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PubMed2005

Optimization of codon usage enhances the immunogenicity of a DNA vaccine encoding mycobacterial antigen Ag85B.

In spite of its many other benefits, DNA vaccine is limited in its application by its insufficient immunogenicity. One promising approach for enhancing its immunogenicity is to maximize its expression in the immunized host. In the current study, we investigated whether codon optimization of the mycobacterial antigen Ag85B gene could enhance the expression and immunogenicity of the Ag85B DNA vaccine. We generated a synthetic humanized Ag85B (hAg85B) gene in which codon usage was optimized for expression in human cells. DNA plasmids with codon-optimized hAg85B increased the level of protein expression in vitro and in vivo. DNA vaccine with hAg85B induced stronger Th1-like and cytotoxic T-cell immune responses in BALB/c mice and generated higher protective immunity in a BALB/c mouse model of Mycobacterium tuberculosis aerosol infection than did the DNA vaccine with wild-type Ag85B. Therefore, our results suggest that codon optimization of mycobacterial antigens (e.g., Ag85B) could improve protein expression and thereby enhance the immunogenicity of DNA vaccines against M. tuberculosis.

Acyltransferases↗
PubMed1985

Leucine regulation of the ilvGEDA operon of Serratia marcescens by attenuation is modulated by a single leucine codon.

The effect of leucine limitation and of restricted leucine tRNA charging on the expression of the ilvGEDA operon of Serratia marcescens was examined. In this organism, the ilv leader region specifies a putative peptide containing only a single leucine codon that could be involved in leucine-mediated control by attenuation (E. Harms, J.-H. Hsu, C. S. Subrahmanyam, and H. E. Umbarger, J. Bacteriol. 164:207-216, 1985). A plasmid (pPU134) containing the DNA of the S. marcescens ilv control region and three of the associated structural genes was studied as a single chromosomal copy in an Escherichia coli strain auxotrophic for all three branched-chain amino acids. The S. marcescens ilv genes responded to a multivalent control similar to that found in other enteric organisms. Furthermore, the S. marcescens ilv genes were derepressed when the charging of leucine tRNA was restricted in a leuS derivative of E. coli that had been transformed with pPU134. It was concluded that ribosome stalling leading to deattenuation is not dependent on either tandem or a consecutive series of codons for the regulatory amino acid. However, the fact that the single leucine codon is a less frequently used codon (CUA) may be important. The procedure for obtaining the cloned ilv genes in single chromosomal copy exploited the dependence of ColE1 replicons on the polA gene. The cloning experiments also revealed a branched-chain amino acid-glutamate transaminase in S. marcescens that is different from transaminase B.

Codon↗
PubMed1988

Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation.

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

Base Sequence↗
PubMed1990

A minor arginine tRNA mutant limits translation preferentially of a protein dependent on the cognate codon.

The Escherichia coli argU gene encodes a rare arginine tRNA (anticodon UCU) that translates the similarly rare AGA codon. The argU10(Ts) mutation is a transition that changes the first nucleotide of the mature tRNA from G to A, presumably destabilizing the acceptor stem. This mutation, when present in haploid condition in the chromosome, reduces the growth rate at 30 degrees C and results in cessation of growth after 60 to 90 min at 43 degrees C. The mutation also preferentially limits (compared with total protein synthesis) translation of an induced gene that depends on five AGA codons, i.e., the lambda cI repressor gene. Translation of another inducible protein, beta-galactosidase, which does not involve AGA codons, was inhibited to a much lesser extent. The chromosomal argU(Ts) mutation also confers the Pin phenotype, that is, loss of ability of the host, as a P2 lysogen, to inhibit growth of bacteriophage lambda, probably the result of reduced translation of the P2 old gene, which contains five AGA codons (E. Haggård-Ljungquist, V. Barreiro, R. Calendar, D. M. Kurnit, and H. Cheng, Gene 85:25-33, 1989).

Alleles↗
PubMed1991

Transcription attenuation-mediated control of leu operon expression: influence of the number of Leu control codons.

Four adjacent Leu codons within the leu leader RNA are critically important in transcription attenuation-mediated control of leu operon expression in Salmonella typhimurium and Escherichia coli (P. W. Carter, D. L. Weiss, H. L. Weith, and J. M. Calvo, J. Bacteriol. 162:943-949, 1985). The leader region from S. typhimurium was altered by site-directed mutagenesis to produce constructs having between one and seven adjacent Leu codons, all CUA. leu operon expression was measured in strains containing six of these constructs, each integrated into the chromosome in a single copy. Operon expression was sufficiently high that all strains grew in minimal medium unsupplemented by leucine. Expression of the operon was measured in strains cultured in such a way that their growth was limited by the intracellular concentration of either leucine or of leucyl-tRNA. In general, the leu operon for each construct responded similarly to the parent construct in terms of the degree of expression as a function of the degree of limitation. However, a strain containing (CUA)1 and, to a certain extent, a strain having (CUA)2 responded somewhat more sluggishly and strains containing (CUA)6 and (CUA)7 responded more sensitively to limitations than did the parent construct. In addition, DNA fragments containing the leu promoter and leader region were used as templates in in vitro transcription reactions employing purified RNA polymerase. With nucleoside triphosphate concentrations of 200 microM, RNA polymerase paused during transcription of the leu leader region at a site about 95 bp downstream from the site of transcription initiation. The halftimes of the pause were 1 min at 37 degrees C and 3 min at 22 degrees C. The pause was lengthened substantially when the GTP concentration was lowered to 20 micromoles. Our results are interpreted most easily in terms of an all-or-none model. Given two Leu control codons, the operon responds with nearly maximum output over a wide range of leucine limitation, and that outcome does not change much with increasing numbers of control codons.

Base Sequence↗
PubMed1995

Molecular cloning of a 46-kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of CGG codon usage for arginine.

The DNA sequence of the gene encoding the early and specific 46-kDa surface antigen (P46) of Mycoplasma hyopneumoniae has been determined. The P46 gene, encoding a putative lipoprotein, contained three TGA codons and a single CGG codon in a 1,257-bp open reading frame. Edman degradation of peptide fragments showed that at least one TGA codon encodes tryptophan and that the CGG codon, which has been reported to be nonsense or unassigned in other mycoplasmas, is used for arginine in M. hyopneumoniae.

Amino Acid Sequence↗
PubMed2006

Reduction of the rate of poliovirus protein synthesis through large-scale codon deoptimization causes attenuation of viral virulence by lowering specific infectivity.

Exploring the utility of de novo gene synthesis with the aim of designing stably attenuated polioviruses (PV), we followed two strategies to construct PV variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type (wt) poliovirus codons (PV-SD). Despite 934 nucleotide changes in the capsid coding region, PV-SD RNA produced virus with wild-type characteristics. In contrast, no viable virus was recovered from PV-AB RNA carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level. After subcloning of smaller portions of the AB capsid coding sequence into the wt background, several viable viruses were obtained with a wide range of phenotypes corresponding to their efficiency of directing genome translation. Surprisingly, when inoculated with equal infectious doses (PFU), even the most replication-deficient viruses appeared to be as pathogenic in PV-sensitive CD155tg (transgenic) mice as the PV(M) wild type. However, infection with equal amounts of virus particles revealed a neuroattenuated phenotype over 100-fold. Direct analysis indicated a striking reduction of the specific infectivity of PV-AB-type virus particles. Due to the distribution effect of many silent mutations over large genome segments, codon-deoptimized viruses should have genetically stable phenotypes, and they may prove suitable as attenuated substrates for the production of poliovirus vaccines.

Animals↗
PubMed1983

Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons.

The role which post-translational modification plays in the genesis of herpes simplex virus-induced polypeptides was investigated. Two-dimensional gel electrophoresis was used to identify those polypeptides (i) synthesized in vitro, (ii) labeled in vivo during a pulse, and (iii) labeled after a chase. Excluding glycoproteins, we detected 36 precursor or short-lived polypeptides, 8 polypeptides which were generated by post-translational modification, 46 polypeptides which were apparently not modified after synthesis, and 19 polypeptides which were either transient intermediates or not modified. Comparison of polypeptides synthesized in vitro and during an in vivo pulse showed that translation in vitro resembles quite closely translation in vivo and that amounts of protein synthesized in vivo are determined largely by the levels of mRNA. This analysis provided the basis for an investigation of the suggestion (C.M. Preston and D.J. McGeoch, J. Virol. 38:593-605, 1981) that the two polypeptides of apparent molecular weights of 43,000 (VI 43) and 39,000 (VI 39) encoded by the herpes simplex virus type 1 thymidine kinase gene are translated from a single mRNA by two in-phase initiation codons. Hybrid arrest was used to identify in vitro translation products encoded by the thymidine kinase gene. Two-dimensional gel electrophoresis showed that VI 39 was more acidic than VI 43, consistent with the predicted amino acid composition of a polypeptide whose synthesis was initiated at the second AUG codon, located 135 bases downstream from the first. Furthermore, two-dimensional gels revealed a third polypeptide whose synthesis was arrested by the same fragment. Its pI and apparent molecular weight (38,000) were compatible with initiation of translation at a third AUG codon an additional 42 bases downstream. Our findings provide strong evidence that downstream initiation codons within the thymidine kinase mRNA are used.

Codon↗
PubMed1987

Expression of the Rous sarcoma virus env gene from a simian virus 40 late-region replacement vector: effects of upstream initiation codons.

Expression of the Rous sarcoma virus envelope gene (env) from a simian virus 40 (SV40) late-region replacement vector is dependent on the position of env within the SV40 late-region sequences. The difference in expression levels appeared to be due to differences in the efficiency with which the env-specific transcripts were translated, because transcription levels from different constructions were similar. Deletion of the nucleotides encoding the agnoprotein initiator codon, located upstream of the env sequences in the poorly expressed construct, resulted in high levels of env expression. The agnoprotein initiator codon and overlapping open reading frame thus act as strong barriers to further ribosome scanning and prevent initiation at the env AUG codon. We conclude that AUG codons present in the late region of SV40 can reduce expression of inserted genes positioned downstream. Nevertheless, intrinsic properties of the gene may determine its ultimate level of expression.

Animals↗
PubMed1988

Retroviral gag gene amber codon suppression is caused by an intrinsic cis-acting component of the viral mRNA.

In some type C retroviruses, translation of the pol gene appears to require translational suppression of the proximal gag amber codon. To identify the region of the viral nucleic acid responsible for synthesis of the pol gene products, a 300-base-pair DNA fragment containing the stop codon from a type C murine virus (AK virus) was inserted into the Escherichia coli lacZ gene such that the translational reading frame was maintained. Introduction of the resulting fusion gene into cells resulted in the suppression of the viral stop codon. As measured by beta-galactosidase production, suppression occurred at a frequency of approximately 10%. Suppression could occur in at least several vertebrate cell types and was not augmented by virus replication or the expression of viral gene products. This indicates that gag amber codon suppression does not require augmented levels of suppressor tRNA species.

Acetyltransferases↗
PubMed1988

Translational control of cytomegalovirus gene expression is mediated by upstream AUG codons.

Human cytomegalovirus regulates gene expression at both the transcriptional and posttranscriptional levels. We have characterized posttranscriptional signals carried on the RNA of one beta (or delayed-early) gene, signals that we show delay the translation of an actively transcribed gene. The cis-acting components of this signal map to two upstream AUG codons in the 5' leader of the beta gene transcript that act to delay the kinetics and reduce the levels of gene expression. Both upstream AUG codons are part of short open reading frames, and the presence of either is sufficient to suppress expression of a downstream open reading frame. These upstream natural AUG codons appear to act in cis at the level of translation, consistent with the scanning hypothesis of eucaryotic translation. The occurrence of natural upstream AUG codons in other cytomegalovirus transcripts suggests their physiological significance in influencing the kinetics and steady-state levels of gene expression during viral growth.

Cells, Cultured↗
PubMed1989

Significance of premature stop codons in env of simian immunodeficiency virus.

The location of the translational termination codon for the transmembrane protein (TMP) varies in three infectious molecular clones of simian immunodeficiency virus from macaques (SIVmac). The SIVmac251 and SIVmac142 infectious clones have premature stop signals that differ in location by one codon; transfection of these DNAs into human HUT-78 cells yielded virus with a truncated TMP (28 to 30 kilodaltons [kDa]). The SIVmac239 infectious clone does not have a premature stop codon in its TMP-coding region. Transfection of HUT-78 cells with this clone initially yielded virus with a full-length TMP (41 kDa). At 20 to 30 days posttransfection, SIVmac239 virus with a 41-kDa TMP gradually disappeared coincident with the emergence of a virus with a 28-kDa TMP. Virus production dramatically increased in parallel with the emergence of a virus with a 28-kDa TMP. Sequence analysis of viral DNAs from these cultures showed that premature stop codons arising by point mutation were responsible for the change in size of the TMP with time. A similar selective pressure for truncated forms of TMP was observed when the SIVmac239 clone was transfected into human peripheral blood lymphocytes (PBL). In contrast, no such selective pressure was observed in macaque PBL. When the SIVmac239 clone was transfected into macaque PBL and the resultant virus was serially passaged in macaque PBL, the virus replicated very well and maintained a 41-kDa TMP for 80 days in culture. Macaque monkeys were infected with SIVmac239 having a 28-kDa TMP; virus subsequently recovered from T4-enriched lymphocytes of peripheral blood showed only the 41-kDa form of TMP. These results indicate that the natural form of TMP in SIVmac is the full-length 41-kDa TMP, just as in human immunodeficiency virus type 1. Viruses with truncated forms of TMP appear to result from mutation and selection during propagation in unnatural human cells.

Amino Acid Sequence↗
PubMed1992

The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.

Animals↗
PubMed1994

Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity.

It has been shown that two of the three adeno-associated virus type 2 capsid proteins, B and C, are synthesized from a single spliced transcript. Protein C arises from an AUG codon at nucleotide 2810, whereas protein B is initiated by a unique eucaryotic initiation codon (ACG) that lies 65 triplets upstream from the C origin. The third capsid component, protein A, is synthesized from a second spliced transcript which uses an alternative 3' acceptor site. In this study we used oligonucleotide-directed mutagenesis to confirm the positions of the B initiation codon and the 3' acceptor sites for the alternatively spliced B/C and A protein messages. We also located definitively the protein A initiation codon, an AUG triplet mapping to nucleotide 2203. Mutagenesis of the B initiator permitted a direct test of the effect of increased B initiator strength on the translational efficiencies of the B and C proteins. It was found that conversion of the relatively inefficient protein B initiator (ACG) to an AUG enhanced the level of B synthesis while abolishing the synthesis of C from its downstream AUG initiator. Protein C synthesis thus depends on the strength of the B initiator, i.e., the relatively higher levels of C (approximately 20-fold greater than B) must result from frequent readthrough of the weak B initiator. Finally, we examined the abilities of mutants deficient in the synthesis of A, B, or C to produce infectious virions. We found that at least two of the structural proteins, B and C, are required for the production of infectious virions and that sequestration of single-stranded adeno-associated virus genomes from the pool of replicating DNA molecules does not occur in the absence of either of these proteins.

Blotting, Northern↗
PubMed1994

Mutagenic study of codons 74 and 215 of the human immunodeficiency virus type 1 reverse transcriptase, which are significant in nucleoside analog resistance.

Mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) at codon 215 has been shown to play a significant role in resistance to zidovudine (AZT). Substitution of threonine with tyrosine or phenylalanine alone confers decreased susceptibility to the inhibitor. In this study we constructed a panel of 10 viruses with different amino acids at this codon, including 7 novel mutants, and assessed their susceptibilities to AZT. The majority of the new mutants were AZT sensitive, whereas the Thr-215-->Trp mutant was partially resistant (threefold less susceptible). A combination of the Thr-215-->Trp with the other AZT resistance mutations Lys-70-->Arg and Met-41-->Leu gave additive resistance. The Thr-215-->Phe virus was less AZT resistant than the Thr-215-->Tyr mutant, both on its own and when each was combined with the Met-41-->Leu mutant. These observations confirm the general hypothesis that increased bulk of the amino acid side chains at this position confers decreased AZT sensitivity. A leucine-to-valine substitution at codon 74 has previously been found to confer dideoxynucleoside resistance. We constructed mutants with five novel amino acid substitutions (Ala, Gly, Glu, Met, and Asp) at codon 74. Of these, only one (that with the Met substitution) retained enough RT activity to yield viable virus. It thus appears that there are severe structure-function constraints on the amino acid side chains at this position in the enzyme. The activities of the Leu-74-->Ala and Leu-74-->Met RT enzymes expressed in Escherichia coli appeared to have reduced susceptibility to ddGTP compared with the wild-type enzyme. The mutants described in this work may prove useful for correlation with structural studies of the human immunodeficiency virus type 1 RT.

Codon↗
PubMed1994

The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons.

Respiratory syncytial (RS) virus synthesizes two mature forms of its attachment glycoprotein G: an anchored type II integral membrane form and a smaller form that is secreted into the medium. Here we demonstrate that these two forms are synthesized as distinct primary translation products of a single species of G protein mRNA by initiation at either of two different AUGs. Mutant cDNAs which eliminated one of the other of the two AUG codons near the 5' end of the G gene open reading frame were constructed. Analysis of the proteins synthesized from these cDNAs, either by translation of transcripts in a cell-free system or in cells infected with recombinant vaccinia viruses containing either one of the mutant cDNAs, showed that elimination of either the first or the second of these AUG codons abrogated the synthesis of the membrane-anchored or the secreted form of the protein, respectively. Additionally, two unglycosylated forms of G protein which comigrated with the unglycosylated G proteins expressed by these recombinant viruses were detected in RS virus-infected cells. Since the second AUG encodes a methionine residue that lies near the middle of the signal/anchor domain, initiation at this codon resulted in a protein with a hydrophobic amino terminus. This form of the glycoprotein was efficiently secreted from cells infected with the vaccinia virus recombinant, and the amino-terminal sequence of this protein was identical to that of G protein secreted from RS virus-infected cells. Our results demonstrate that the secreted form of RS virus G protein is produced by initiation at the second AUG codon of the G open reading frame, followed by proteolytic removal of the signal/anchor domain.

Amino Acid Sequence↗
PubMed1998

Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage.

DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination.

AIDS Vaccines↗
PubMed2006

RNA secondary structure in the coding region of dengue virus type 2 directs translation start codon selection and is required for viral replication.

Dengue virus is a positive-strand RNA virus and a member of the genus Flavivirus, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.

Animals↗