Reverse Transcriptase Domain Research Articles

Sequences in the terminal protein and reverse transcriptase domains of the hepatitis B virus polymerase contribute to RNA binding and encapsidation

Hepatitis B virus (HBV) antiviral therapy is plagued by limited efficacy and resistance to most nucleos(t)ide analog drugs. We have proposed that the complex RNA binding mechanism of the HBV reverse transcriptase (P) may be a novel target for antivirals. We previously found that RNA binds to the duck HBV (DHBV) P through interactions with the T3 and RT1 motifs in the viral terminal protein and reverse transcriptase domains, respectively. Here, we extended these studies to HBV P. HBV T3 and RT1 synthetic peptides bound RNA in a similar manner as did analogous DHBV peptides. The HBV T3 motif could partially substitute for DHBV T3 during RNA binding and DNA priming by DHBV P, whereas replacing RT1 supported substantial RNA binding but not priming. Substituting both the HBV T3 and RT1 motifs restored near wild-type levels of RNA binding but supported very little priming. Alanine-scanning mutations to the HBV T3 and RT1 motifs blocked HBV ε RNA binding in vitro and pgRNA encapsidation in cells. These data indicate that both the HBV T3 and RT1 motifs contain sequences essential for HBV ε RNA binding and encapsidation of the RNA pregenome, which is similar to their functions in DHBV. Small molecules that bind to T3 and/or RT1 would therefore inhibit encapsidation of the viral RNA and block genomic replication. Such drugs would target a novel viral function and would be good candidates for use in combination with the nucleoside analogs to improve efficacy of antiviral therapy.

Discovery and analysis of an active long terminal repeat-retrotransposable element in Aspergillus oryzae

Wild-type Aspergillus oryzae RIB40 contains two copies of the AO090005001597 gene. We previously constructed A. oryzae RIB40 strain, RKuAF8B, with multiple chromosomal deletions, in which the AO090005001597 copy number was found to be increased significantly. Sequence analysis indicated that AO090005001597 is part of a putative 6,000-bp retrotransposable element, flanked by two long terminal repeats (LTRs) of 669 bp, with characteristics of retroviruses and retrotransposons, and thus designated AoLTR (A. oryzae LTR-retrotransposable element). AoLTR comprised putative reverse transcriptase, RNase H, and integrase domains. The deduced amino acid sequence alignment of AoLTR showed 94% overall identity with AFLAV, an A. flavus Tf1/sushi retrotransposon. Quantitative real-time RT-PCR showed that AoLTR gene expression was significantly increased in the RKuAF8B, in accordance with the increased copy number. Inverse PCR indicated that the full-length retrotransposable element was randomly integrated into multiple genomic locations. However, no obvious phenotypic changes were associated with the increased AoLTR gene copy number.

Antiviral efficacy of adefovir dipivoxil in the treatment of chronic hepatitis B subjects from Indian subcontinent

Adefovir is one of the therapeutic options for the treatment of chronic hepatitis B. A total of 30 adefovir-experienced subjects with the median treatment duration of 12 (interquartile range (IQR) 6-18) months were studied. Virological response was measured by hepatitis B virus deoxyribonucleic acid (HBV DNA) levels. HBV reverse transcriptase (rt) domains were sequenced for the identification of resistance mutations. Among the 30 subjects, two (7%) showed virological response and 19 (63%) were non-responders. The virological response for the remaining nine (30%) subjects was not determined. On sequence analysis, two subjects were identified with rtI169L and rtA181V mutation after 9 months and 18 months of adefovir treatment, respectively. Though the frequencies of adefovir resistance mutations are low, a large majority of subjects showed non-response. Therefore, adefovir in the management of HBV should be used judiciously.

Comparative Analysis of Hepatitis B Virus Polymerase Sequences Required for Viral RNA Binding, RNA Packaging, and Protein Priming

Hepatitis B virus replicates a DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific association with a viral RNA signal, termed ε (Hε), located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. HP is made up of four domains, including the terminal protein (TP), the spacer, the reverse transcriptase (RT), and the RNase H domains. A recently developed, Hε-dependent, in vitro protein priming assay was used in this study to demonstrate that almost the entire TP and RT domains and most of the RNase H domain were required for protein priming. Specific residues within TP, RT, and the spacer were identified as being critical for HP-Hε binding and/or protein priming. Comparison of HP sequence requirements for Hε binding, pgRNA packaging, and protein priming allowed the classification of the HP mutants into five groups, each with distinct effects on these complex and related processes. Detailed characterization of HP requirements for these related and essential functions of HP will further elucidate the mechanisms of its multiple functions and aid in the targeting of these functions for antiviral therapy.

Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

We reported that 3′-azido-2′,3′-dideoxyguanosine (3′-azido-ddG) selected for the L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain of HIV-1 reverse transcriptase (RT). In this study, we have defined the molecular mechanisms of 3′-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V, F77L, V106I, L214F, R277K, and K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3′-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows RT to effectively discriminate between the natural nucleotide (dGTP) and 3′-azido-ddG-triphosphate (3′-azido-ddGTP). 3′-azido-ddGTP discrimination was primarily driven by a decrease in 3′-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT’s ability to excise the chain-terminating 3′-azido-ddG-monophosphate (3′-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme’s ability to excise 3′-azido-ddGMP on an RNA/DNA, but not on a DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Collectively, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3′-azido-2′,3′-dideoxynucleosides.

LINE class retroposon is a component of DNA polymorphic fragments of trematode Himasthla elongata parthenitae

We found that the S-SAP (Sequence-Specific Amplification Polymorphism) method revealed clonal variability in the genomes of flatworm Himasthla elongata (Trematoda, Echinostomatidae) larvae. The larvae are parthenogenetic and have been considered genetically homogeneous. We performed cloning and sequencing of an about 500-bp conservative fragment (B1). B1 sequence analysis showed that this fragment had maximal homology with LINE elements from the CR1 family of Hydra and sparrow. In situ hybridization (FISH) revealed B1 dispersed distribution. Several other fragments cloned from the same agarose electrophoresis band correspond to the conservative domain of reverse transcriptase (RT) from CR1 family. Thus, it has been shown that (1) cercariae of trematode H. elongata have clonal variability, (2) the S-SAP method allows patterns to be obtained of fragment distribution characteristic for individual cercariae, and (3) RT conservative domain of the CR1 family participates in the pattern of polymorphic fragments generation. Identification of the CR1 transcripts in H. elongata cercariae transcriptom will be the aim of a future work. Cloning of variable fragments from the pattern of fragments is in progress.

A Polymorphism at Position 400 in the Connection Subdomain of HIV-1 Reverse Transcriptase Affects Sensitivity to NNRTIs and RNaseH Activity

Reverse transcriptase (RT) plays an essential role in HIV-1 replication, and inhibition of this enzyme is a key component of HIV-treatment. However, the use of RT inhibitors can lead to the emergence of drug-resistant variants. Until recently, most clinically relevant resistance mutations were found in the polymerase domain of RT. Lately, an increasing number of resistance mutations has been identified in the connection and RNaseH domain. To further explore the role of these domains we analyzed the complete RT sequence of HIV-1 subtype B patients failing therapy. Position A/T400 in the connection subdomain is polymorphic, but the proportion of T400 increases from 41% in naïve patients to 72% in patients failing therapy. Previous studies suggested a role for threonine in conferring resistance to nucleoside RT inhibitors. Here we report that T400 also mediates resistance to non-nucleoside RT inhibitors. The susceptibility to NVP and EFV was reduced 5-fold and 2-fold, respectively, in the wild-type subtype B NL4.3 background. We show that substitution A400T reduces the RNaseH activity. The changes in enzyme activity are remarkable given the distance to both the polymerase and RNaseH active sites. Molecular dynamics simulations were performed, which provide a novel atomistic mechanism for the reduction in RNaseH activity induced by T400. Substitution A400T was found to change the conformation of the RNaseH primer grip region. Formation of an additional hydrogen bond between residue T400 and E396 may play a role in this structural change. The slower degradation of the viral RNA genome may provide more time for dissociation of the bound NNRTI from the stalled RT-template/primer complex, after which reverse transcription can resume.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei

BackgroundAbdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp.ResultsA new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3′ and 5′ RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival.ConclusionsOur results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.

DIRS and Ngaro Retrotransposons in Fungi.

Retrotransposons with a tyrosine recombinase (YR) have been discovered recently and lack thorough annotation in fungi. YR retrotransposons are divided into 3 groups: DIRS, Ngaro and VIPER (known only from kinetoplastida). We used comparative genomics to investigate the evolutionary patterns of retrotransposons in the fungal kingdom. The identification of both functional and remnant elements provides a unique view on both recent and past transposition activity. Our searches covering a wide range of fungal genomes allowed us to identify 2241 YR retrotransposons. Based on CLANS clustering of concatenated sequences of the reverse transcriptase (RT), RNase H (RH), DNA N-6-adenine-methyltransferase (MT) and YR protein domains we propose a revised classification of YR elements expanded by two new categories of Ngaro elements. A phylogenetic analysis of 477 representatives supports this observation and additionally demonstrates that DIRS and Ngaro abundance changed independently in Basidiomycota and Blastocladiomycota/Mucoromycotina/Kixellomycotina. Interestingly, a single remnant Ngaro element could be identified in an Ascomycota genome. Our analysis revealed also that 3 Pucciniomycotina taxa, known for their overall mobile element abundance and big genome size, encode an elevated number of Ngaro retrotransposons. Considering the presence of DIRS elements in all analyzed Mucoromycotina , Kickxellomycotina and Blastocladiomycota genomes one might assume a common origin of fungal DIRS retrotransposons with a loss in Dicarya. Ngaro elements described to date from Opisthokonta, seem to have invaded the common ancestor of Agaricomycotina and Pucciniomycotina after Ustilagomycotina divergence. Yet, most of analyzed genomes are devoid of YR elements and most identified retrotransposons are incomplete.

Reverse Transcription of Spliced psbA mRNA in Chlamydomonas spp. and Its Possible Role in Evolutionary Intron Loss

Reverse transcription of mRNA is thought to be an important first step in a model that explains certain evolutionary changes within genes, such as the loss of introns or RNA editing sites. In this model, reverse transcription of mRNA produces cDNA molecules that replace part of the parental gene by homologous recombination. In vivo evidence of reverse transcription of physiologically relevant mRNAs is generally lacking, however, except in genetically engineered cells. Here, we provide in vivo evidence for reverse transcription of the chloroplast psbA mRNA in two naturally occurring species of Chlamydomonas (raudensis and subcaudata) that is based on the presence of spliced cDNAs in both organisms. The psbA cDNAs, which lack the group II intron of the genomic gene, are nearly full length, and the majority of them--though not all--are in the form of RNA-cDNA hybrids. Moreover, the presence in these species of psbA cDNAs is correlated with the loss of an early group I intron from the same psbA gene. The group II intron that interrupts psbA in C. raudensis and C. subcaudata potentially encodes a protein with a reverse transcriptase domain, and the C. raudensis protein was shown to have reverse transcriptase activity in vitro. These results provide strong evidence for reverse transcription of a physiologically important mRNA (psbA) in two species of Chlamydomonas that have also lost an intron from the same gene, possibly through recombination with the cDNA.

Lamivudine Monotherapy in Chronic Hepatitis B Patients from the Indian Subcontinent: Antiviral Resistance Mutations and Predictive Factors of Treatment Response

Management of chronic hepatitis B is a global public health challenge. There are several updated guidelines proposed based on treatment outcome data from the respective study populations. In this study, we aim to characterize the antiviral resistance mutations to lamivudine monotherapy in patients diagnosed with chronic hepatitis B from the Indian subcontinent. A total of 147 lamivudine-treated patients with a median treatment duration of 13 (interquartile range 8-24) months were studied. Virological response was measured by hepatitis B virus (HBV) DNA levels. Antiviral resistance mutations were identified by sequencing HBV reverse transcriptase domains. Factors associated with virological response and antiviral resistance mutations were analyzed. Virological response was observed in 50 (35%) patients while 84 (57%) were non-responders. The virological response for the remaining 13 (9%) patients was undetermined. Forty patients (27%) developed lamivudine-resistant mutations. HBV genotypes, subgenotypes and hepatitis B surface antigen subtypes did not show significant association with virological response or lamivudine-resistant mutations. High HBV DNA levels and increased treatment duration were strongly associated with the development of lamivudine-resistant mutations (p=0.002 and p<0.001). Patients who continued to be positive for hepatitis B e antigen have an increased risk for treatment failure (p=0.010). High baseline aspartate transaminase levels were significantly associated with subsequent lamivudine response (p=0.037). Considering the limited potency and high resistance rates to lamivudine therapy, our study emphasizes the use of more potent drugs in the management of chronic hepatitis B in the Indian subcontinent.

Structural characterization of copia-type retrotransposons leads to insights into the marker development in a biofuel crop, Jatropha curcasL.

BackgroundRecently, Jatropha curcas L. has attracted worldwide attention for its potential as a source of biodiesel. However, most DNA markers have demonstrated high levels of genetic similarity among and within jatropha populations around the globe. Despite promising features of copia-type retrotransposons as ideal genetic tools for gene tagging, mutagenesis, and marker-assisted selection, they have not been characterized in the jatropha genome yet. Here, we examined the diversity, evolution, and genome-wide organization of copia-type retrotransposons in the Asian, African, and Mesoamerican accessions of jatropha, then introduced a retrotransposon-based marker for this biofuel crop.ResultsIn total, 157 PCR fragments that were amplified using the degenerate primers for the reverse transcriptase (RT) domain of copia-type retroelements were sequenced and aligned to construct the neighbor-joining tree. Phylogenetic analysis demonstrated that isolated copia RT sequences were classified into ten families, which were then grouped into three lineages. An in-depth study of the jatropha genome for the RT sequences of each family led to the characterization of full consensus sequences of the jatropha copia-type families. Estimated copy numbers of target sequences were largely different among families, as was presence of genes within 5 kb flanking regions for each family. Five copia-type families were as appealing candidates for the development of DNA marker systems. A candidate marker from family Jc7 was particularly capable of detecting genetic variation among different jatropha accessions. Fluorescence in situ hybridization (FISH) to metaphase chromosomes reveals that copia-type retrotransposons are scattered across chromosomes mainly located in the distal part regions.ConclusionThis is the first report on genome-wide analysis and the cytogenetic mapping of copia-type retrotransposons of jatropha, leading to the discovery of families bearing high potential as DNA markers. Distinct dynamics of individual copia-type families, feasibility of a retrotransposon-based insertion polymorphism marker system in examining genetic variability, and approaches for the development of breeding strategies in jatropha using copia-type retrotransposons are discussed.

Ty3 reverse transcriptase complexed with an RNA/DNA hybrid shows structural and functional asymmetry

Retrotransposons are mobile genetic elements that replicate through an RNA intermediate. They are divided into two groups, depending on the presence of flanking long-terminal repeat (LTR) sequences. Approximately 40% of the human genome is derived from retroelements with 8% corresponding to the LTR class. Retroviruses, such as human immunodeficiency virus (HIV-1) evolved from LTR elements through acquiring the envelope genes which allow them to leave the cell and infect other cells. Reverse transcriptase (RT) is a critical enzyme of retroelements, combining DNA polymerase and RNase H activities to convert the (+) strand RNA genome into double-stranded DNA that is competent for integration. However, In contrast to the extensive structural characterization of retroviral RTs, detailed information on the enzymes from LTR-containing retrotransposons such as Ty3 of Saccharomyces cerevisiae, is lacking. Ty3 belongs to the Gypsy family and its RT is perhaps the most extensively characterized LTR-retrotransposon enzyme with respect to its catalytic activities, as well as the nucleic acid duplexes with which it interacts. Although structural motifs ascribed to substrate recognition and catalysis are generally similar to those of vertebrate retroviral RTs, a notable difference is replacement of the highly-conserved active site His with Tyr in the RNase H domain of Ty3 RT. More significantly Ty3 RT lacks the connection, or tether, between its DNA polymerase and RNase H active sites, with the consequence that on the nucleic acid substrate they are separated by ~13 bp as opposed to the 17-18 bp observed for lentiviral and gammaretroviral enzymes. Structural similarity between the HIV-1 connection and its RNase H domain originally suggested the latter arose through domain duplication, while an alternative theory proposes the functional RNase H domain was acquired as a new, and more efficient element from a source outside the LTR retrotransposons. Structural and biochemical information on subdomain organization of Ty3 RT is therefore important in understanding the evolutionary divergence between these essential vertebrate retroviral and LTR-retrotransposon enzymes. To this end, we provide the first structure of Ty3 RT in complex with an RNA/DNA hybrid at 3.1 A resolution. The fully-active enzyme is an asymmetric homodimer of 55 kDa subunits, and is formed only in the presence of the RNA/DNA substrate. Modeling the spatial separation between the DNA polymerase and RNase H active sites, coupled with site-directed mutagenesis, suggests that one active site of each subunit contributes enzyme activity.

Exploiting Drug-Resistant Enzymes as Tools To Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3',4'-dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5 °C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy.

Isolation and characterization of reverse transcriptase fragments of LTR retrotransposons from the genome of Chenopodium quinoa (Amaranthaceae)

Key message High heterogeneity was observed among conserved domains of reverse transcriptase ( rt ) isolated from quinoa. Only one Ty1- copia rt was highly amplified. Reverse transcriptase sequences were located predominantly in pericentromeric region of quinoa chromosomes. The heterogeneity, genomic abundance, and chromosomal distribution of reverse transcriptase (rt)-coding fragments of Ty1-copia and Ty3-gypsy long terminal repeat retrotransposons were analyzed in the Chenopodium quinoa genome. Conserved domains of the rt gene were amplified and characterized using degenerate oligonucleotide primer pairs. Sequence analyses indicated that half of Ty1-copia rt (51 %) and 39 % of Ty3-gypsy rt fragments contained intact reading frames. High heterogeneity among rt sequences was observed for both Ty1-copia and Ty3-gypsy rt amplicons, with Ty1-copia more heterogeneous than Ty3-gypsy. Most of the isolated rt fragments were present in quinoa genome in low copy numbers, with only one highly amplified Ty1-copia rt sequence family. The gypsy-like RNase H fragments co-amplified with Ty1-copia-degenerate primers were shown to be highly amplified in the quinoa genome indicating either higher abundance of some gypsy families of which rt domains could not be amplified, or independent evolution of this gypsy-region in quinoa. Both Ty1-copia and Ty3-gypsy retrotransposons were preferentially located in pericentromeric heterochromatin of quinoa chromosomes. Phylogenetic analyses of newly amplified rt fragments together with well-characterized retrotransposon families from other organisms allowed identification of major lineages of retroelements in the genome of quinoa and provided preliminary insight into their evolutionary dynamics.

Isolation, identification, and characterization of genomic LTR retrotransposon sequences from masson pine (Pinus massoniana)

Long terminal repeat (LTR) retrotransposons are ubiquitous in the plant kingdom and play an important role in plant genome evolution. Masson pine (Pinus massoniana) is one of the most economically important forest trees. However, the masson pine genome is poorly understood. Conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy LTR retrotransposons were amplified from masson pine using degenerate primers. Sequence analysis showed less heterogeneity among Ty1-copia group retrotransposons than among Ty3-gypsy group retrotransposons. Phylogenetic analysis demonstrated that Ty1-copia and Ty3-gypsy RT sequences had considerable homology with other species, indicating that both vertical transmission and horizontal transmission may probably be the source of LTR retrotransposons in masson pine. Southern dot blot hybridization results suggested that both types of LTR retrotransposons are present in the genome of masson pine with high copy number. These results contribute to further understanding about the organization and evolution of the masson pine genome and provide fundamental genetic information for the utilization of retrotransposons in masson pine.

Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416-Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining α-helix M', Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors. Based on our structural data, we analyzed the role of the p51 C terminus by evaluating selectively mutated p66/p51 heterodimers carrying (i) p51 truncations that encroach on α-M', (ii) alterations that interrupt the Asn-348-Tyr-427 interaction, and (iii) alanine substitutions throughout the region Phe-416-Pro-421. Collectively, our data support the notion that the p51 C terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.

The Major Reverse Transcriptase–Incompetent Splice Variant of the Human Telomerase Protein Inhibits Telomerase Activity but Protects from Apoptosis

Human telomerase reverse transcriptase (hTERT; the catalytic protein subunit of telomerase) is subjected to numerous alternative splicing events, but the regulation and function of these splice variants is obscure. Full-length hTERT includes conserved domains that encode reverse transcriptase activity, RNA binding, and other functions. The major splice variant termed α+β- or β-deletion is highly expressed in stem and cancer cells, where it codes for a truncated protein lacking most of the reverse transcriptase domain but retaining the known RNA-binding motifs. In a breast cancer cell panel, we found that β-deletion was the hTERT transcript that was most highly expressed. Splicing of this transcript was controlled by the splice regulators SRSF11, HNRNPH2, and HNRNPL, and the β-deletion transcript variant was associated with polyribosomes in cells. When ectopically overexpressed, β-deletion protein competed for binding to telomerase RNA (hTR/TERC), thereby inhibiting endogenous telomerase activity. Overexpressed β-deletion protein localized to the nucleus and mitochondria and protected breast cancer cells from cisplatin-induced apoptosis. Our results reveal that a major hTERT splice variant can confer a growth advantage to cancer cells independent of telomere maintenance, suggesting that hTERT makes multiple contributions to cancer pathophysiology.

Virological response and antiviral resistance mutations in chronic hepatitis B subjects experiencing entecavir therapy: An Indian subcontinent perspective

Entecavir is one of the therapeutic options currently available for the management of chronic hepatitis B. In this study, we aimed to analyse the virological response and antiviral resistance mutations in chronic hepatitis B subjects experiencing entecavir therapy from the Indian subcontinent. A total of 45 chronic hepatitis B subjects were studied at baseline and were followed up on entecavir treatment. Among these subjects, 25 (56%) were HBeAg-positive at baseline. Virological response was measured by hepatitis B virus (HBV) DNA levels. HBV reverse transcriptase (rt) domains were sequenced for the identification of resistance mutations. Three-Dimensional (3D) model of HBV polymerase/rt protein, docking and molecular dynamics simulation (MDS) studies were performed for characterization of antiviral resistance mutations. At the median treatment duration of 6 (IQR 6–11) months, 38 (84%) showed virological response. Subjects who showed anti-HBe response demonstrated significant association with virological response (p=0.034). On sequence analysis, none of the subjects were identified with signature entecavir resistance mutations. However, one subject was exclusively detected with rtV173L mutation. Molecular modeling, docking and MDS studies revealed that the rtV173L mutation cannot confer resistance to entecavir independently. Our findings also showed that the prevailing HBV genotypes, subgenotypes and HBsAg subtypes in this population does not influence treatment outcome to entecavir therapy. In conclusion, entecavir is a potent drug in terms of viral DNA suppression. In addition, none of the subjects developed antiviral resistance mutations to entecavir. Therefore entecavir is a suitable drug of choice in the management of chronic HBV.

Controllable inhibition of hepatitis B virus replication by a DR1-targeting short hairpin RNA (shRNA) expressed from a DOX-inducible lentiviral vector

As a highly efficient delivery system, lentiviral vectors (LVs) have become a powerful tool to assess the antiviral efficacy of RNA drugs such as short hairpin RNA (shRNA) and decoys. Furthermore, recent advanced systems allow controlled expression of the effector RNA via coexpression of a tetracycline/doxycycline (DOX) responsive repressor (tTR-KRAB). Herein, this system was utilized to assess the antiviral effects of LV-encoded shRNAs targeting three conserved regions on the pregenomic RNA of hepatitis B virus (HBV), namely the region coding for the reverse transcriptase (RT) domain of the viral polymerase (LV-HBV-shRNA1), the core promoter (CP; LV-HBV-shRNA2), and the direct repeat 1 (DR1; LV-HBV-shRNA3). Transduction of just the LV-HBV-shRNA vectors into the stably HBV expressing HepG2.2.15 cell line showed significant reductions in secreted HBsAg and HBeAg, intracellular HBcAg as well as HBV RNA and DNA replicative intermediates for all vectors, however, most pronouncedly for the DR1-targeting shRNA3. The corresponding vector was therefore applied in the DOX-controlled system. Notably, strong interference with HBV replication was found in the presence of the inducer DOX whereas the antiviral effect was essentially ablated in its absence; hence, the silencing effect of the shRNA and consequently HBV replication could be strictly regulated by DOX. This newly established system may therefore provide a valuable platform to study the antiviral efficacy of RNA drugs against HBV in a regulated manner, and even be applicable in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s11262-013-0886-2) contains supplementary material, which is available to authorized users.