NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.
DNA Damage ; drug effects ; DNA Repair ; genetics ; DNA Replication ; genetics ; DNA-Binding Proteins ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Endopeptidases ; genetics ; Gene Knockout Techniques ; Humans ; Hydrogen Peroxide ; toxicity ; NEDD8 Protein ; genetics ; Oxidative Stress ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ubiquitin-Conjugating Enzymes ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Ubiquitination ; genetics ; Ultraviolet Rays

OBJECTIVETo investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.

METHODSForty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.

RESULTSCompared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.

CONCLUSIONCompared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

Adult ; Antiviral Agents ; pharmacology ; China ; DNA, Viral ; genetics ; metabolism ; Drug Resistance, Viral ; genetics ; Female ; HIV Infections ; drug therapy ; HIV-1 ; drug effects ; genetics ; metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Middle Aged ; Mutation ; Proviruses ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; RNA-Directed DNA Polymerase

BACKGROUND/AIMS: To investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B. METHODS: Seventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations. RESULTS: The median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response. CONCLUSIONS: The baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.
Adenine/administration & dosage/*analogs & derivatives/therapeutic use ; Adult ; Aged ; Aged, 80 and over ; Antiviral Agents/administration & dosage/*therapeutic use ; DNA-Directed DNA Polymerase/genetics ; Drug Resistance, Viral/genetics ; Drug Therapy, Combination ; Female ; Hepatitis B virus/*genetics ; Hepatitis B, Chronic/*drug therapy/virology ; Humans ; Lamivudine/administration & dosage/*therapeutic use ; Male ; Middle Aged ; Organophosphonates/administration & dosage/*therapeutic use ; Treatment Outcome ; Viral Load/drug effects ; Young Adult

OBJECTIVETo study resistance evolution pathway of HIV-1 CRF_BC under drug selection pressure, and compare with B subtype.

METHODSBased on the reverse transcriptase region of CRF_ 97BC HIV-1 from 588 treatment-naive and 274 treatment patients, selection pressure based method was used to select resistance-associated mutations, and Bayesian network was used to construct the resistance evolutionary pathway under antiretroviral therapy. Meanwhile, it was constructed that the resistance evolutionary pathway for B subtype with the same regimens using the data from HIV resistance database, and made a comparison with CRF_07BC.

RESULTSThe major resistance mutations for CRF_07BC were identified including K103N, Q197K, V179D and Y188L. While for B subtype, the major resistance mutations include M184V, K103N,Y181C, T69N,G190A, K238T,Y188H and P225H. Much difference was observed between these two classes. However, the classical TMA1 (41L, 210W and 215Y) and TMA2 (67N, 70R and 219E/Q) pathways exist in both pathways. As different from B subtype, the predicted major drug resistance mutations for CRF_07BC did not contain TAM-related mutations, and nucleoside reverse transcriptase inhibitor-related mutations and non-nucleoside reverse transcriptase inhibitor-related mutations were mutually depending on each other.

CONCLUSIONHIV-1 CRF_07BC showed distinctive resistance evolutionary pathway, the mutations K103N,Q197K,V179D and Y188L were the major resistance mutations, and different resistance evolutionary pathways were observed between HIV-1 CRF_07BC and B subtype.

Anti-HIV Agents ; pharmacology ; Bayes Theorem ; Drug Resistance, Viral ; genetics ; Evolution, Molecular ; HIV-1 ; drug effects ; enzymology ; genetics ; Humans ; Mutation ; RNA-Directed DNA Polymerase ; genetics

Adult ; Aged ; Antiviral Agents ; pharmacology ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Genes, Viral ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Lamivudine ; pharmacology ; Male ; Middle Aged ; Nucleotides ; pharmacology ; Young Adult

OBJECTIVETo explore the mechanism for adefovir dipivoxil (ADV) resistance occurred in chronic hepatitis B patients of a series of phase III clinical trails.

METHODS30 resistant HBV strains were selected out from 177 cases of ADV treated chronic hepatitis B patients. HBV polymerase RT region were amplified by nested PCR and analyzed with the standard nucleotide sequence of HBV strains deposited in GeneBank.

RESULTS21 out of 30 HBV strains were primary resistant strains, among them 5 HBV strains (23.8%, 5/21) had the polymorphism site of rtN118H. While the other 9 HBV strains showed secondary resistance, variations in conservative region C (rtM207V) and other non-conservative regions were found. The classic mutation sites such as rtN236T and rtA181V/T were not found.

CONCLUSIONSPolymorphism site of rtN118H might be responsible for HBV primary resistance to ADV therapy. rtM207V variation in HBV RT C domain and other variation sites might play a role in HBV secondary resistance to ADV treatment, and natural resistant quasispecies may be the basis for the ADV quick resistance. These conclusions await further confirmation by phenotype test.

Adenine ; analogs & derivatives ; pharmacology ; therapeutic use ; Adult ; Alanine Transaminase ; blood ; Amino Acid Sequence ; Antiviral Agents ; pharmacology ; therapeutic use ; Base Sequence ; DNA Primers ; DNA, Viral ; blood ; Drug Resistance, Viral ; Female ; Genotype ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; genetics ; virology ; Humans ; Male ; Molecular Sequence Data ; Organophosphonates ; pharmacology ; therapeutic use ; Polymorphism, Genetic ; genetics ; RNA-Directed DNA Polymerase ; drug effects ; genetics ; Reverse Transcriptase Inhibitors ; pharmacology ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

BACKGROUND/AIMS: Adefovir dipivoxil (ADV) is a nucleotide analogue that inhibits wild-type hepatitis B virus (HBV) and lamivudine (LMV)-resistant HBV mutants. The aim of this study was to elucidate the efficacy of ADV monotherapy and the incidence of genotypic resistance to ADV in patients with LMV-resistant chronic HBV infection. METHODS: This study involved 124 patients with chronic HBV infection who had received ADV monotherapy due to the presence of LMV-resistant HBV mutants. The efficacy of ADV was evaluated by the normalization of serum alanine aminotransferase (ALT) level and by the reduction of serum HBV DNA level (with cutoff levels of 2x10(4) IU/mL and 2x10(2) IU/mL). The cumulative rate of HBeAg loss or seroconversion was assessed in HBeAg-positive patients. The development of mutations in the reverse trancriptase region of HBV DNA polymerase was evaluated by direct sequencing analysis during ADV monotherapy. RESULTS: The mean serum HBV DNA level was 5.94 log10IU/mL. At 12 and 24 months after ADV monotherapy, the cumulative rates of serum ALT normalization were 69.4% and 75.5%, respectively, and those of serum HBV DNA reduction were 79.8% and 89.2% for a cutoff level of 2x10(4) IU/mL, and 44.2% and 59.0% for a cutoff of 2x10(2) IU/mL. The mean serum HBV DNA levels at 12 and 24 months were significantly lower than baseline, at 3.24 and 3.04 log10IU/mL, respectively (P<0.001). At 12 months after ADV treatment, the cumulative rates of HBeAg loss and seroconversion were 15.8% and 10.5%, respectively, and the rtN236T and rtA181T/V mutants in HBV DNA polymerase were identified in 25% and 64% of patients, respectively. CONCLUSIONS: Although ADV monotherapy is effective, it leads to a high rate of mutations of HBV DNA reverse transcriptase gene in patients with chronic HBV infections who have LMV-resistant HBV mutants.
Adenine/*analogs & derivatives/therapeutic use ; Adult ; Amino Acid Substitution ; Antiviral Agents/*therapeutic use ; DNA, Viral/analysis ; DNA-Directed DNA Polymerase/drug effects/genetics ; Drug Resistance, Viral ; Female ; Genotype ; Hepatitis B e Antigens/blood ; Hepatitis B virus/*drug effects ; Hepatitis B, Chronic/diagnosis/*drug therapy ; Humans ; Lamivudine/*therapeutic use ; Male ; Middle Aged ; Mutation ; Phosphonic Acids/*therapeutic use ; Sequence Analysis, DNA

OBJECTIVETo detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates.

METHODSUsing the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC).

RESULTSAmong the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively.

CONCLUSIONNot only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.

Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; genetics ; Humans ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; Phosphoric Acids ; Polymerase Chain Reaction ; Rifampin ; pharmacology

OBJECTIVETo evaluate the prophylactic efficacy of adefovir dipivoxil (ADV) for post-transplant recurrence of hepatitis B virus (HBV) with lamivudine-resistant YMDD mutation in liver recipients.

METHODSFrom March 2004 to May 2006, 20 patients with chronic hepatitis B associated with YMDD mutant HBV prior to liver transplantation received treatment with ADV and additional intramuscular hepatitis B immunoglobulin (HBIG) for prevention of post-transplant graft reinfection. The liver function, serum HBsAg, anti-HBs (HBIG), HBeAg, anti-HBc, anti-HBe, HBV DNA and creatinine were examined in all the patients before and after the transplantation.

RESULTSThe median follow-up duration of these patients after the transplantation was 33.5 months. Nineteen patients survived and one patient died of recurrent hepatocellular carcinoma. There was significant difference in YMDD mutation rate between the patients with HBV-DNA over 10(6) copies/ml and those with HBV-DNA less than 10(6) copies/ml (12.4% vs 2.5%, P < 0.05). HBV-DNA was undetectable at 4 weeks after the transplantation in 95.0% of the patients (19/20) and at 6 months in one case. No recurrence of hepatitis B was detected by long-term regular testing of HBsAg, HBeAg and HBV-DNA. Serum creatinine increased in 1 case 1 year after the use of ADV.

CONCLUSIONADV offers protection against recurrence of HBV with YMDD mutation after liver transplantation with only mild nephrotoxicity, but renal function monitoring during the use of ADV is still necessary.

Adult ; Aged ; Amino Acid Motifs ; Antiviral Agents ; therapeutic use ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; Female ; Hepatitis B ; prevention & control ; Hepatitis B virus ; genetics ; Humans ; Lamivudine ; therapeutic use ; Liver Cirrhosis ; surgery ; virology ; Liver Transplantation ; adverse effects ; Male ; Middle Aged ; Mutation ; Recurrence

BACKGROUNDThe most frequently used therapy for post-transplantation recurrence of hepatitis B virus (HBV) infection is lamivudine, but this drug is associated with a high resistance rate due to YMDD mutant. In preliminary reports, adefovir dipivoxil (ADV) has been shown to have activity against lamivudine-resistant strains of HBV. However, clinical experience in treatment of HBV infection after liver transplantation (LT) is still not entirely clear. This study was aimed to evaluate the prophylactic efficacy of ADV plus hepatitis B immunoglobulin (HBIG) in patients with YMDD mutant before LT.

METHODSFrom March 2004 to March 2006, 16 patients with chronic hepatitis B had lamivudine-resistant YMDD mutants detected prior to liver transplantation and received treatment with ADV plus additional intramuscular HBIG after LT as prophylaxis against graft reinfection. Tests for liver function, serum HBsAg, anti-HBs (HBIG), HBeAg, anti-HBc, anti-HBe, HBV-DNA, and creatinine were assessed pre- or post-liver transplantation.

RESULTSThe median follow-up of these patients post-liver transplantation was 19.4 months. Fifteen patients survived and one patient died of recurrence of hepatocellular carcinoma (HCC). There was significant difference (10.98% vs. 2.26%, P < 0.05) in YMDD mutant rate between the patients with HBV-DNA over 10(6) copies/ml and those with HBV-DNA less than 10(6) copies/ml. Fifteen patients (93.8%) had undetectable HBV-DNA at 4 weeks and 1 (6.3%) at 6 months after LT. No hepatitis B recurrence was detected by persistent testing of HBsAg, HBeAg, and HBV-DNA and no increase of serum creatinine level associated with ADV was observed in any of the patients.

CONCLUSIONADV combined with intramuscular HBIG can effectively prevent patients with pre-transplantation YMDD mutant from HBV recurrence after LT.

Adenine ; analogs & derivatives ; therapeutic use ; Adult ; Aged ; Antiviral Agents ; therapeutic use ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; Hepatitis B ; prevention & control ; Humans ; Lamivudine ; therapeutic use ; Liver Transplantation ; adverse effects ; Male ; Middle Aged ; Mutation ; Organophosphonates ; therapeutic use ; Recurrence