Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

Human immunodeficiency virus (HIV) reverse transcriptase substitutes for temperature-sensitive DNA polymerase I (Pol Its) in Escherichia coli, providing a screen for anti-HIV reverse transcriptase nucleoside analogs in bacteria. Since phosphorylation of nucleosides in E. coli is limited to thymidine and its derivatives, we coexpressed herpes simplex virus thymidine kinase, an enzyme that phosphorylates a wide variety of nucleoside analogs, together with HIV reverse transcriptase. Coexpression of herpes simplex virus thymidine kinase and HIV reverse transcriptase rendered Pol Its cells sensitive to dideoxycytidine. Studies with different nucleoside analogs indicate that this bacterial screening system is able to select and identify nucleoside analogs that specifically target HIV reverse transcriptase.
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PMID:A screen in Escherichia coli for nucleoside analogs that target human immunodeficiency virus (HIV) reverse transcriptase: coexpression of HIV reverse transcriptase and herpes simplex virus thymidine kinase. 754 49

This study was initiated to evaluate a role for gamma interferon (IFN-gamma) in herpes simplex virus type 1 (HSV-1) infection. At the acute stage of infection in mice, HSV-1 replication in trigeminal ganglia and brain stem tissue was modestly but consistently enhanced in mice from which IFN-gamma was by ablated monoclonal antibody treatment and in mice genetically lacking the IFN-gamma receptor (Rgko mice). As determined by reverse transcriptase PCR, IFN-gamma and tumor necrosis factor alpha transcripts were present in trigeminal ganglia during both acute and latent HSV-1 infection. CD4+ and CD8+ T cells were detected initially in trigeminal ganglia at day 5 after HSV-1 inoculation, and these cells persisted for 6 months into latency. The T cells were focused around morphologically normal neurons that showed no signs of active infection, but many of which expressed HSV-1 latency-associated transcripts. Secreted IFN-gamma was present up to 6 months into latency in areas of the T-cell infiltration. By 9 months into latency, both the T-cell infiltrate and IFN-gamma expression had cleared, although there remained a slight increase in macrophage levels in trigeminal ganglia. In HSV-1-infected brain stem tissue, T cells and IFN-gamma expression were present at 1 month but were gone by 6 months after infection. Our hypothesis is that the persistence of T cells and the sustained IFN-gamma expression occur in response to an HSV-1 antigen(s) in the nervous system. This hypothesis is consistent with a new model of HSV-1 latency which suggests that limited HSV-1 antigen expression occurs during latency (M. Kosz-Vnenchak, J. Jacobson, D.M. Coen, and D.M. Knipe, J. Virol. 67:5383-5393, 1993). We speculate that prolonged secretion of IFN-gamma during latency may modulate a reactivated HSV-1 infection.
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PMID:Gamma interferon expression during acute and latent nervous system infection by herpes simplex virus type 1. 760 58

A leptomeningeal cell line (LM7) harbouring an unknown retrovirus was recently isolated from the cerebrospinal fluid of a patient with multiple sclerosis. However, spontaneous expression of the LM7 retrovirus in this primary culture is quite low. We present results showing that in vitro infection of LM7 cells with herpes simplex virus type 1 (HSV-1), but not that of control cells, results in (i) potent stimulation of the specific reverse transcriptase (RT) activity detected in the culture supernatant and (ii) co-expression of both typical HSV-1 virions and abundant retrovirus-like particles. Transfection of LM7 cells with plasmids expressing HSV-1 immediate early (IE) ICP0 and ICP4 proteins produced a similar enhancement of RT activity in culture supernatants with retrovirus-like particles being identifiable by electron microscopy. These effects were not observed with a plasmid expressing ICP27 or with the parental plasmid in LM7 cells, nor with any of these four plasmids in control cells. These results show that HSV IE trans-activating proteins strongly enhance the expression of the latent retrovirus present in LM7 cells. The possible role in vivo of herpesviruses as 'triggering' cofactors in the retrovirus hypothesis for multiple sclerosis aetiology is also discussed.
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PMID:Herpes simplex virus ICP0 and ICP4 immediate early proteins strongly enhance expression of a retrovirus harboured by a leptomeningeal cell line from a patient with multiple sclerosis. 767 35

Competitive quantitative PCR and reverse transcriptase-PCR were used to quantitate DNA and RNA from an attenuated ribonucleotide reductase-deleted herpes simplex virus type 1 (HSV-1) mutant in the rat trigeminal ganglion after peripheral inoculation following corneal scarification. Amplification of ganglionic DNA with oligonucleotide primers specific for the HSV-1 glycoprotein B (gB) gene and for the latency-associated transcript (LAT) gene indicated that there were approximately 2 x 10(5) genome equivalents per ganglion at 2 days, 7 days, and 8 weeks after inoculation. Amplification of ganglionic RNA with primers specific for HSV-1 LAT indicated that the amount of LAT RNA was also stable over 8 weeks, with 10(7) LAT molecules per ganglion at 2 days and at 7 days postinoculation and 1.4 x 10(7) LAT molecules per ganglion at 8 weeks. In situ hybridization with a digoxigenin-labeled riboprobe specific for LAT detected an average of one to two LAT-positive cells in each positive 6-microns section of trigeminal ganglion. In situ PCR detection of HSV-1 genomes in similar sections, using digoxigenin-labeled nucleotides with primers specific for HSV-1 gB, identified as many as 120 genome-positive cells per section. These results indicate that there are approximately 50 LAT molecules per latent HSV-1 genome in the trigeminal ganglion, compared with 15 LAT molecules per latent HSV-1 genome in the central nervous system (R. Ramakrishnan, D. J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994), but that cells with detectable LATs by in situ hybridization represent only a small proportion of those ganglionic neurons containing HSV-1 genomes. The presence of latent HSV-1 genomes in a large number of neurons suggests that HSV-1 may be more efficient in establishing the latent state than would be anticipated from previous reports.
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PMID:PCR-based analysis of herpes simplex virus type 1 latency in the rat trigeminal ganglion established with a ribonucleotide reductase-deficient mutant. 793 90

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.
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PMID:Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain. 810 47

Intracerebral inoculation of the MS strain of herpes simplex virus type 2 (HSV-2) into mice causes an acute encephalitis associated with multifocal demyelination and necrotizing retinitis. We have studied the distribution of latent virus in mice that had recovered from the acute encephalitis. Four weeks or longer after inoculation, HSV-2 could be recovered from the trigeminal ganglia of all mice examined by co-culture of explants in roller tubes. The virus could not be recovered from explants of retina or brain stem. HSV-2 latency associated transcript (LAT) was readily detected in the trigeminal ganglia by reverse transcriptase-PCR more than 4 months after inoculation. LAT was also demonstrated in the brain but this required nested PCR for consistent detection. Both LAT and ICP0 mRNA were detected in brain tissue during the acute encephalitis but, unlike LAT, ICP0 mRNA could not be amplified from the trigeminal ganglia or brain beyond 4 weeks after inoculation of the virus. In situ hybridisation with a double-stranded DNA probe to the ICP0/LAT overlap region of HSV-2 revealed signal in trigeminal ganglion neurons and occasional cells in the brain stem. These findings indicate that HSV-2 introduced by intracerebral inoculation becomes latent in the trigeminal ganglia and that transcription of LAT also persists within the brain.
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PMID:Latent infection with the MS strain of herpes simplex virus type 2 in the mouse following intracerebral inoculation. 828 71

A study was undertaken to assess the etiology, optimal diagnostic method, and incidence of healing of perianal ulcers in HIV-seropositive men. Between March 1990 and December 1991, 26 HIV-seropositive homosexual or bisexual males were referred with perianal ulcerations. According to CDC criteria, three (12 percent) were Class II, six (23 percent) were Class III, and 17 (65 percent) were Class IV. Eighteen patients had one ulcer, five had two ulcers, and two had three ulcers. In one patient the ulcer was circumanal. Patients with superficial erosions were not included. Biopsies were obtained in 23 patients for routine microscopy, HIV, cytomegalovirus, herpes simplex virus, and acid-fast bacilli. Biopsy revealed an immunoblastic lymphoma in one patient. A comparison of microscopy and culture results revealed culture to be more helpful in determining the etiology of these ulcers. Medical treatment included reverse transcriptase inhibitors (zidovudine, dideoxyinosine, and dideoxycytosine), oral and topical Zovirax (Burroughs Wellcome, Research Triangle Park, NC), ganciclovir, and oral broad-spectrum antibiotics. Surgical treatment included lateral internal sphincterotomy in three patients and seton placement in one patient. Follow-up for at least four weeks was obtained in 22 patients. Overall, healing occurred in 15 patients (68 percent): three (20 percent) were Class II, four (27 percent) were Class III, and eight (53 percent) were Class IV. Healing occurred in all four patients who underwent surgical treatment. In conclusion, aggressive diagnostic maneuvers allow the use of both medical and conservative surgical measures to successfully treat the majority of perianal ulcers in this patient population.
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PMID:Is aggressive management of perianal ulcers in homosexual HIV-seropositive men justified? 844 27

The role of putative promoter or activator sequences downstream of the herpes simplex virus type 2 latency-associated transcript (LAT) promoter and upstream of the LAT intron was investigated in vivo by constructing and evaluating mutant viruses with deletions in this region. The deletion of LAT promoter sequences upstream of the primary LAT transcript reduced levels of LAT expression during productive infections, compared with the LAT expression level of wild-type virus, and abolished LAT expression during latency. The deletion of the putative downstream regulatory elements reduced but did not eliminate LAT expression during productive and latent infections. The deletion of both regions almost completely eliminated acute LAT transcription, although additional acute LAT-region transcription directed by sequences upstream of either region was detected by reverse transcriptase PCR. The deletion of the downstream elements did not influence the ability of the virus to reactivate from latently infected guinea pigs relative to the ability of the wild-type virus to reactivate; thus, decreased LAT expression did not affect the frequency of recurrence. The deletion of both regions did not affect the ability of the virus to establish latency. We conclude that downstream regulatory elements are necessary for maximal acute LAT expression but do not constitute an independent promoter during latency and do not play an obvious role in the establishment of our reactivation from latency.
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PMID:Downstream regulatory elements increase acute and latent herpes simplex virus type 2 latency-associated transcript expression but do not influence recurrence phenotype or establishment of latency. 862 72

The "bystander effect" refers to the death of unmodified tumor cells when in contact with ganciclovir (GCV)-exposed, herpes simplex virus-thymidine kinase (HSV-TK)-modified tumor cells. Although the exact mechanism or mechanisms involved in mediating the bystander effect in vivo are unknown, our findings suggest that an intact host immune system is required for the phenomenon to occur. The present study was designed to establish the effect of HSV-TK-modified tumor cells and GCV on the tumor and its microenvironment in vivo. In sublethally irradiated and immunodeficient Balb/c mice, the bystander effect was observed to be diminished or abrogated. Histopathologic examination of the tumor mass from immunocompetent mice demonstrated centralized hemorrhagic tumor necrosis (38%) after inoculation of the HSV-TK-modified tumor cells and GCV in tumor-bearing mice compared with the control mice (5%), indicating that cytokines such as tumor necrosis factor-alpha (TNF-alpha) were being released locally. This hypothesis was underscored using reverse transcriptase polymerase chain reaction (RT-PCR), by the demonstration of cytokine mRNA expression in mice treated with HSV-TK-expressing tumors and GCV. Semiquantitative PCR analysis for TNF-alpha using PCR-MIMIC on tumor samples from mice treated on days 1 and 4 showed a two-fold increase in the level on mRNA expression. Also, immunohistochemical staining for TNF-alpha showed that mononuclear inflammatory cells infiltrating the tumor were its source. Finally, characterization of tumor-infiltrating lymphocytes (TIL) in experimental animals demonstrated a two- to three-fold increase in the number of macrophages and T cells compared with control animals. These results demonstrate that, in vivo, the bystander effect is mediated in part by an antitumor response through the release of cytokines. Further, the cytokine milieu and tumor microenvironment can be modulated following injection of HSV-TK cells and GCV to enhance the host immune response, which is of potential use in clinical trials.
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PMID:In vivo analysis of the 'bystander effect': a cytokine cascade. 864 34


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