at the 3′ splice site (43
). These sequences are not found within the ADE3-HCV C120-lacZ
bicistronic mRNA. Furthermore, full-length bicistronic transcript was visualized by Northern analysis, and the results of reverse transcription-PCR revealed the presence of the IRES between ADE3
(Fig. ). These findings demonstrate that internal initiation mediated by the HCV IRES is not a result of splicing of the bicistronic transcript.
The presence of a cryptic promoter in HCV-specific DNA sequences might lead to the production of short, capped lacZ monocistronic mRNAs that are translated by 5′-end-dependent initiation. If such cryptic promoters were present, then deletion of the ADH1 promoter, which directs transcription of the bicistronic mRNA, should not affect the level of β-galactosidase. Deletion of the ADH1 promoter for production of bicistronic mRNAs abolished synthesis of β-galactosidase.
It is not known why the HCV IRES mediates internal initiation in yeast when RNA encoding 120 amino acids of the viral polyprotein but not 5 amino acids was included in the bicistronic mRNA. Nonviral open reading frames may influence the efficiency of internal initiation, possibly by affecting the secondary structure surrounding the initiating codon (58
). Such an influence has been reported for another virus. The intergenic IRES of Plautia stali
virus is more efficient at mediating internal initiation when viral RNA downstream of the 3′ end of the IRES is included with the downstream cistron of the bicistronic mRNA (61
A number of cellular proteins appear to be required for efficient internal initiation mediated by the HCV IRES. La protein binds the 5′ untranslated region of hepatitis C virus around the initiating AUG codon (3
). Addition of recombinant La protein to rabbit reticulocyte lysates stimulates HCV IRES-dependent internal initiation (57
), and depletion of La protein from Huh7 cells by SELEX RNA decreased the ability of the HCV IRES to mediate internal initiation (1
). La protein also binds the poliovirus IRES (47
), and the addition of recombinant La protein to rabbit reticulocyte lysates increases the efficiency of internal initiation mediated by this IRES (48
). Reduction of La protein in HeLa cells by treatment with siRNA decreased both translation and replication of poliovirus RNA, and replication of recombinant poliovirus dependent upon the HCV IRES (16
La protein is a chaperone for small RNAs transcribed by RNA polymerase III. It binds the UUUOH
sequence of the 3′ ends of these RNAs and protects them from degradation (75
). La protein also effects the translation of mRNAs containing 5′-terminal oligopyrimidine tracts (14
). The three genes in S. cerevisiae
that encode homologs of La protein are LPH1
, and SLF1
. Similar to the mammalian ortholog, Lph1p is a chaperone for small RNAs transcribed by RNA polymerase III, binds the UUUOH
sequence of the 3′ ends of these RNAs, protects them from degradation, and facilitates the formation of RNA-protein complexes of these and other small RNAs in the cell. Lph1p is required for endonucleolytic cleavage of the 3′ trailer sequence of the tRNASer
CGA. In cells lacking Lhp1p, the 3′ trailer sequence is removed by exonucleases (75
). Lhp1p is required for growth when the unfolded protein response is induced by elevated temperatures (28
). Sro9p and Slf1p, two cytoplasmic proteins that contain La motifs, bind RNA associated with translating ribosomes. Strains deficient in either protein are less sensitive than wild-type strains to some protein synthesis inhibitors (63
The role of yeast La protein orthologs in HCV IRES-mediated internal initiation was determined in strains deleted for the genes encoding one or all three of these proteins. The results indicate that the efficiency of internal initiation is not affected in any of these strains. In contrast, depletion of La protein from mammalian cells reduces HCV IRES-mediated initiation (1
). The reason for the difference between yeast and mammalian cells is not clear. Yeast proteins other than La protein may act as RNA chaperones for the HCV IRES, or the IRES may fold properly in yeast without the need for La protein. Furthermore, the synthesis of human La protein in yeast did not stimulate internal initiation dependent upon the HCV IRES. This result was unexpected, because supplementing reticulocyte lysates with La protein stimulates HCV IRES-mediated initiation (57
). La protein may act as an RNA chaperone for the HCV IRES only in a complex with other proteins that are not encoded in the yeast genome.
The human cellular protein Pcbp2, which binds poly(rC), poly(rG), and poly(rU) tracts in both pre-mRNAs and mRNAs (41
), interacts with the 5′UTR of HCV (19, 35) and poliovirus (9
). Depletion of Pcbp2 from HeLa cell lysates reduces both translation and replication of poliovirus RNA, and addition of recombinant Pcbp2 to the depleted extract restores translation and replication to wild-type levels (10
). It has been suggested that Pcbp2 plays a role in HCV IRES-meditated initiation (64
), but depletion of the protein from HeLa cell lysates had no effect on HCV IRES-mediated initiation (15
). In agreement with the results of depletion experiments, HCV IRES-dependent initiation was observed in S. cerevisiae
in the absence of Pcbp2. Furthermore, the synthesis of human Pcbp2 in yeast did not affect the efficiency of HCV IRES-mediated β-galactosidase production. It seems unlikely that Pcbp2 is required for HCV IRES-dependent internal initiation.
Polypyrimidine tract-binding protein binds three sites within the poliovirus IRES, and sites within the IRES, polyprotein coding region, and the variable X region of the 3′UTR of HCV mRNA (7, 59). Ptb binds pyrimidine-rich sequences and is involved in differential splicing of RNA polymerase II transcripts (20
). Depletion of Ptb from both reticulocyte lysates and HuH7 cells by SELEX RNA reduced internal initiation mediated by the HCV IRES, and addition of recombinant Ptb restored the ability of the HCV IRES to direct internal initiation (7
). In agreement with these findings, HCV IRES-mediated translation occurred in yeast in the absence of Ptb, and production of human Ptb in yeast modestly stimulated the efficiency of HCV IRES-dependent β-galactosidase production. It is not known why production of human Ptb reduced 5′-end-dependent translation in yeast.
Other human proteins believed to be required for internal initiation of translation include upstream of N-ras (unr), murine proliferation-associated protein 1 (Mpp1), and Nsap1 (25
). HCV IRES-mediated initiation in yeast is independent of these proteins, as no sequences encoding unr, Mpp1, or Nsap1 are found in the yeast genome.
Internal initiation of translation has been demonstrated both in cell-free yeast extracts (4
) and in living yeast cells (38
). The intergenic IRES of cricket paralysis virus and sequences from four genes of S. cerevisiae
have been to shown to mediate internal initiation in yeast in vivo (38
). In yeast the cricket paralysis intergenic IRES is efficient at mediating internal initiation only in a mutant strain in which the ternary complex of eIF2-GTP-Met-tRNAiMet
is made artificially low (65
). The IRES within the coding region of Ure2p mRNA efficiently mediates internal initiation only in a mutant strain of yeast in which the interaction between the 5′-cap structure and eIF4E is severely compromised (38
). In contrast to the results obtained with the HCV IRES, internal initiation was not observed on bicistronic mRNAs containing the 5′UTRs of poliovirus, rhinovirus type 14, and the yeast gene TFIID
(data not shown). Internal initiation of translation dependent upon the IRES of poliovirus and TFIID has been demonstrated in extracts of yeast cells (26
), emphasizing the importance of using an in vivo assay to determine the requirements for internal initiation.
The unique ability of the HCV IRES to mediate internal initiation in wild-type yeast permits a genetic approach to identify cell proteins required for this process. The differences in the requirements for La and PTB in HCV IRES-mediated initiation in yeast and mammalian systems does not exclude the possibility that other cell proteins may be needed for internal initiation. Novel proteins required for HCV IRES-dependent translation might be revealed by studies in yeast. Furthermore, the core mechanism of translation can be elucidated in yeast. For example, the requirement for specific subunits of eIF2 and eIF3 during internal initiation can be evaluated. These studies may elucidate the mechanism of internal initiation and may lead to the discovery of new antiviral agents against HCV.