HIV-1 Rev is an accessory protein that plays a key role in nuclear exportation,stabilization,and translation of the viral mRNAs.Rev of HIV-1 clade BC often shows a truncation of 16 AAs due to a premature stop codon at...HIV-1 Rev is an accessory protein that plays a key role in nuclear exportation,stabilization,and translation of the viral mRNAs.Rev of HIV-1 clade BC often shows a truncation of 16 AAs due to a premature stop codon at residue 101.This stop codon presents the highest frequency in clade BC and the lowest frequency in clade B.In order to discover the potential biological effect of this truncation on Rev activity and virus replication of clade BC,we constructed Rev expression vectors of clade BC with or without 16 AAs within C-terminal separately,and replaced the stop codon by Q in a CRF07_BC infectious clone.We found that 16 AAs truncation had no effect on expression and activity of Rev in clade BC.Also,the mutation from the stop codon to Q had no effect on virus replication of clade BC.Next,to investigate the effect of this truncation on Rev activity and replication capacity of clade B,Rev expression vectors of clade B carrying or lacking 16 AAs in C-terminal were constructed respectively,and residue Q at position 101 within Rev was substituted by the stop codon in a clade B infectious clone.It was found that 16 AAs truncation significantly down-regulated Rev expression and impaired clade B Rev activity.Furthermore,a Q-to-stop codon substitution within Rev significantly reduced viral replication fitness of clade B.These results indicate that the premature stop codon at residue 101 within Rev exerts diverse impact on viral replication among different HIV-1 clades.展开更多
Background:A key step in gene expression is the recognition of the stop codon to terminate translation at the correct position.However,it has been observed that ribosomes can misinterpret the stop codon and continue t...Background:A key step in gene expression is the recognition of the stop codon to terminate translation at the correct position.However,it has been observed that ribosomes can misinterpret the stop codon and continue the translation in the 3′UTR region.This phenomenon is called stop codon read-through(SCR).It has been suggested that these events would occur on a programmed basis,but the underlying mechanisms are still not well understood.Methods:Here,we present a strategy for the comprehensive identification of SCR events in the Drosophila melanogaster transcriptome by evaluating the ribosomal density profiles.The associated ribosomal leak rate was estimated for every event identified.A statistical characterization of the frequency of nucleotide use in the proximal region to the stop codon in the sequences associated to SCR events was performed.Results:The results show that the nucleotide usage pattern in transcripts with the UGA codon is different from the pattern for those transcripts ending in the UAA codon,suggesting the existence of at least two mechanisms that could alter the translational termination process.Furthermore,a linear regression models for each of the three stop codons was developed,and we show that the models using the nucleotides at informative positions outperforms those models that consider the entire sequence context to the stop codon.Conclusions:We report that distal nucleotides can affect the SCR rate in a stop-codon dependent manner.展开更多
Molecular virology methods including polymerase chain reaction, cloning and sequencing have revolutionised our understanding of viral genome variation. In the case of hepatitis B virus (HBV), sequencing studies have i...Molecular virology methods including polymerase chain reaction, cloning and sequencing have revolutionised our understanding of viral genome variation. In the case of hepatitis B virus (HBV), sequencing studies have identified a number of virus variants normally found during the natural course of chronic infection. The appearance of the precore stop codon (with G-for-A substitution at position 1896) and basal core promoter (BCP) (with A-for-T and G-for-A, at positions 1762 and 1764, respectively) variants which reduce or abrogate hepatitis B e antigen (HBeAg) production, heralds the initiation of the seroconversion phase from HBeAg to anti-HBe positivity. The gradual removal of the tolerogenic effect of HBeAg leads to the awakening of the immune response (immune clearance phase). Most patients after HBeAg seroconversion become “inactive HBsAg carriers”. However during the course of infection precore and/or BCP variants may emerge and be selected leading to HBeAg negative chronic hepatitis B (CHB) with high viremia levels (reactivation phase). The prevalence of HBeAg negative CHB has been increasing over the last few decades and has become the commonest type of HBV infection in many countries of the world. This probably reflects the aging of existing HBV carriers and the effective prevention measures restricting new HBV infections. Frequent acute exacerbations accompanied by high viral replication, elevated alanine aminotransferase levels and histological activity are a common feature of HBeAg negative CHB leading to cirrhosis much faster than in HBeAg positive CHB patients.展开更多
文摘HIV-1 Rev is an accessory protein that plays a key role in nuclear exportation,stabilization,and translation of the viral mRNAs.Rev of HIV-1 clade BC often shows a truncation of 16 AAs due to a premature stop codon at residue 101.This stop codon presents the highest frequency in clade BC and the lowest frequency in clade B.In order to discover the potential biological effect of this truncation on Rev activity and virus replication of clade BC,we constructed Rev expression vectors of clade BC with or without 16 AAs within C-terminal separately,and replaced the stop codon by Q in a CRF07_BC infectious clone.We found that 16 AAs truncation had no effect on expression and activity of Rev in clade BC.Also,the mutation from the stop codon to Q had no effect on virus replication of clade BC.Next,to investigate the effect of this truncation on Rev activity and replication capacity of clade B,Rev expression vectors of clade B carrying or lacking 16 AAs in C-terminal were constructed respectively,and residue Q at position 101 within Rev was substituted by the stop codon in a clade B infectious clone.It was found that 16 AAs truncation significantly down-regulated Rev expression and impaired clade B Rev activity.Furthermore,a Q-to-stop codon substitution within Rev significantly reduced viral replication fitness of clade B.These results indicate that the premature stop codon at residue 101 within Rev exerts diverse impact on viral replication among different HIV-1 clades.
基金LIE is funded by CONICET Ph.D.Fellowship.AMA and LD are researchers of CONICET(Argentina).JRR is Full Professor at the UNLP(Argentina).This work was supported by CONICET,Argentina(PIP2017-00059).
文摘Background:A key step in gene expression is the recognition of the stop codon to terminate translation at the correct position.However,it has been observed that ribosomes can misinterpret the stop codon and continue the translation in the 3′UTR region.This phenomenon is called stop codon read-through(SCR).It has been suggested that these events would occur on a programmed basis,but the underlying mechanisms are still not well understood.Methods:Here,we present a strategy for the comprehensive identification of SCR events in the Drosophila melanogaster transcriptome by evaluating the ribosomal density profiles.The associated ribosomal leak rate was estimated for every event identified.A statistical characterization of the frequency of nucleotide use in the proximal region to the stop codon in the sequences associated to SCR events was performed.Results:The results show that the nucleotide usage pattern in transcripts with the UGA codon is different from the pattern for those transcripts ending in the UAA codon,suggesting the existence of at least two mechanisms that could alter the translational termination process.Furthermore,a linear regression models for each of the three stop codons was developed,and we show that the models using the nucleotides at informative positions outperforms those models that consider the entire sequence context to the stop codon.Conclusions:We report that distal nucleotides can affect the SCR rate in a stop-codon dependent manner.
文摘Molecular virology methods including polymerase chain reaction, cloning and sequencing have revolutionised our understanding of viral genome variation. In the case of hepatitis B virus (HBV), sequencing studies have identified a number of virus variants normally found during the natural course of chronic infection. The appearance of the precore stop codon (with G-for-A substitution at position 1896) and basal core promoter (BCP) (with A-for-T and G-for-A, at positions 1762 and 1764, respectively) variants which reduce or abrogate hepatitis B e antigen (HBeAg) production, heralds the initiation of the seroconversion phase from HBeAg to anti-HBe positivity. The gradual removal of the tolerogenic effect of HBeAg leads to the awakening of the immune response (immune clearance phase). Most patients after HBeAg seroconversion become “inactive HBsAg carriers”. However during the course of infection precore and/or BCP variants may emerge and be selected leading to HBeAg negative chronic hepatitis B (CHB) with high viremia levels (reactivation phase). The prevalence of HBeAg negative CHB has been increasing over the last few decades and has become the commonest type of HBV infection in many countries of the world. This probably reflects the aging of existing HBV carriers and the effective prevention measures restricting new HBV infections. Frequent acute exacerbations accompanied by high viral replication, elevated alanine aminotransferase levels and histological activity are a common feature of HBeAg negative CHB leading to cirrhosis much faster than in HBeAg positive CHB patients.
