Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plas...Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plasmid pGEM-4Z; Homologous substrate RNA was transcribed for pRz277B and labelled withα-32 P-UTP. The trans-cleaving activity was studied by mixing the ribozymes and substrate in appropriate who under certain conditions. Results: The trans-cleaved products were generated after mixing the ribozyme with the substrate for 30 min, and accumulated more for 90 min. Conclusion: Both g. Rz74 and g. Rz55 possess trans-cleaving activity.展开更多
Recent reports show that many cellular RNAs are processed to form circular species that are relatively abundant and resistant to host nucleases.In some cases,such circles actually bind host microRNAs.Such depletion of...Recent reports show that many cellular RNAs are processed to form circular species that are relatively abundant and resistant to host nucleases.In some cases,such circles actually bind host microRNAs.Such depletion of available microRNAs appears to have biological roles;for instance,in homeostasis and disease.These findings regarding host RNA circles support a speculative reappraisal of the origin and mode of replication of hepatitis delta virus,hepatitis delta virus(HDV),an agent with a small circular RNA genome;specifically,it is proposed that in hepatocytes infected with hepatitis B virus(HBV),some viral RNA species are processed to circular forms,which by a series of chance events lead to an RNA that can be both replicated by host enzymes and assembled,using HBV envelope proteins,to form particles some of which are infectious.Such a model also may provide some new insights into the potential pathogenic potential of HDV infections.In return,new insights into HDV might provide information leading to a better understanding of the roles of the host RNA circles.展开更多
Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hep...Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.展开更多
Gene mutations influence the folding kinetics of hepatitis delta virus （HDV） ribozyme. In this work, we study the effect of the double mutation on the folding kinetics of HDV ribozyme. By using the master equation m...Gene mutations influence the folding kinetics of hepatitis delta virus （HDV） ribozyme. In this work, we study the effect of the double mutation on the folding kinetics of HDV ribozyme. By using the master equation method combined with RNA folding free energy landscape, we predict the folding kinetics of C13A：G82U and A16U：U79A mutated HDV sequences. Their folding pathways are identified by recursively searching the states with high net flux-in（out） population starting from the native state. The results indicate that the folding kinetics of C 13A：G82U mutation sequence is bi-phasic, which is similar to the wild type （wtHDV） sequence. While the folding kinetics of A16U：U79A mutation sequence is mono-phasic, it quickly folds to the native state in 30 s. Thus, the folding kinetics of double mutated HDV ribozyme depends on the mutation sites.展开更多
基金Supported by National Natural Science Foundation of China, NO.3960003l
文摘Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plasmid pGEM-4Z; Homologous substrate RNA was transcribed for pRz277B and labelled withα-32 P-UTP. The trans-cleaving activity was studied by mixing the ribozymes and substrate in appropriate who under certain conditions. Results: The trans-cleaved products were generated after mixing the ribozyme with the substrate for 30 min, and accumulated more for 90 min. Conclusion: Both g. Rz74 and g. Rz55 possess trans-cleaving activity.
文摘Recent reports show that many cellular RNAs are processed to form circular species that are relatively abundant and resistant to host nucleases.In some cases,such circles actually bind host microRNAs.Such depletion of available microRNAs appears to have biological roles;for instance,in homeostasis and disease.These findings regarding host RNA circles support a speculative reappraisal of the origin and mode of replication of hepatitis delta virus,hepatitis delta virus(HDV),an agent with a small circular RNA genome;specifically,it is proposed that in hepatocytes infected with hepatitis B virus(HBV),some viral RNA species are processed to circular forms,which by a series of chance events lead to an RNA that can be both replicated by host enzymes and assembled,using HBV envelope proteins,to form particles some of which are infectious.Such a model also may provide some new insights into the potential pathogenic potential of HDV infections.In return,new insights into HDV might provide information leading to a better understanding of the roles of the host RNA circles.
文摘Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.
基金Supported by the National Natural Science Foundation of China(31270761)
文摘Gene mutations influence the folding kinetics of hepatitis delta virus （HDV） ribozyme. In this work, we study the effect of the double mutation on the folding kinetics of HDV ribozyme. By using the master equation method combined with RNA folding free energy landscape, we predict the folding kinetics of C13A：G82U and A16U：U79A mutated HDV sequences. Their folding pathways are identified by recursively searching the states with high net flux-in（out） population starting from the native state. The results indicate that the folding kinetics of C 13A：G82U mutation sequence is bi-phasic, which is similar to the wild type （wtHDV） sequence. While the folding kinetics of A16U：U79A mutation sequence is mono-phasic, it quickly folds to the native state in 30 s. Thus, the folding kinetics of double mutated HDV ribozyme depends on the mutation sites.
