Core Amino Acid Substitutions Research Articles

Amino acid substitutions in core and HBsAg regions of hepatitis B virus in patients with monoinfection and HBV/HIV-coinfection in the Republic of Guinea

The aim of this study was to characterize the genetic variants of HBV currently circulating in the Republic of Guinea, based on the nucleotide sequences of the complete virus genome, and to analyze clinically significant mutations in the Core and HBsAg regions during HBV monoinfection and HBV/HIV coinfection.Materials and methods. The study material was represented by 2616 blood serum samples collected from residents of the Republic of Guinea. The subjects were examined for the presence of HBV markers with a qualitative detection of HBsAg, HBs IgG, and HBCore IgG. HBV complete genome nucleotide sequences were obtained for 298 samples including HIV/HBV coinfected patients. Amplification and subsequent sequencing of HBV were performed using nested PCR with pair’s overlapping primers jointly flanking the complete HBV genome (S, P, C, X genes).Results. HBV serological markers were detected in 80.77% samples, while HBsAg was detected in 16.01% of the examined group. HBV DNA we detected in 22.36%. The prevalence of HBsAg-negative HBV in patients with HIV RNA is 45.16%, which is significantly higher than 6.07% found in the group without HIV infection. Phylogenetic analysis of HBV in the examined samples showed that HBV genotype E (75.5%) predominates in the group compared to HBV genotype D1 (9.39%), D2 (4.02%), D3 (6.37%), and A2 (4.7%). In the tested group, the variability of amino acids among the HBV samples was higher in the PreCore/Core region than in the PreS1/PreS2/S region. SHB mutations were detected in 83,89%, Core mutations in 94.29%, PreCore amino acid substitutions in 16.77% of the patients, respectively.The results obtained in this work demonstrate a high prevalence of HBV in the region and indicate the need for further largescale studies of HBV mutations in order to improve strategies for disease control and prevention in the Republic of Guinea.

Core amino acid substitutions in HCV-3a isolates from Pakistan and opportunities for multi-epitopic vaccines

Hepatitis C virus (HCV), which infected 71 million worldwide and about 5%–6% are from Pakistan, is an ssRNA virus, responsible for end‐stage liver disease. To date, no effective therapy is available to cure this disease. Hence, it is important to study the most prevalent genotypes infecting human population and design novel vaccine or small molecule inhibitors to control the infections associated with HCV. Therefore, in this study clinical samples (n = 35; HCV-3a) from HCV patients were subjected to Sanger sequencing method. The sequencing of the core gene, which is generally considered as conserved, involved in the detection, quantitation and genotyping of HCV was performed. Multiple mutations, that is, R46C, R70Q, L91C, G60E, N/S105A, P108A, N110I, S116V, G90S, A77G and G145R that could be linked with response to antiviral therapies were detected. Phylogenetic analysis suggests emerging viral isolates are circulating in Pakistan. Using ab initio modelling technique, we predicted the 3D structure of core protein and subjected to molecular dynamics simulation to extract the most stable conformation of the structure for further analysis. Immunoinformatic approaches were used to propose a multi-epitopes vaccine against HCV by using core protein. The vaccine constructs consist of nine CTL and three HTL epitopes joined by different linkers were docked against the two reported Toll-like receptors (TLR-3 and TLR-8). Docking of vaccine construct with TLR-3 and TLR-8 shows proper binding and in silico expression of the vaccine resulted in a CAI value of 0.93. These analyses suggest that specific immune responses may be produced by the proposed vaccine. Communicated by Ramaswamy H. Sarma

Anti-HBV activity of the HBV capsid assembly modulator JNJ-56136379 across full-length genotype A-H clinical isolates and core site-directed mutants in vitro.

To characterize antiviral activity of the capsid assembly modulator (CAM-N) JNJ-56136379 against HBV genotypes and variants carrying amino acid substitutions in the core protein. Anti-HBV activity of JNJ-56136379 was investigated against a diverse panel of 53 HBV clinical isolates (genotypes A-H). The impact of core amino acid substitutions using site-directed mutants (SDMs) was assessed in a transient replication assay. JNJ-56136379 median 50% effective concentration (EC50) values across all genotypes were 10-33 nM versus 17 nM (genotype D reference). JNJ-56136379 remained active against isolates carrying nucleos(t)ide analogue resistance mutations (median EC50 2-25 nM) or basal core promoter (BCP) ± precore (PC) mutations (median EC50 13-20 nM) or PC mutations (median EC50 11 nM), representing activity against isolates from HBeAg-positive and -negative hepatitis B patients. Core amino acid substitutions in the CAM-binding pocket, when tested as SDMs at positions 23, 25, 30, 33, 37, 106, 110, 118, 124, 127 and 128, reduced JNJ-56136379 anti-HBV activity; EC50 fold increases ranged from 3.0 (S106T) to 85 (T33N). All substitutions were rare in a public database of >7600 HBV core sequences (frequencies 0.01%-0.3%). Nucleos(t)ide analogues retained full activity against these core SDMs. JNJ-56136379, a potent HBV CAM-N, currently in Phase 2 clinical development, was generally fully active against an extensive panel of genotype A-H clinical isolates, regardless of the presence of nucleos(t)ide analogue resistance or BCP/PC mutations. JNJ-56136379 activity was reduced by some core amino acid substitutions in the CAM-binding pocket.

Single-cell PCR of the luciferase conserved catalytic domain reveals a unique cluster in the toxic bioluminescent dinoflagellate Pyrodinium bahamense

AB Aquatic Biology Contact the journal Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AB 25:139-150 (2016) - DOI: https://doi.org/10.3354/ab00664 Single-cell PCR of the luciferase conserved catalytic domain reveals a unique cluster in the toxic bioluminescent dinoflagellate Pyrodinium bahamense Kathleen D. Cusick1,6,*, Steven W. Wilhelm1,2, Paul E. Hargraves4,5, Gary S. Sayler1,2,3,4,5 1Center for Environmental Biotechnology, The University of Tennessee, 676 Dabney Hall, Knoxville, TN 37996, USA 2Department of Microbiology, The University of Tennessee, Knoxville, TN 37996, USA 3Department of Ecology and Evolutionary Biology, The University of Tennessee, Knoxville, TN 37996, USA 4Harbor Branch Oceanographic Institute of Florida Atlantic University, Ft Pierce, FL 34946, USA 5UT-ORNL Joint Institute of Biological Sciences, Oak Ridge, TN 37831, USA 6Present address: US Naval Research Lab, Washington, DC 20375, USA *Corresponding author: kdcusick@gmail.com ABSTRACT: Pyrodinium bahamense is a toxic, bioluminescent dinoflagellate with a record of intense bloom formation in both the Atlantic-Caribbean and Indo-Pacific regions. To date, limited genetic information exists for P. bahamense in comparison to other closely related harmful algal bloom taxa such as Alexandrium, or other bioluminescent taxa such as Pyrocystis. This study utilized single-cell PCR to explore the molecular diversity of P. bahamense within the Indian River Lagoon (IRL), Florida, USA, and a bioluminescent bay in Puerto Rico. Pyrodinium-specific primers targeting a ca.1.2-kb region of the 18S rRNA gene and degenerate primers targeting the conserved catalytic domain of the luciferase gene (lcf) were applied to single cells isolated from both geographic regions as well as single cells of clonal isolates from the IRL. Phylogenetic analysis revealed that while P. bahamense is more closely related to Alexandrium spp. at the 18S rRNA gene level, its lcf sequences are more closely related to Pyrocystis spp. than Alexandrium spp. Pyrodinium bahamense lcf sequences from the Western Atlantic formed 2 distinct clusters. These clusters were defined by a set of core amino acid substitutions, and the extent of variation was greater than that recorded between the established variants of Pyrocystis lcf. lcf sequences from an Indo-Pacific strain formed a third distinct cluster. Based on these results, the potential of lcf for use in tracking sub-populations of P. bahamense is discussed. KEY WORDS: Indian River Lagoon · Dinoflagellate · Pyrodinium bahamense · Saxitoxin · Single‑cell PCR · Luciferase · Bioluminescence Full text in pdf format Supplementary material Supplementary material PreviousNextCite this article as: Cusick KD, Wilhelm SW, Hargraves PE, Sayler GS (2016) Single-cell PCR of the luciferase conserved catalytic domain reveals a unique cluster in the toxic bioluminescent dinoflagellate Pyrodinium bahamense. Aquat Biol 25:139-150. https://doi.org/10.3354/ab00664 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AB Vol. 25. Online publication date: September 21, 2016 Print ISSN: 1864-7782; Online ISSN: 1864-7790 Copyright © 2016 Inter-Research.

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System.

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment.

Low-dose pegylated interferon-alpha-2a plus ribavirin therapy for elderly and/or cirrhotic patients with hepatitis C virus genotype-1b and high viral load

Pegylated interferon (PEG-IFN) plus ribavirin therapy is still recommended for elderly and/or cirrhotic patients. This study examined whether sustained virological response (SVR) to low-dose PEG-IFN-α2a plus ribavirin therapy for elderly and/or cirrhotic patients could be predicted based on viral reduction within 2 weeks after therapy initiation or interleukin IL-(28B) polymorphism and viral mutations. Participants comprised 115 elderly (≥65 years) and/or cirrhotic patients with genotype-1b and high viral load. Reduced doses of PEG-IFN-α2a (90 μg/kg/week) and ribavirin (400-800 mg/day) were administered for 48-72 weeks based on virological response of each patient. SVR was achieved in 34% (39/115), and treatment was discontinued in 15% (17/115). Univariate analysis identified age, α-fetoprotein, fibrosis marker, interferon sensitivity-determining region (ISDR), IL-28B polymorphism and level of viral reduction within 2 weeks as factors contributing significantly to SVR. However, no significant differences were noted in core amino acid substitutions. Multivariate analysis identified age, hyaluronic acid, ISDR and viral reduction as factors independently associated with SVR. Positive predictive value (PPV) and negative predictive value (NPV) of SVR based on the level of viral reduction at 2 weeks (cutoff level, 1.7 log IU/ml) were 83% and 84%, respectively. The PPV of SVR based on IL-28B major and ISDR mutant was 70%, and the NPV of SVR based on IL-28B minor and wild-type ISDR was 89%. Evaluations of viral reduction at 2 weeks or both IL-28B and ISDR are useful to predict SVR to low-dose PEG-IFN-α2a plus ribavirin therapy for elderly and/or cirrhotic patients.

Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production

The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV.

Hepatitis C virus: How genetic variability affects pathobiology of disease

As an RNA virus, hepatitis C virus (HCV) shows a characteristically high level of nucleotide diversity. Accumulation of nucleotide substitutions in the virus has resulted in diversification into quasispecies, subtypes and distinct genotypes. Pathobiological studies linking nucleotide and amino acid sequences with clinical findings have identified relationships between certain genotypes and characteristic biological properties. Genotype 3 HCV infection was found to be associated with a high level of liver steatosis. Genotypes 1 and 4 were found to be more resistant to interferon (IFN) based therapies than genotypes 2 and 3. Studies of genotype 1 sequences obtained from patients treated with IFN have identified a relationship between favorable response to interferon therapy and amino acid substitutions in the NS5A region (interferon response determining region; ISDR). Further studies have identified a relationship between the effect of IFN therapy and other regions of the NS5A protein. More recently, a relationship has been found between poor response to peg-IFN plus ribavirin combination therapy and substitutions at amino acid 70 and 91 in the core protein. Furthermore, a correlation between human genetic variation in the IL28B (IFN-lamda 3) locus and core amino acid substitutions has been characterized. In this review we briefly summarize the discovery, classification and nomenclature of HCV genotypes and subtypes. We also discuss amino acid substitutions within specific regions that have been reported to be associated with outcome of IFN and peg-IFN plus ribavirin combination therapy.

Amino acid substitutions in core and NS5A regions of the HCV genome can predict virological decrease with pegylated interferon plus ribavirin therapy

The current standard therapy for chronic hepatitis C is pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. Recently, it has been reported that amino acid (aa) substitutions in the core region, as well as the IFN-sensitivity-determining region (ISDR), were predictive of non-virological response (NVR), sustained virological response (SVR) and early virological response. Despite the importance of these two predictive factors for combination therapy, their interaction is poorly understood. A total of 117 patients who were treated with PEG-IFN-α2b plus RBV combination therapy were selected for participation in this study. We determined the aa sequences in the core region and ISDR by direct sequencing and analysed them along with clinical data to identify predictive factors for therapeutic response. The aa sequences in the core region and γ-glutamyl transpeptidase (GTP) levels were associated with SVR and NVR, but aa sequences in the ISDR were not. However, substitutions at both aa 70 and aa 91 in the core region without substitutions in the ISDR and higher levels of γ-GTP were independent predictive factors for NVR. Wild-type aa 70 and aa 91 in the core region, higher platelet counts and lower levels of γ-GTP were independent predictive factors for SVR. These results indicate that analyses of aa substitutions in both the core region and the ISDR are useful for predicting the effectiveness of combination therapy, and could help to avoid therapy exposure for patients who have a low probability of SVR.

A PAL for Schistosoma mansoni PHM

Parasitic helminth neuromuscular function is a proven target for chemotherapeutic control. Although neuropeptide signalling plays a key role in helminth motor function, it has not yet provided targets for known anthelmintics. The majority of biologically active neuropeptides display a C-terminal amide (NH2) motif, generated exclusively by the sequential action of two enzymes, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidylglycine α-amidating lyase (PAL). Further to our previous description of a monofunctional PHM enzyme (SmPHM) from the human blood fluke Schistosoma mansoni, here we describe a cDNA encoding S. mansoni PAL (SmPAL). SmPAL is a monofunctional enzyme which, following heterologous expression, we find to have functionally similar catalytic activity and optimal pH values, but key catalytic core amino acid substitutions, when compared to other known PALs including those found in humans. We have used in situ hybridisation to demonstrate that in adult schistosomes, SmPAL mRNA (Sm-pal-1) is expressed in neuronal cell bodies of the central nervous system, consistent with a role for amidated neuropeptides in S. mansoni neuromuscular function. In order to validate SmPAL as a putative drug target we applied published RNA interference (RNAi) methods in efforts to trigger knockdown of Sm-pal-1 transcript in larval schistosomula. Although transcript knockdown was recorded on several occasions, silencing was variable and inconsistent and did not associate with any observable aberrant phenotype. The inconsistent outcomes of RNAi suggest that there may be tissue-specific differences in the applicability of RNAi methods for S. mansoni, with neuronal targets proving more difficult or refractory to knockdown. The key role played by schistosome amidating enzymes in neuropeptide maturation make them appealing as drug targets; their validation as such will depend on the development of more robust reverse genetic tools to facilitate efficient neuronal gene function studies.

Quantification of hepatitis C amino acid substitutions 70 and 91 in the core coding region by real-time amplification refractory mutation system reverse transcription-polymerase chain reaction

Objective. The effects of hepatitis C virus (HCV) sequence variations on the success of antiviral therapy or the development of hepatocellular carcinoma (HCC) are complex for many reasons. Recently, there have been several reports on the effects of genotype 1b HCV core amino acid substitutions 70 and/or 91 on the outcome of antiviral therapies and the clinical course. The purpose of this study was to establish real-time amplification refractory mutation system (ARMS) reverse transcription (RT)-polymerase chain reaction (PCR) assays for easy detection of these HCV mutations. Material and methods. Plasmids p-core-W, including the wild-type HCV core coding region (70R and 91L), and p-core-M, including the mutant-type HCV core (70Q and 91M), were constructed by cloning and PCR-based mutagenesis for control vector of the wild-type core and that of the mutant core, respectively. Using serially diluted forms of these vectors, SyBr Green-based real-time ARMS RT-PCR detection with each of the specific primer pairs was performed. Results. Each primer could clearly distinguish the difference between p-core-W and p-core-M at the same copy numbers. Concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-W versus p-core-M could be distinguished, while for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. Conclusions. This method could be a useful alternative for the detection of genotype 1b HCV core amino acid substitutions 70 and 91 and be reliably applied for rapid screening.

Selection of hepatitis B virus variants with aminoacid substitutions inside the core antigen during interferon-alpha therapy.

The hepatitis B virus (HBV) core antigen carries many epitopes relevant for B and T cell response that show aminoacid variation during viral infection. In a longitudinal analysis, sequential serum samples of 15 patients that suffered from chronic HBV infection were collected before, during, and after high-dose IFN-alpha treatment. The HBV preCore/Core (preC/C) sequence of the selected samples in each patient was determined and analysed for sequence variations compared to the pretreatment sample. The positions of HBV core aminoacid substitutions were assigned to immunodominant B, CD4(+) and CD8(+) cell epitopes. Seventy-five percent of all aminoacid substitutions were found within immunodominant T and B cell epitopes (12.5% were inside known HBV core mutation cluster regions) that show an increased number of clustered aminoacid substitutions during chronic HBV infection and overlap partially with the immunodominant epitopes. Only 12.5% of the detected core antigen aminoacid substitutions could not be assigned to any epitope or mutation cluster region. Stable HBV core antigen aminoacid substitutions, which were found between pretreatment sequence and the last sequence analysed during therapy, were found most frequently inside T helper cell epitopes. This longitudinal analysis of aminoacid substitutions inside the HBV core antigen in patients with chronic HBV infection shows that core aminoacid variations occur most frequently inside immunodominant HBV core epitopes, possibly due to an immuneselective pressure of the host against the virus. The data also suggest that stable HBV variants with aminoacid substitutions in immunodominant core epitopes can be selected during high-dose IFN-alpha therapy and persist after the end of treatment.

Susceptibility of beta-lactamase to core amino acid substitutions.

To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases. It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.