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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 Mg 2+ and Mn 2+ , 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: MT, IA, KM, PM, HJ, EA, AW, SB, DI, GE, KB, DF, BC, DA, MR, JB, JL No competing interests declared, (© 2018, Taylor et al.)

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., Competing Interests: The authors have no conflicts to disclose.

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., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

β-L-enantiomer of 2',3'-didehydro-2',3'-dideoxyadenosine-5'-triphosphate (β-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 β-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 β-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 β-L-D4A-TP inhibited the replication of HBV DNA by inactivating the reverse transcriptase (RT) activity in a concentration-dependent manner. The kinetics of β-L-D4A-TP inhibition of the RT activity was the result of an apparent competitive inhibition with dATP.

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.

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.

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.

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.

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.

Background: Current regulations and recommendations proposed for the production of vaccines in continuous cell lines of any origin demand that these be free of exogenous viruses, particularly retroviruses. Recently, the ultra-sensitive product-enhanced reverse transcriptase (PERT) assay can be used to detect minute of reverse transcriptase (RTase) in single retroviral particle and is 10(6) times more sensitive than the conventional RTase assays. However, coincidental with this increase in sensitivity is an increase in false-positive reactions derived from contaminating cellular DNA polymerases, which are known to have RTase-like activities., Objectives: To develop a modified single-tube one-step PERT (mSTOS-PERT) assay with improvements on decreasing significantly the level of false-positive reactions, and to evaluate the mSTOS-PERT assay for sensitivity and specificity., Study Design: Ampliwaxtrade mark was used to compartmentalize the reverse transcription (RT) and PCR step in the same micro-tube with more efficiency and reproducibility, while maintaining the high sensitivity. The DNA amplification products were separated by 2% agarose gel electrophoresis, and then analyzed by non-isotopic Southern blot hybridization. A wide variety of cell lines used in biologicals production were detected to validate the improved mSTOS-PERT assay., Results: The detection limit for the mSTOS-PERT assay was at least 10(-9)U, when using AMV-RTase as a positive control. Furthermore, heparin involvement in the RT step can eliminate completely the false-positive PERT signals which are exhibited by cellular polymerases such as DNA-dependent DNA polymerase alpha, gamma released by cell death. Most mammalian cells (MRC-5, Vero, WISH, 2BS, RK-13, MDCK, etc.) are PERT-negative in cell supernatants. Some PERT-positive signals in cell lysates were found to be introduced by the cellular DNA polymerases and could be inhibited specifically by heparin. Chick cells derived from either chick embryo fibroblasts (CEF) or allantoic fluid from SPF embryonated eggs, murine hybridoma cell SP2/0, etc., contained authentic RTase activities, which could not be inactivated by heparin., Conclusions: The improved mSTOS-PERT assay described here may distinguish the genuine RTase activity from cellular polymerases with high sensitivity and specificity, and is rapid and easy to perform to screen for the possible contamination of minute retroviruses in the cell substrates used in vaccine production.

A growing number of cellular processes originally thought not to involve the nucleolus now seem to be associated with this organelle. In recent years, a variety of RNAs and proteins with no apparent function in ribosome genesis have been discovered in this nuclear compartment. This paper reports the presence in the nucleolus of a reverse transcriptase (RT) previously found to be associated with telomeres in Chironomus. Immunofluorescence detection using a specific antibody against conserved domains shared by RTs showed a distinct pattern of staining in the giant nucleoli of polytenized cells. This nucleolar localization was confirmed in a number of larval tissues and embryonic cells of Chironomus thummi and C. pallidivitatus; its distribution showed a definite necklace pattern that did not completely colocalize with fibrillarin or nucleolin and appeared to be different to that of typical nucleolar components. There is evidence that both telomerase RT and RNA template subunits are present in the nucleoli of mammalian and yeast cells. However, chironomids do not have typical telomeres or telomerase. As in other Diptera, telomeres lack the short, simple repeats maintained by telomerase and instead have more complex sequences in the range of hundreds of nucleotides. It has been suggested that the RT associated with these telomeres might be involved in their maintenance, perhaps involving a mechanism similar to that of telomerase retrotranscription and retrotransposition in Drosophila. The present results indicate that the putative Chironomus telomere elongation machinery and telomerase share a nucleolar localization. This reinforces the idea that nucleoli are functionally linked to telomere maintenance irrespective of the differences in their molecular organization and therefore in the strategy adopted for their elongation.

To elucidate the mechanism of cellulose synthesis, we isolated a mutant of Arabidopsis (changed sensitivity to cellulose synthesis inhibitors 1, css1) that showed changed sensitivity to cellulose biosynthesis inhibitor. The analysis of phenotypes indicated that the css1 mutation influenced various fundamental metabolic pathways including amino acid metabolism, triacylglycerol degradation and polysaccharide synthesis (cellulose and starch) during the early stage of plant growth. Unexpectedly, the map-based cloning of the gene responsible for the css1 mutation identified a protein (At-nMat1a) that was assumed to be a splicing factor of the mitochondrial group II intron. In accordance with this result, this mutant exhibited improper splicing of the mitochondrial NAD4 transcript. We noticed that the phenotypes of the css1 mutant are similar to the responses to anoxia that hinders mitochondrial aerobic respiration. It seems that the defect in the function of mitochondria influences various aspects of fundamental cellular metabolism including cellulose synthesis. Our results suggested that sucrose synthase (SuSy), an enzyme involved in the biosynthesis of cellulose, plays key roles in the connection between mitochondria and cellulose synthesis. The isolation of the css1 mutant also provides a useful resource in the study of post-transcriptional gene regulation in mitochondria.

Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that persistently infects horses and causes a disease that is characterized by periodic episodes of fever, thrombocytopenia, and viremia. EIAV encodes only four regulatory/accessory genes, (tat, rev, ttm, and S2) and is the least genetically complex of all known lentiviruses. We sought to determine the role of the EIAV S2 accessory gene of EIAV by introducing mutations that would prevent S2 expression on the p19/wenv17 infectious molecular clone. Virus derived from the p19/wenv17 molecular clone is highly virulent and routinely fatal when given in high doses (J. Virol. 72 (1998) 483). In contrast, an S2 deletion mutant on the p19/wenv17 background is unable to induce acute disease and plasma virus loads were reduced by 2.5 to 4.0 logs at 15 days post-infection. The S2 deleted virus failed to produce any detectable clinical signs during a 5-month observation period. These results demonstrate that S2 gene expression is essential for disease expression of EIAV.

Prions as causative agents of transmissible spongiform encephalopathies have been well investigated in experimental and modelling work. However, little is known about the molecular pathogenesis of prion-induced encephalopathies, the role of co-factors, and the interaction of prions with cellular components. We investigated the influence of prion infection on expression of murine endogenous retroviruses (ERVs), which compose approximately 10% of the mouse genome. Hypothalamic neuronal cells (GT1) and neuroblastoma cells (N2a) were examined. Both cell lines can be persistently infected with mouse adapted prion strains, i.e., RML. Using a mammalian retrovirus-specific DNA microarray and quantitative PCR methods, we compared the expression profiles of ERVs in prion-infected, uninfected, and anti-prion compound-treated murine neuronal cell lines, including clonal cell populations. The results suggest that prion infection influences ERV expression in neuronal cell lines, that this influence is cell line-specific, ERV-specific, and responsive to anti-prion compound treatment.

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

The Gag-Pol polyprotein of the human immunodeficiency virus type 1 (HIV-1) is the precursor of the virus enzymatic activities and is produced via a programmed -1 translational frameshift. In this study, we altered the frameshift efficiency by introducing mutations within the slippery sequence and the frameshift stimulatory signal, the two elements that control the frameshift. These mutations decreased the frameshift efficiency to different degrees, ranging from approximately 0.3% to 70% of the wild-type efficiency. These values were mirrored by a reduced incorporation of Gag-Pol into virus-like particles, as assessed by a decrease in the reverse transcriptase activity associated to these particles. Analysis of Gag processing in infectious mutant virions revealed processing defects to various extents, with no clear correlation with frameshift decrease. Nevertheless, the observed frameshift reductions translated into equivalently reduced viral infectivity and replication kinetics. Our results show that even moderate variations in frameshift efficiency, as obtained with mutations in the frameshift stimulatory signal, reduce viral replication. Therapeutic targeting of this structure may therefore result in the attenuation of virus replication and in clinical benefit.

Product safety testing for retroviruses can be achieved by a panel of screening assays, including electron microscopy, viral gene specific PCRs, virus propagation, and detection of reverse transciptase activity. The application of PCR-based reverse transcriptase assays (PERT) that are approximately a million-fold more sensitive than conventional nucleotide incorporation assays in the testing of biologicals is described. Use of PERT assays can be applied to three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load and (iii) monitoring levels of infectious retrovirus generation in cell lines that contain endogenous retroviruses.

The product-enhanced reverse transcriptase (PERT) assay has been used to detect reverse transcriptase (RT) activity associated with retroviruses. Although the PERT assay has been proposed as a method for detection of replication-competent retrovirus (RCR) and lentivirus (RCL), it has not been rigorously compared with existing methods for RCR and RCL detection. We have assessed the PERT assay for detection of RCL and RCR that may contaminate lentiviral and retroviral vectors and compared it with published methods for RCL (p24gag ELISA/gag PCR) and RCR (S+/L-) detection. Our results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication-competent HIV-1 in an RCL detection assay. Comparison of detection of replication-competent retroviruses, GALV and RD114, by extended S+/L- and PERT assays indicates that both assays can detect 1 IU of each virus. Our findings suggest that the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

The simian immunodeficiency virus (SIV) capsid protein (CA), a constituent of the Pr55Gag polyprotein, is phosphorylated in virions but not in virus-producing cells (Rue, S.M., Roos, J.W., Tarwater, P.M., Clements, J.E., Barber, S.A., 2005. Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus. J. Virol. 79 (4), 2484-2492.). Using phosphoamino acid analysis of CA, we show that serine is the primary phosphate acceptor. A series of substitution mutants of serines in the CA domain of Pr55Gag were constructed in the infectious viral clone SIVmac239. These virus mutants were examined for defects in virus replication and virion infectivity, release, and morphology, as well as alterations in phosphorylation of CA-containing proteins. Although the virus mutants exhibited a number of replication defects, none of these defects could be directly attributed to aberrant CA phosphorylation. A novel defect was a block in early budding, which was common among several virus mutants with substitutions in the CA N terminus. Together, these results indicate that certain residues in the CA N terminus are crucial for early budding events.

Background: Stimulated by earlier reports on the presence of retroviruses in mouse and hamster melanoma cell lines, we addressed the question as to whether human melanoma cell lines might also harbour a retrovirus., Methods and Results: The melanoma cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO, MML-I, MeWo, A-375, Colo-38, BS-780 were confirmed to be human by human leucocyte antigen (HLA) typing, and supernatants were tested by the product-enhanced reverse transcriptase (PERT) assay for reverse transcriptase (RT) activity. Cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO and MML-I were positive, whereas cell lines MeWo, A-375, Colo-38 and BS-780 were negative. The RT activity peaked at a buoyant density in sucrose typical for retroviruses. From this peak fraction an R-U5 sequence indistinguishable from murine leukemia virus (MLV) was identified by particle-associated retrovirus RNA amplification (PARRA). Semiquantitative MLV-specific RNA-PCR demonstrated colocalization of the MLV-like RNA and RT activity on the sucrose gradient of SK-Mel-25. MLV RNA and DNA were also detectable in culture supernatants of SK-MEL-28, MEL-JUSO and MML-I, but not of MeWo, A-375, Colo-38 and BS-780 by semiquantitative polymerase chain reaction (PCR). Sequence comparison revealed highest homology with the RET sequence previously identified in mouse myeloma SP2/0-AG14 cells. Taken together, our data strongly suggest that certain human melanoma cell lines are productively infected by a MLV which was probably introduced during tumour passage in mice or by laboratory contamination many years ago and subsequently spread to other lines., Conclusion: We recommend mandatory testing of melanoma and other human cell lines for contamination with infectious MLV or other animal retroviruses, similar to mycoplasma screening, in order to avoid artificial experimental data.

Objective: Porcine endogenous retroviruses (PERVs) pose a risk for xenotransplantations using pig materials as they are present in the genome of all pigs and are able to infect human cells in vitro. Until recently, transmission of PERVs in vivo was only described in severe combined immunodeficient (SCID) and nude mice inoculated with PERV-producing cells. However, in this series of experiments microchimerism could not be excluded. To overcome this problem, the risk of PERV infection was addressed in a similar way but using cell-free inoculation of mouse cells in vitro and SCID mice in vivo., Methods: Mouse cell lines and primary cells were incubated in vitro with PERV-A, with a recombinant PERV-A/C and with PERV-B. Provirus integration was assessed by PCR. Reverse transcriptase activity was measured in the cell supernatants. SCID mice were inoculated in vivo with cell-free virus at high titers., Results: None of the mouse cell lines and primary cells could be infected by PERV and no provirus integration was observed in different organs of the inoculated SCID mice., Conclusion: The data indicate that PERV-A, PERV-A/C and PERV-B could not infect different mouse cells. These data correlate with the recent finding that mouse cells lack a functional receptor for PERV-A. Although the receptor for PERV-B is still unknown, these data suggest that previously reported PERV transmissions to SCID and nude mice in vivo might be due to microchimerism or pseudotyping with murine viruses and indicate that normal mice are an inappropriate model for the study of PERV infection and pathogenesis., (Copyright (c) 2005 S. Karger AG, Basel.)

It was recently found that the Duck hepatitis B virus (DHBV) reverse transcriptase is primarily a non-encapsidated cytoplasmic molecule that is rapidly translated and has a very short half-life. Here, a non-encapsidated reverse transcriptase from the human Hepatitis B virus (HBV) was characterized. HBV polymerase accumulated in the cytoplasm in a manner similar to non-encapsidated DHBV polymerase. However, the HBV polymerase accumulated at an apparently lower concentration and had a longer half-life than the DHBV enzyme, and it displayed no evidence of the post-translational modifications observed for DHBV. Unlike the DHBV polymerase, immunofluorescence detection of the HBV polymerase in cells was suppressed by the core protein, and this suppression occurred independently of encapsidation. This implies an interaction between the polymerase and core in addition to encapsidation, but the polymerase and core did not co-immunoprecipitate, so the interaction might not be direct. These data indicate that production of cytoplasmic, non-encapsidated polymerase is conserved among the hepadnaviral genera. Furthermore, conservation of the cytoplasmic form of the polymerase suggests that it might have function(s) in virus replication or pathology beyond copying the viral genome.

We previously characterized a series of small in-frame deletions within the C-terminal third of the simian immunodeficiency virus (SIV) gp41 cytoplasmic domain that significantly impair the incorporation of the envelope (Env) glycoprotein into particles and Env-mediated virus entry. Among these mutations, removal of Env residues 832-837 caused the most drastic defective phenotype. In the present study, we introduced the Delta832-837 deletion into the PBj1.9 molecular clone and investigated the effect of this env mutation on virus replication in the CEMx174 cell line. This in-frame deletion was found to severely compromise virus replication. Interestingly, long-term culture of the PBjEnvDelta832-837 mutant led to the emergence of two independent populations of revertant viruses that, while differing in the time point at which they appear, encode truncated gp41 cytoplasmic tails of similar lengths. The first emergent virus population contained a premature stop codon mutation at Env residue 778, whereas the late-appearing population harbored a stop codon mutation at Env residue 774, which results in the truncation of the gp41 cytoplasmic tail to 52 and 48 amino acids, respectively. Analysis of derivatives of PBjEnvDelta832-837 containing either the Tyr778stop or the Trp774stop mutations demonstrated that these second-site changes were sufficient to reverse the Env incorporation and infectivity defects imposed by the original Delta832-837 deletion, as well as to confer to the Env double mutants essentially wild-type replication kinetics. Our results thus provide further insight into the mechanisms underlying SIV adaptation to novel selective forces.

Determining the subcellular localization of the L1 ORF2 protein (ORF2p) has been impossible to date because of technical limitations in detecting either endogenous or overexpressed forms of the protein. Here we report visualization of the full-length ORF2p in cultured human cells following expression in a modified vaccinia virus/T7 RNA polymerase (MVA/T7RP) system. The MVA/T7RP system was used to ascertain subcellular localization of L1 ORF1p and ORF2p both as fusions with green fluorescent protein and by immunocytochemistry. Full-length ORF2p was predominantly cytoplasmic, while carboxy-terminal-deleted ORF2p localized additionally to the nucleolus. We mapped a functional nucleolar localization signal in ORF2p. ORF1p appeared in the cytoplasm with a speckled pattern and colocalized with ORF2p in nucleoli in a subset of cells. These findings help explain the presence of chimeras between L1s and small RNA gene sequences recently discovered in the human genome.

CD8(+) T lymphocytes have the potential ability to inhibit human immunodeficiency virus (HIV) replication, by secreting soluble(s) factor(s) known as CD8(+) T lymphocyte antiviral factor (CAF). A panel of CD8(+) and CD4(+) T cell clones from HIV1-infected and uninfected donors were generated to better define the phenotype of CAF-producing cells. We first verified that the different CD4(+) T cell subsets (Th0, Th1, and Th2) were productively infected by X4 and R5 virus strains. X4 viral replication in CD4(+) T cells was controlled by the three CD8(+) T cell subsets (Tc0, Tc1, and Tc2); however, the frequency of Tc clones controlling R5 strain was much lower with a dramatic absence of this activity among Tc clones from uninfected donor. Finally, capacity to control viral replication showed an heterogeneity: some clones could control both virus strains, some controlled only the X4 virus, whereas the majority exerted no suppressive activity.

Rodent cells are used widely to manufacture recombinant proteins for pharmaceutical use in humans and animals. However, all rodent cell lines express endogenous retroviruses that require appropriate testing regimes for identification and characterisation. In this communication we report the results of transmission electron microscopy, reverse transcriptase assay and infectious virus assays for retrovirus in 185 manufacturer cell banks of mouse, rat or hamster origin. The results indicated considerable variability of retroviral expression levels by transmission electron microscopy and reverse transcriptase assay, but nevertheless characteristic features of each cell type were observed. Infectious retrovirus was detected in mouse myeloma and hybridoma cell lines, but not in cell lines of hamster or rat origin. There was no evidence of contamination of cell banks with exogenous retrovirus. The results of retroviral characterisation of the parental mouse cell lines NS0, NS-1 and Sp2/0Ag14 by the above assays were consistent with the results of the survey. Co-cultivation of the above parental mouse cell lines with mouse and human cell lines suggested that the ability to infect human cells was related to threshold susceptibility of cell types and the levels of expression of infectious xenotropic retrovirus by mouse cells.

Chemokines play critical roles in HIV-1 infection, serving both to modulate viral replication and to recruit target cells to sites of infection. Interferon-gamma-inducible protein 10 (IP-10/CXCL10) is a C-X-C chemokine that acts specifically upon activated T cells and macrophages and attracts T cells into the cerebrospinal fluid (CSF) in HIV-associated neurological disease. We now demonstrate that IP-10 stimulates HIV-1 replication in monocyte-derived macrophages and peripheral blood lymphocytes. We further demonstrate that neutralization of endogenous IP-10 or blocking the function of its receptor, CXCR3, reduces HIV-1 replication in these same cells. Therefore, blocking the interaction between IP-10 and CXCR3 represents a possible new target for anti-retroviral therapy.

The antiviral activity of recombinant feline interferon-gamma (rFeIFN-gamma) against feline immunodeficiency virus (FIV) was investigated. A persistently FIV(Bang)-infected feline T cell line (FeT-J/Bang) was treated with either rFeIFN-omega, rFeIFN-gamma, or recombinant human IFN-alpha2 (rHuIFN-alpha2), and the culture fluids were tested for antiviral activity by reverse transcriptase (RT) assay. FeT-J/Bang cell cultures treated with rFeIFN-omega showed dose-dependent inhibition of RT activity. In contrast, rFeIFN-gamma treatment had no antiviral effect on FIV replication but instead caused a statistically significant enhancement on day 9 of culture. Antiviral activity of rFeIFN-gamma was also tested on feline peripheral blood mononuclear cells (PBMC). PBMC cultures were inoculated with FIV(Bang) and simultaneously treated with either rFeIFN-omega, rFeIFN-gamma, or rHuIFN-alpha2. FeIFN-gamma had no effect on FIV replication, unlike the rFeIFN-omega and rHuIFN-alpha2, which had strong anti-FIV effects. In another study, rFeIFN-gamma treatment was initiated 3 days before FIV(Bang) infection, the day of FIV(Bang) infection, or 3 days post-FIV(Bang) infection and then tested for antiviral activity. The time of initiating rFeIFN-gamma treatment had no effect on the antiviral activity. Hence, these results suggest that unlike rHuIFN-alpha2 and rFeIFN-omega, rFeIFN-gamma has no inhibitory effect on FIV replication in PBMC but causes a slight enhancement in a feline T cell line.

Plasma from macaques inoculated with macrophage-tropic SIVmac were tested for neutralization/enhancement of macrophage-tropic SIVmac239-17EBR in normal rhesus macrophages. The plasma of a macaque (R71) that developed a highly virulent neuro-tropic (macrophage-tropic) strain of SIVmac in neuro-adaptation studies significantly enhanced infection of SIVmac239-17EBR as well as infection of dual-tropic SIVmac251. Plasma from other macaques that were inoculated with macrophage-tropic virus, neutralized SIVmac239-17EBR. Also, plasma from macaques infected with lymphocyte-tropic SIVmac239 or dual-tropic SIVmac251 neutralized SIVmac251 infection. When the effect of R71 plasma on the early stages of the SIV life cycle was investigated, this plasma significantly increased binding of 35S-methionine labeled SIVmac239-17EBR and increased binding of 35S-SIVmac251 to primary macrophages, unlike plasma from other macaques infected with either macrophage-tropic or lymphocyte-tropic SIV. A single-cycle infection assay showed higher percentages of positive cells and more intense fluorescence in cultures treated with R71 plasma compared with control plasma. By in situ hybridization, SIV RNA transcripts were detected earlier (12 hr post-infection) and in higher percentages, in cultures treated with R71 plasma than in cultures treated with control plasma (18 hr post-infection). These results indicate that enhancing activity in infected macaque plasma may be associated with severe infection by highly virulent macrophage-tropic (neuro-tropic) SIV. The enhancing effect occurs early in infection and results in increased transcription of SIV RNA.

The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.

Telomerase activity is believed to be crucial for cell immortalization and cancerization, and is proven to be induced by c-myc protein. Telomerase reverse transcriptase (TERT) has been recently identified as a catalytic subunit of telomerase, whose expression is closely correlated with telomerase activity. We estimated telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and examined the immunohistochemical expression of TERT and c-myc protein in 21 ameloblastoma tissues. All ameloblastoma samples were positive for telomerase activity, and TERT expression was detected in the nuclei of neoplastic cells but not in those of stromal cells. Numerous peripheral columnar or cuboidal cells, sporadic central polyhedral cells and some granular cells in ameloblastomas reacted with anti-TERT antibody. These results suggest that telomerase activity is associated with the oncogenesis or proliferative potential of odontogenic epithelium. The expression of c-myc protein showed a similar distribution pattern to that of TERT, suggesting that c-myc protein might induce telomerase activity in ameloblastomas.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

Objective: Hypertrophy of the thyroid gland in Graves' disease is related to an autoimmune response directed against TSH receptors found in thyroid cells. Recently, investigators have suggested that autoimmune diseases, including thyroid diseases may, at least in part, correlate with the expression of proteins encoded by the retroviral genome. In the present study, to confirm the correlation between thyroid autoimmune disorders and retroviral infections, we examined reverse transcriptase (RT) activity in thyroid tissues as a marker of retroviral infection., Patients and Measurements: Thyroid tissues obtained at surgery from patients with various thyroid disorders (normal thyroid adjacent to adenoma, six cases; Graves' disease thyroid tissue, 25 cases; adenoma, eight cases; papillary carcinoma, 12 cases; Graves' disease peripheral blood lymphocytes, 11 cases) were used for RT assay, using a specific, improved assay system., Results: Thyroid tissue extracts from patients with Graves' disease contained high RT activity which resembled that demonstrated in retroviruses. The RT existed in the thyroid tissue as a complex, with endogenous template RNA, and the activity was confirmed not to be due to other DNA polymerases., Conclusion: Retroviral RT distinguished from known cellular DNA polymerases is expressed in the thyroids of patients with Graves' disease. In a permissive genetice and immunological environment, retroviral DNA integrated into genomic DNA could precipitate the onset of Graves' disease.

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for determining the activity of HIV reverse transcriptase (HIV-RT). By using a sequential capping strategy, the assay has been configured to utilize only generic reagents such as biotinylated dUTP, streptavidin-allophycocyanin, and streptavidin-europium. The assay was optimized for a HIV-RT high-throughput screen. Under optimized conditions, a signal-to-background ratio of approximately 10:1 and a Z' factor of 0.8 were obtained. The titration curves of several known HIV-RT inhibitors have been evaluated with this assay format. This HTRF format can be used for high-throughput assays with other nucleotide polymerases.

The detection of adventitious retroviruses has always been critical for assessing the safety concerns associated with viral vaccines. Assays for the enzymatic activity of reverse transcriptase (RT) are used as general methods for the detection of both known and unknown retroviruses. Several studies using newly-developed ultrasensitive PCR-based RT assays reported RT activity in viral vaccines grown in chicken cells. Here, we have assessed the performances of such a PCR-based RT assay--PERT assay--for the quantitative detection of RT activity in vaccines. Sensitivity, linearity and reproducibility of the method were studied on purified RT and viral vaccines treated to release RT from potentially contaminant retroviruses. The level of RT activity detected in chicken cell-derived vaccines was higher for live attenuated vaccines compared to inactivated ones. Contrary to other studies, RT activity was found in some mammalian cell-derived vaccines. AZT-TP sensitivity of RT activities detected in these vaccines and discrimination between retroviral and RT-like activities was further investigated. Feasibility and limits of PERT assay as a broad-spectrum retroviruses detection method in vaccines are discussed.

Five methods for the assessment of plasma viral load (VL) were evaluated in 103 seropositive patients infected with various subtypes of HIV-1. The methods included three RNA-based assays (Amplicor Monitor 1.5, Quantiplex version 2.0, NucliSens), one ultrasensitive reverse transcriptase (PERT) assay and one "boosted" p24 antigen (Ag) enzyme immunoassay (EIA). Subtyping was based on sequencing in env. The sensitivities were, in decreasing order, Amplicor > PERT > p24 Ag > NucliSens > Quantiplex. The low sensitivity of NucliSens was related to the missing of several non-B (A, E, F, G) or recombinant strains, whereas that of Quantiplex did not depend on subtype. In the 1 group O sample and 4 group M samples, only PERT assay or p24Ag EIA produced a positive result. In the quantitative range, correlation was best between Amplicor and Quantiplex (r = 0.8848), fair between Amplicor and NucliSens (r = 0.7064) or PERT assay (r = 0.7266), lowest between Amplicor and p24Ag EIA (r = 0.3989). Amplicor underestimated VL in 1 subtype E sample. Thus, Amplicor performed best in terms of sensitivity (compared with all other assays) and accuracy (compared with NucliSens, PERT assay, and p24Ag) for non-B subtypes in group M samples. PERT assay appears useful for VL assessment in infections by group O or other highly divergent viruses.

To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating reverse transcription. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection. Only one of these species is able to start and complete reverse transcription in the cell cytoplasm. This RTC is composed of at least the viral genome, capsid, integrase, and reverse transcriptase proteins. The RTC becomes permeable to micrococcal nuclease but not to antibodies. Shortly after initiation of reverse transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion. The sedimentation velocity of the RTC decreases during reverse transcription. After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further.

The localization of a reverse transcriptase-related protein in salivary gland polytene chromosomes was investigated by immunohistochemistry in two species of Chironomus. The antibodies used were raised against a recombinant protein containing phylogenetically conserved motifs of reverse transcriptases and derived from an abundant non-LTR element previously identified in Chironomus. Immunoreactive protein was found in some telomeres, in a centromeric region, in a few interstitial bands and in Balbiani ring 3. The telomeric signal was probably dependent on transcription and increased dramatically when telomeric heat shock puffs were induced. A correlation with transcription was also seen in Balbiani ring 3, the immunobinding of which disappeared after inhibition of transcription with actinomycin D.

The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new 32P-based, Mg2+-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID50s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immunodeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of recombinant HIV-1 RT enzyme indicated that 10(-5) U, which is equivalent to 4.25 x 10(4) virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg2+-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans.