Fluorescence-based biochemical analysis of human hepatitis B virus reverse transcriptase activity.

Although the unique mechanism by which hepatitis B virus (HBV) polymerase primes reverse transcription is now well-characterized, the subsequent elongation process remains poorly understood. Reverse transcriptase (RT)-RNase H sequences from polymerase amino acid 304 (the C-terminal part of spacer domain) to 843 were expressed in Escherichia coli and purified partially. RT elongation activity was investigated using the fluorescent-tagged primer and homopolymeric RNA templates. RT elongation activity depended on both Mg2+ and Mn2+, and had low affinity for purine deoxynucleotides, which may be related with the success of adefovir, tenofovir, and entecavir. However, the polymerization rate was lower than that of human immunodeficiency virus RT. All HBV genotypes displayed similar RT activity, except for genotype B, which demonstrated increased elongation activity.

A Possible Role for Long Interspersed Nuclear Elements-1 (LINE-1) in Huntington's Disease Progression.

BACKGROUND Recent studies have shown that increased mobilization of Long Interspersed Nuclear Elements-1 (L1) can promote the pathophysiology of multiple neurological diseases. However, its role in Huntington's disease (HD) remains unknown. MATERIAL AND METHODS R6/2 mice - a common mouse model of HD - were used to evaluate changes in L1 mobilization. Pyrosequencing was used to evaluate methylation content changes. L1-ORF1 and L1-ORF2 expression analysis were evaluated by RT-PCR and immunoblotting. Changes in pro-survival signaling were evaluated by L1-ORF overexpression studies and validated in the mouse model by immunohistochemistry and immunoblotting. RESULTS We found an increased mobilization of L1 elements in the caudate genome of R6/2 mice (p<0.05) - a common mouse model of HD - but not in wild-type mice. Subsequent pyrosequencing and expression analysis showed that the L1 elements were hypomethylated and their respective ORFs were overexpressed in the affected tissues. In addition, a significant decrease in the pro-survival proteins such as the phosphoproteins of AKT target proteins, mTORC1 activity, and AMPK alpha levels was observed with the increase in the expression L1-ORF2. CONCLUSIONS These findings indicate that hyperactive retrotransposition of L1 triggers a downstream signaling pathway affecting the neuronal survival pathways via downregulation of mTORC1 activity and AMPKalpha and increasing apoptosis in neurons.

Dissection of affinity captured LINE-1 macromolecular complexes.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription.

MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal Mucosa and SHIV-RT in Rectal Mucosa.

The Population Council's microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad-spectrum microbicide against HIV, herpes simplex virus (HSV), and human papillomavirus. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection, and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and human papillomavirus in murine models. Recently, we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 coinfection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC, and compared quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay readouts for monitoring SHIV-RT infection. MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosas and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by 1-step simian immunodeficiency virus gag quantitative RT-PCR and p27 enzyme-linked immunosorbent assay demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of quantitative RT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

Preparation and evaluation of MS2 bacteriophage-like particles packaging hepatitis E virus RNA.

Hepatitis E virus (HEV) is the pathogen causing hepatitis E (HE). It arouses global public health concern since it is a zoonotic disease. The objective of this letter is to report a cost-effective internal control prepared for monitoring procedures of HEV reverse transcriptase (RT)-PCR detection. A selected conserved HEV RNA fragment was integrated into the downstream of the truncated MS2 bacteriophage genome based on Armored RNA technology. The resulting MS2-HEV gene harbored by the pET-28b-MS2-HEV plasmid was transformed into E. coli BL21(DE3) for expression analysis by SDS-PAGE. The expression products were purified and concentrated by ultrasonication and ultrafiltration separation. The morphology and stability properties of the virus-like particles (VLPs) were evaluated by electron microscopy scanning and nuclease challenges, respectively. SDS-PAGE results showed that the constructed MS2-HEV gene expressed efficiently and the purity of the VLPs was highly consistent with the result in electron microscopy. Stability evaluation results demonstrated that the prepared VLPs exhibited strong resistance to DNase I and RNase A attacks and also performed long-lasting protection of coated HEV RNA for at least 4 months at -20°C. These data revealed that the prepared VLPs meet the basic requirements of use as internal control material in the HEV RNA amplification assay.

Searching for Common Mammalian Retroviruses in Pediatric Idiopathic Diseases.

Mammalian retroviruses cause a variety of diseases in their hosts, including hematological and immunodeficiency disorders. Both human T-cell leukemia (HTLV) and human immunodeficiency (HIV) viruses originated from several independent zoonotic transmissions, indicating that cross-species transmissions from animal to humans may still occur. Thus, as the risk for retroviral transmissions from animals to humans increase, we investigated whether mammalian retroviruses are involved in selected pediatric idiopathic diseases whose symptoms evoke retroviral infections. Blood samples, sera, and synovial fluids, or bone marrow cells were collected from pediatric patients under 18 years of age with different autoimmune idiopathic diseases. Overall, we screened clinical samples from 110 children using sensitive nested and semi-nested PCR strategies targeting env genes, and a C-type retrovirus reverse transcriptase (RT) activity kit. All clinical samples were free of retroviral signatures, indicating the unlikelihood of an etiological role of the retroviruses we assessed in the pediatric diseases we tested.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes.

BACKGROUND: A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. METHODS: Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. RESULTS: PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. CONCLUSION: No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

ERVK polyprotein processing and reverse transcriptase expression in human cell line models of neurological disease.

Enhanced expression of the reverse transcriptase (RT) protein encoded by human endogenous retrovirus-K (ERVK) is a promising biomarker for several inflammatory and neurological diseases. However, unlike RT enzymes encoded by exogenous retroviruses, little work has been done to identify ERVK RT isoforms, their expression patterns, and cellular localization. Using Western blot, we showcase the ERVK gag-pro-pol polyprotein processing leading to the production of several ERVK RT isoforms in human neuronal (ReNcell CX) and astrocytic (SVGA) models of neuroinflammatory disease. Since the pro-inflammatory cytokine IFNγ plays a key role in the pathology of several ERVK-associated neurological diseases, we sought to determine if IFNγ can drive ERVK RT expression. IFNγ signalling markedly enhanced ERVK polyprotein and RT expression in both human astrocytes and neurons. RT isoforms were expressed in a cell-type specific pattern and the RT-RNase H form was significantly increased with IFNγ treatment. Fluorescent imaging revealed distinct cytoplasmic, perinuclear and nuclear ERVK RT staining patterns upon IFNγ stimulation of astrocytes and neurons. These findings indicate that ERVK expression is inducible under inflammatory conditions such as IFNγ exposure-and thus, these newly established in vitro models may be useful in exploring ERVK biology in the context of neuroinflammatory disease.

Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing.

Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR amplification, Illumina adapters and index sequences are introduced, thereby allowing amplicons to be pooled and sequenced on the standard Illumina platform for genomic DNA sequencing. Moreover, we demonstrate how to map sequencing reads and perform analysis of the sequencing data with freely available tools that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells.

[Detection methods for retroviruses and reverse transcriptase: commentary].

Retroviruses are often used as a carrier for expression of target protein or chimeric target gene. Although non-infectious viruses are selected in the laboratory, it does not exclude harms form these viruses. The monitoring and detection of retroviruses has a very important significance. Reverse transcriptase activity is an important indicator of retrovirus replication. Herein, methods for detection of retroviruses and reverse transcriptase are reviewed for further research references.

Epizootic neoplasia of the lateral line system of lake trout (Salvelinus namaycush) in New York's Finger Lakes.

This article documents an epizootic of inflammation and neoplasia selectively affecting the lateral line system of lake trout (Salvelinus namaycush) in 4 Finger Lakes in New York from 1985 to 1994. We studied more than 100 cases of this disease. Tumors occurred in 8% (5/64) of mature and 21% (3/14) of immature lake trout in the most severely affected lake. Lesions consisted of 1 or more neoplasm(s) in association with lymphocytic inflammation, multifocal erosions, and ulcerations of the epidermis along the lateral line. Lesions progressed from inflammatory to neoplastic, with 2-year-old lake trout showing locally extensive, intense lymphocytic infiltrates; 2- to 3-year-old fish having multiple, variably sized white masses up to 3 mm in diameter; and fish over 5 years old exhibiting 1 or more white, cerebriform masses greater than 1 cm in diameter. Histologic diagnoses of the tumors were predominantly spindle cell sarcomas or benign or malignant peripheral nerve sheath neoplasms, with fewer epitheliomas and carcinomas. Prevalence estimates did not vary significantly between sexes or season. The cause of this epizootic remains unclear. Tumor transmission trials, virus isolation procedures, and ultrastructural study of lesions failed to reveal evidence of a viral etiology. The Finger Lakes in which the disease occurred did not receive substantially more chemical pollution than unaffected lakes in the same chain during the epizootic, making an environmental carcinogen an unlikely primary cause of the epizootic. A hereditary component, however, may have contributed to this syndrome since only fish of the Seneca Lake strain were affected.

Group II intron protein localization and insertion sites are affected by polyphosphate.

Mobile group II introns consist of a catalytic intron RNA and an intron-encoded protein with reverse transcriptase activity, which act together in a ribonucleoprotein particle to promote DNA integration during intron mobility. Previously, we found that the Lactococcus lactis Ll.LtrB intron-encoded protein (LtrA) expressed alone or with the intron RNA to form ribonucleoprotein particles localizes to bacterial cellular poles, potentially accounting for the intron's preferential insertion in the oriC and ter regions of the Escherichia coli chromosome. Here, by using cell microarrays and automated fluorescence microscopy to screen a transposon-insertion library, we identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) whose disruption results in both an increased proportion of cells with more diffuse LtrA localization and a more uniform genomic distribution of Ll.LtrB-insertion sites. Surprisingly, we find that a common factor affecting LtrA localization in these and other disruptants is the accumulation of intracellular polyphosphate, which appears to bind LtrA and other basic proteins and delocalize them away from the poles. Our findings show that the intracellular localization of a group II intron-encoded protein is a major determinant of insertion-site preference. More generally, our results suggest that polyphosphate accumulation may provide a means of localizing proteins to different sites of action during cellular stress or entry into stationary phase, with potentially wide physiological consequences.

Inhibition of reverse transcriptase activity of hepatitis B virus polymerase by ß-l-D4A-TP.

ß-L-enantiomer of 2',3'-didehydro-2',3'-dideoxyadenosine-5'-triphosphate (ß-L-D4A-TP) has previously been proven to inhibit the replication of viral DNA in the Hep G2 2.2.15 cells and in transgenic mouse harboring 1.3-fold-overlength genome of Hepatitis B virus (HBV). To study the inhibition mechanism of the nucleoside analog ß-L-D4A-TP, a polymerase reaction in vitro with the recombinant HBV nucleocapsids was conducted to determine the exact mode of inhibition of the HBV replication by ß-L-D4A-TP. The HBV viral DNA and viral DNA-polymerase complex formed in the polymerase reaction were assayed. The results of this study showed that ß-L-D4A-TP inhibited the replication of HBV DNA by inactivating the reverse transcriptase (RT) activity in a concentration-dependent manner. The kinetics of ß-L-D4A-TP inhibition of the RT activity was the result of an apparent competitive inhibition with dATP.

Quantitative analysis of the interactions between HIV-1 integrase and retroviral reverse transcriptases.

The RT (reverse transcriptase) of HIV-1 interacts with HIV-1 IN (integrase) and inhibits its enzymatic activities. However, the molecular mechanisms underling these interactions are not well understood. In order to study these mechanisms, we have analysed the interactions of HIV-1 IN with HIV-1 RT and with two other related RTs: those of HIV-2 and MLV (murine-leukaemia virus). All three RTs inhibited HIV-1 IN, albeit to a different extent, suggesting a common site of binding that could be slightly modified for each one of the studied RTs. Using surface plasmon resonance technology, which monitors direct protein-protein interactions, we performed kinetic analyses of the binding of HIV-1 IN to these three RTs and observed interesting binding patterns. The interaction of HIV-1 RT with HIV-1 IN was unique and followed a two-state reaction model. According to this model, the initial IN-RT complex formation was followed by a conformational change in the complex that led to an elevation of the total affinity between these two proteins. In contrast, HIV-2 and MLV RTs interacted with IN in a simple bi-molecular manner, without any apparent secondary conformational changes. Interestingly, HIV-1 and HIV-2 RTs were the most efficient inhibitors of HIV-1 IN activity, whereas HIV-1 and MLV RTs showed the highest affinity towards HIV-1 IN. These modes of direct protein interactions, along with the apparent rate constants calculated and the correlations of the interaction kinetics with the capacity of the RTs to inhibit IN activities, are all discussed.

Quantification of reverse transcriptase in ALS and elimination of a novel retroviral candidate.

BACKGROUND: Retroviral involvement in amyotrophic lateral sclerosis (ALS) has been suspected for several years since the recognition that both murine and human retroviruses can cause ALS-like syndromes. Nonquantitative studies have demonstrated the retroviral enzyme reverse transcriptase (RT) in ALS patients' sera, but the amount and source of RT activity are unknown. We therefore developed a quantitative assay to study RT levels in ALS and examined the possibility that the recently discovered human gammaretrovirus XMRV (xenotropic MuLV-related virus) might be the source of the RT activity. METHODS: A quantitative product-enhanced RT assay was used to measure RT activity levels in serum and CSF. XMRV sequences were sought by PCR analysis of DNA and RNA extracted from blood. RESULTS: Fifty percent of ALS patients' sera contained >6 x 10(-8) RT units/mL as opposed to 7% of control sera (p = 0.008). The levels of RT activity in ALS patients were comparable to the levels observed in patients infected with HIV. RT activity was detected in only 1 of 25 CSF samples tested. XMRV sequences were not found in any of 25 nucleic acid extracts obtained from ALS patients' blood. CONCLUSIONS: These findings further support the concept of retroviral involvement in amyotrophic lateral sclerosis (ALS) and demonstrate that serum is more suitable than CSF for assay of reverse transcriptase (RT) activity in this disease. The levels of serum RT activity detected are comparable to those found in HIV infection. XMRV is not detectable in the blood of ALS patients, and the agent responsible for ALS-associated RT activity therefore remains unidentified.

Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR.

BACKGROUND: In functional genomics, transcript measurement is of fundamental importance. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays are the most popular technology and depend on the initial molecular step, the reverse transcription (RT). This study provides a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used RT systems and measured the production of PCR-amplifiable products and the influence of PCR inhibitor contents. RESULTS: The qRT-PCR assays were conducted using the TaqMan system, although we also tested the SYBR Green I chemistry, which is not compatible with all the RT systems. When dealing with low-abundance transcripts, the SuperScript II system generated more detectable molecules than the four other systems tested: Sensiscript, Omniscript, SuperScript III and PowerScript (P < 0.05). However, the Sensiscript and PowerScript systems were more efficient for detecting high-abundance transcripts in the presence of 1 to 2 mug background RNA (P < 0.05). The most striking aspect was the influence of the dilution of the RT reaction on the subsequent PCR. Indeed, some inhibition was released when diluted RT reactions were used for the quantitative PCR measurements. Furthermore, the amount of background RNA in the RT reaction was also a major component influencing a downstream step in qRT-PCR, the PCR reaction. Whereas Sensiscript was less biased, the other systems contained an important source of PCR inhibitors, interfering as much as 70% with the qRT-PCR. CONCLUSION: This study provides a complex overview of the influence of elements such as RT systems, qRTPCR chemistry, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Whereas the most significant influencing factor is the presence of inhibitors in the RT systems, total background RNA is also a major influencing component that affects the PCR reaction. Whenever the aim of a study is to obtain a precise gene expression measurement or to profile the global transcriptome (e.g. microarray), the RT step is critical and should be examined with care.

A reverse transcriptase assay for early diagnosis of infant HIV infection in resource-limited settings.

Early diagnosis of pediatric HIV infection is confounded by persistence of maternal antibodies until 18 months, necessitating the use of expensive assays such as HIV-1 DNA PCR, an untenable option in resource-limited settings. This is the first report of a low-cost, commercial, reverse transcriptase (RT) assay for the diagnosis of HIV-1 infection in infants. RT assays were performed on 42 samples from 30 HIV-exposed Kenyan infants under 15 months of age. When correlated with serologic testing conducted after 18 months, the sensitivity, specificity, positive and negative predictive values of the RT assay were 92%, 93%, 87% and 96%. A low-cost assay for infant HIV diagnosis is urgently needed, and these results merit further evaluation.

A controlled study of reverse transcriptase in serum and CSF of HIV-negative patients with ALS.

Reverse transcriptase has been detected in the serum of HIV-negative patients with amyotrophic lateral sclerosis (ALS). An ALS-like disorder in HIV-positive patients can remit with antiretroviral therapy. Using the product enhanced assay technique, we measured reverse transcriptase activity in the serum and CSF of 23 HIV-negative patients with ALS and 21 neurologic disease controls. Results for CSF were not significant, whereas reverse transcriptase was detected in 56% of ALS sera vs 19% of controls.

Antigen-driven HIV expansion in allergen-specific T cells.

In industrialized countries there is a high prevalence of allergy toward nickel ions. The exposure of affected individuals to nickel leads to a delayed-type hypersensitivity reaction, which is induced by antigen-specific CD4 and CD8 T cells. Beside this antigenic potential, immunomodulatory properties of nickel ions were described. To dissect the role of both mechanisms for HIV replication, we studied HIV expansion in PBMC of nickel-allergic and nonallergic donors. Nickel ions promote HIV replication in PBMC as efficiently as protein antigens. The nickel-mediated virus expansion strictly required the presence of nickel-specific T cells. Data obtained with nickel-specific CD4 T cell clones showed that antigen-mediated proliferation is an absolute prerequisite for HIV expansion. However, the previously suggested immunomodulatory properties of nickel ions do not seem to contribute to HIV expansion. As a widely distributed antigen with increasing numbers of allergic people, nickel may be an important and underestimated factor of HIV expansion in vivo.

A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein.

The HIV-1 capsid (CA) protein plays an important role in virus assembly and infectivity. Previously, we showed that Ala substitutions in the N-terminal residues Trp23 and Phe40 cause a severely defective phenotype. In searching for mutations at these positions that result in a non-lethal phenotype, we identified one candidate, W23F. Mutant virions contained aberrant cores, but unlike W23A, also displayed some infectivity in a single-round replication assay and delayed replication kinetics in MT-4 cells. Following long-term passage in MT-4 cells, two second-site mutations were isolated. In particular, the W23F/V26I mutation partially restored the wild-type phenotype, including production of particles with conical cores and wild-type replication kinetics in MT-4 cells. A structural model is proposed to explain the suppressor phenotype. These findings describe a novel occurrence, namely suppression of a mutation in a hydrophobic residue that is critical for maintaining the structural integrity of CA and proper core assembly.