A Shine-Dalgarno-like Sequence Mediates in Vitro Ribosomal Internal Entry and Subsequent Scanning for Translation Initiation of Coxsackievirus B3 RNA

AbstractTranslation initiation of coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5′ untranslated region. However, the details of ribosome-template recognition and subsequent translation initiation are still poorly understood. In this study, we have provided evidence to support the hypothesis that 40S ribosomal subunits bind to CVB3 RNA via basepairing with 18S rRNA in a manner analogous to that of the Shine-Dalgarno (S-D) sequence in prokaryotic systems. We also identified a new site within both the 18S rRNA and the polpyrimidine-tract sequence of the IRES that allows them to form stronger sequence complementation. All these data were obtained from in vitro translation experiments using mutant RNAs containing either an antisense IRES core sequence at the original position or site-directed mutations or deletions in the polypyrimidine tract of the IRES. The mutations significantly reduced translation efficiency but did not abolish protein synthesis, suggesting that the S-D-like sequence is essential, but not sufficient for ribosome binding. To determine how ribosomes reach the initiation codon after internal entry, we created additional mutants: when the authentic initiation codon at nucleotide (nt) 742 was mutated, a 180-nt downstream in-frame AUG codon at nt 922 is able to produce a truncated smaller protein. When this mutation was introduced into the full-length cDNA of CVB3, the derived viruses were still infectious. However, their infectivity was much weaker than that of the wild-type CVB3. In addition, when a stable stem-loop was inserted upstream of the initiation codon in the bicistronic RNA, translation was strongly inhibited. These data suggest that ribosomes reach the initiation codon from the IRES likely by scanning along the viral RNA

Recombinant Decorin Fusion Protein Attenuates Murine Abdominal Aortic Aneurysm Formation and Rupture

Abstract Decorin (DCN) is a small-leucine rich proteoglycan that mediates collagen fibrillogenesis, organization, and tensile strength. Adventitial DCN is reduced in abdominal aortic aneurysm (AAA) resulting in vessel wall instability thereby predisposing the vessel to rupture. Recombinant DCN fusion protein CAR-DCN was engineered with an extended C-terminus comprised of CAR homing peptide that recognizes inflamed blood vessels and penetrates deep into the vessel wall. In the present study, the role of systemically-administered CAR-DCN in AAA progression and rupture was assessed in a murine model. Apolipoprotein E knockout (ApoE-KO) mice were infused with angiotensin II (AngII) for 28 days to induce AAA formation. CAR-DCN or vehicle was administrated systemically until day 15. Mortality due to AAA rupture was significantly reduced in CAR-DCN-treated mice compared to controls. Although the prevalence of AAA was similar between vehicle and CAR-DCN groups, the severity of AAA in the CAR-DCN group was significantly reduced. Histological analysis revealed that CAR-DCN treatment significantly increased DCN and collagen levels within the aortic wall as compared to vehicle controls. Taken together, these results suggest that CAR-DCN treatment attenuates the formation and rupture of Ang II-induced AAA in mice by reinforcing the aortic wall