A fluorescent microplate assay quantifies bacterial efflux and demonstrates two distinct compound binding sites in AcrB.

A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.

The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs.

Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 5' viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 5' end of the CAT sequence, or the insertion of a small stable hairpin structure (deltaG = -18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 5' proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.

Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein.

Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.

Core protein-coding sequence, but not core protein, modulates the efficiency of cap-independent translation directed by the internal ribosome entry site of hepatitis C virus.

Among a myriad of putative functions assigned to the hepatitis C virus (HCV) core protein, several studies suggest that it may modulate internal ribosome entry site (IRES)-mediated initiation of translation. We compared the translational activity of dicistronic reporter transcripts containing the HCV IRES within the intercistronic space fused to downstream sequence encoding either 22 amino acids (aa) or 173 aa of the core protein. The inclusion of the nearly full-length core protein-coding sequence significantly suppressed translation in vitro and in transfected HepG2 cells. However, this suppression was not eliminated by frameshift mutations introduced into the core sequence, suggesting that it occurred at the RNA level and not as a result of core protein expression in cis. Similarly, the expression of core protein (aa 1 to 191) in trans from a recombinant baculovirus did not suppress IRES-directed translation from any of these transcripts in transfected Huh-7 cells. While core protein expression did decrease IRES activity in HepG2 cells (up to 79% suppression), the expression of beta-galactosidase from a control baculovirus also suppressed IRES activity (up to 56%), strongly suggesting that this suppression was nonspecific. Finally, the addition of purified recombinant core protein (aa 1 to 179) to in vitro translation reactions at concentrations up to a 10-fold molar excess over the RNA transcripts resulted in no significant reduction in IRES activity. Consistent with these results, a gel retention assay indicated no difference in the affinities of the recombinant HCV core protein and a recombinant Venezuelan equine encephalitis virus capsid protein for HCV IRES-containing RNA transcripts. We conclude that while the inclusion of core protein-coding sequence downstream of the IRES may reduce the efficiency of cap-independent translation on HCV RNA, the core protein itself has no biologically relevant activity in modulating HCV IRES activity.

Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo.

The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

Mutational analysis of the GB virus B internal ribosome entry site.

GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5' nontranslated GBV-B RNA (5'NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5'NTR (M. Honda, et al. RNA 2:955-968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5' end by structural domain II, a location analogous to the 5' limit of the IRES in both the HCV and pestivirus 5'NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3' limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

Natural variation in translational activities of the 5' nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: evidence for a long-range RNA-RNA interaction outside of the internal ribosomal entry site.

The 5' nontranslated RNA (5'NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5'NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31-42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5'NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5'NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K+ concentrations, with a sevenfold difference evident at 130 to 150 mM K+. These results suggest that there is an RNA-RNA interaction involving 5'NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N.

Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon.

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR in IRES-mediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5' NTR, ribosomal scanning for the initiation codon is limited to a small region.

The influence of AUG codons in the hepatitis C virus 5' nontranslated region on translation and mapping of the translation initiation window.

The initiation of translation of hepatitis C virus (HCV) is cap-independent and mediated by an internal ribosome entry site (IRES) that is located in the 5' nontranslated region (5' NTR) of the viral genome. This 5' NTR is relatively long and folds into a complex structure involving multiple hairpins and a pseudoknot. Within the sequence encompassing the IRES there are several AUG triplets. Some of these AUG codons are conserved between HCV genotypes and the related pestiviruses. In this study the 5 AUG codons (positions 13, 32, 85, 96, and 215) that are present in the 5' NTR of the HCV H-strain have been mutagenized to determine their influence on HCV cap-independent translation. The effect of these mutations on the expression of a chloramphenicol acetyl transferase (CAT) gene was tested in vaccinia virus. vTF7-3 infected Hep2 cells transfected with plasmids for the expression of a monocistronic HCV 5' NTR-CAT mRNA. Mutating the AUG codons at positions 13, 32, and 215 does not have a significant effect on CAT expression, inactivating the AUG codons at either position 85 or position 96 severely impaired IRES function. To determine whether ribosomes scan the RNA to select the initiation site, AUG codons were inserted up- and downstream of the authentic HCV polyprotein translation initiation codon (position 342). Analysis of these mutants has revealed that the ribosome is unable to use an AUG codon that is placed either 7 nucleotides upstream or 8 nucleotides downstream of the inactivated AUG at position 342. These results indicate that when scanning is involved in the recognition of the translation initiating AUG, it is limited to a narrow region between nucleotides 335 and 350.

Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA.

Cap-independent translation of hepatitis C virus (HCV) RNA is mediated by an internal ribosomal entry segment (IRES) located within the 5' nontranslated RNA (5'NTR), but previous studies provide conflicting views of the viral sequences which are required for translation initiation. These discrepancies could have resulted from the inclusion of less than full-length 5'NTR in constructs studied for translation or destabilization of RNA secondary structure due to fusion of the 5'NTR to heterologous reporter sequences. In an effort to resolve this confusion, we constructed a series of mutations within the 5'NTR of a nearly full-length 9.5-kb HCV cDNA clone and examined the impact of these mutations on HCV translation in vitro in rabbit reticulocyte lysates and in transfected Huh-T7 cells. The inclusion of the entire open reading frame in HCV transcripts did not lead to an increase in IRES-directed translation of the capsid and E1 proteins, suggesting that the nonstructural proteins of HCV do not include a translational transactivator. However, in reticulocyte lysates programmed with full-length transcripts, there were multiple aberrent translation initiation sites resembling those identified in some picornaviruses. The deletion of nucleotides (nt) 28-69 of the 5'NTR (stem-loop IIa) sharply reduced capsid translation both in vitro and in vivo. A small deletion mutation involving nt 328-334, immediately upstream of the initiator AUG at nt 342, also resulted in a nearly complete inhibition of translation, as did the deletion of multiple intervening structural elements. An in-frame 12-nt insertion placed within the capsid-coding region 9 nt downstream of the initiator AUG strongly inhibited translation both in vitro and in vivo, while multiple silent mutations within the first 42 nt of the open reading frame also reduced translation in reticulocyte lysates. Thus, domains II and III of the 5'NTR are both essential to activity of the IRES, while conservation of sequence downstream of the initiator AUG is required for optimal IRES-directed translation.

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA.

Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUGLab) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGLab for wild-type and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of eIF-2 was measured over a broad range of mRNA concentrations. In conclusion, eIF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.

Almost the entire 5' non-translated region of hepatitis C virus is required for cap-independent translation.

To investigate which hairpin structures within the 5' untranslated region of hepatitis C virus (HCV) are necessary for cap-independent translation, mutants were constructed that lack one or more hairpin structures. Here we demonstrate, by constructing precisely defined hairpin deletion mutants, that with the exception of the most 5' located hairpin structure, which on deletion shows an increase on translation, each of the predicted hairpins is found to be essential for cap-independent translation. In addition, we demonstrate that HCV 5'UTR driven translation is stimulated by poliovirus 2Apro co-expression.

Enhancement of the vaccinia virus/phage T7 RNA polymerase expression system using encephalomyocarditis virus 5'-untranslated region sequences.

A recombinant vaccinia virus producing the bacteriophage T7 RNA polymerase was used to express foreign genes in eukaryotic cells. Translation efficiency in this expression system was enhanced significantly by employing the encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR) which confers cap-independent translation by directing internal initiation of translation. The enhancement was accomplished by fusing open reading frames (ORFs) to the N terminus of the EMCV polyprotein coding region, thus utilizing its highly efficient translation initiation site. Expression vectors were constructed to allow cloning in all three reading frames. As reporter genes, we used the lacZ gene and a number of genes encoding coronavirus structural proteins: among others the genes encoding glycoproteins with N-terminal signal sequences. The signal sequences of these glycoproteins are located internally in the primary translation product. We demonstrated that this did not interfere with translocation and glycosylation and yields biologically active proteins. The usefulness of sequences that direct internal initiation was extended by using EMCV UTRs to express two and three ORFs from polycistronic mRNAs.

The carboxyl-terminal part of the putative Berne virus polymerase is expressed by ribosomal frameshifting and contains sequence motifs which indicate that toro- and coronaviruses are evolutionarily related.

Sequence analysis of the 3' part (8 kb) of the polymerase gene of the torovirus prototype Berne virus (BEV) revealed that this area contains at least two open reading frames (provisionally designated ORF1a and ORF1b) which overlap by 12 nucleotides. The complete sequence of ORF1b (6873 nucleotides) was determined. Like the coronaviruses, BEV was shown to express its ORF1b by ribosomal frameshifting during translation of the genomic RNA. The predicted tertiary RNA structure (a pseudoknot) in the toro- and coronaviral frameshift-directing region is similar. Analysis of the amino acid sequence of the predicted BEV ORF1b translation product revealed homology with the ORF1b product of coronaviruses. Four conserved domains were identified: the putative polymerase domain, an area containing conserved cysteine and histidine residues, a putative helicase motif, and a domain which seems to be unique for toro- and coronaviruses. The data on the 3' part of the polymerase gene of BEV supplement previously observed similarities between toro- and coronaviruses at the level of genome organization and expression. The two virus families are more closely related to each other than to other families of positive-stranded RNA viruses.