The effects of nucleotide usage in key nucleotide positions +4 and -3 flanking start codon on translation levels mediated by IRES of hepatitis C virus.

Internal ribosomal entry site (IRES) functions as a cis-acting RNA element, which drives an alternative and cap-independent translation initiation pathway. Currently, there are few studies on effects of nucleotide usages at key nucleotide positions +4 and -3 flanking start codon mediated by IRES of hepatitis C virus (HCV). Herein, we focus on the effect of nucleotide usages at -3 and +4 positions mediated by HCV IRES. The nucleotide contexts flanking AUG start codon employed by HCV IRES is firstly analyzed. We found that each position in the six nucleotide positions (-4 to +6) flanking start codon of HCV has a strong tendency to select the specific nucleotide. A set of bicistronic expression vectors containing CAT gene, HCV IRES and EGFP gene were constructed, including 16 different nucleotide combinations at position -3 and +4. Each set, in which nucleotide at the -3 and +4 position has been changed into different nucleotides, included 16 types of bicistronic expression vectors. It was found that the purine nucleotide at the position -3 or +4 obviously impacts on HCV IRES-related expression, and IRES-driven translation is potentially influenced by the Kozak rule. Our results suggest that optimization of nucleotides at positions -3 and +4 is a convenient and efficient way to enhance the level of IRES-mediated translation. Keywords: Cap-independent translation; internal ribosomal entry site; hepatitis C virus; bicistronic expression vector; translation efficiency.

Transcriptional analysis of the porcine TTID gene and association of different TTID genotypes with carcass traits.

The titin immunoglobulin domain (TTID) protein localizes to the Z line in muscle and binds to alpha-actinin and gamma-filamin. It plays an indispensable role in stabilizing and anchoring of thin filaments. In this study, the 5'-regulatory region of the porcine TTID gene was analyzed with bioinformatic methods. Another objective of this study was to further investigate the polymorphism in the intron 6 of the porcine TTID gene. We determined allele frequency among six Chinese porcine purebreds. The polymorphisms were genotyped in a population of 280 F2 pigs representing two Large White x Meishan reference families. Different TTID genotypes were significantly associated with carcass traits, including skin percentage (P < 0.05), loin eye area (P < 0.05), and average skin thickness (P < 0.01). Our study will continue to lay the groundwork for further investigations into the detailed function of the porcine TTID gene.

Genetic features of a translation initiation system composed of IRES element, nucleotide context surrounding the initiation codon, and translation initiation region of classical swine fever virus RNA.

Nucleotide and codon usage are typically examined to investigate viral evolution. In this study, we analyzed the genetic information of 46 strains of classical swine fever virus (CSFV) RNA, nucleotide usage in the internal ribosome entry site (IRES), the nucleotide context surrounding the initiation codon, and synonymous codon usage in the translation initiation region. Phylogenetic analysis of the IRES element indicated that the genetic diversity of this element is generally similar to the phylogenetic clusters of CSFV genotypes. Nucleotides surrounding the initiation codon of CSFV RNA were generally more stable (ACAUGGCACAUGGAGUUG) compared to the internal AUG in the CSFV coding sequence. The second codon position after the initiation codon was generally selected to be GAG, which has lower tRNA abundance in pigs than its synonymous member (GAA). Regarding the synonymous codon usage bias in the CSFV translation initiation region, some codons showing low tRNA abundance in pigs are more frequently located in the translation initiation region than in the open reading frame of CSFV. Although CSFV, similarly to other RNA viruses, has a high mutation rate in nature, the regulatory features of nucleotide and synonymous codon usage of the IRES element, the nucleotide context surrounding the initiation codon and the translation initiation region in CSFV RNA have been 'branded' in the system of translation initiation to accommodate gene expression mediated by the cap-independent translation mechanism.

The characteristics of synonymous codon usage in the initial and terminal translation regions of encephalomyocarditis virus.

The synonymous codon usage patterns in the initial and terminal translation regions (ITR, TTR) of the whole coding sequence of encephalomyocarditis virus (EMCV) were analyzed in relation to those in its natural hosts using the sequences accessible in databases. In general, some low-usage host codons were found over-represented in the ITR and TTR of the virus, while some high-usage host codons were found under-represented in the two viral regions. These relationships are thought to participate in the regulation of the speed of translation of viral proteins and in the suppression of ribosomal traffic jams, both aiming at the increase of virus yields.

Mapping codon usage in sequence regions flanking cleavage positions in the hepatitis A virus polyprotein.

To analyze the synonymous codon usage patterns of sequence regions flanking cleavage sites in the hepatitis A virus (HAV) polyprotein, the codon usage bias at codon positions and the synonymous codon usage in the target contexts of 30 virus strains were estimated by two simple methods that were based on the values for relative synonymous codon usage. In addition, the pattern of synonymous codon usage was compared between the genomic sequences in HAV and those of its human host. Our results indicated that HAV adopts a combination of coincidence and antagonism with the synonymous codon usage in humans. This characteristic may help HAV to efficiently use the translational machinery in its human host. We also observed that codon usage exhibited a strong bias in some specific positions in these contexts, and that the underrepresented synonymous codons, CUA for Leu, ACG for Thr, GUA for Val, and UCG for Ser, are preferentially used in these positions. These underrepresented synonymous codons likely play roles in regulating the rate of protein translation and influencing the secondary structure of the sequence regions flanking the cleavage sites.