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Retroviral and related nucleic acid processing enzymes expression and detection: Applications on clinical studies of HIV and HERVs
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Reverse transcriptase (RT), the expression and detection of the enzyme marking the family Retroviridae, has been studied. A new RT activity assay adapted to 96-well microtitre plates is described. It uses a non- radioactive bromodeoxyuridine triphosphate (BrdUTP) substrate in a solid phase format. The assay was highly sensitive. The assay was also used for the measurement of HIV-1 RT activity blocking ab (RTh-ab), where it also demonstrated high sensitivity and specificity. Cross reactivity studies comparing HIV-1 and HIV-2 RTh-ab proved HIV-1 RTb-ab to be highly type specific, while HIV-2 RTh-ab were type specific to a lower extent. RTb-ab determination is a good complement to current HIV ab assays. Furthermore it can be employed to screen for infection in vaccine trials, when vaccines devoid of pol gene products has been used. The assay has also been used by others for studies of antivirals and therapy resistance, and of SIV infection of macaques.

The human deoxycytidine kinase (dCK) was also investigated. dCK is involved in DNA synthesis and repair pathways. It is also a key enzyme in phosphorylating several clinically important anti-cancer and anti-viral nucleoside analogs. Stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd seemed to bind to the enzyme by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. We concluded that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer. Since UTP has been shown to be the preferred phosphate donor for dCK our studies lead to new information regarding the steps involved in interactions between dCK and its most important ligands. A general model for nucleoside kinase action was suggested.

Expression of retrovirus in the form of RT RNA in serum was also studied using the polymerase chain reaction (RNA). Using general retroviral primers we found RNA related to ERV in a serum from a multiple sclerosis patient. Our sequence (LUMS9) was located to chromosome 3q26.1, while the MSRV variant published by a French group was located to 15q2l.3. Thus, at least two loci of HERV-W sequences can be activated to produce extracellular RNA in MS.

Finally, we studied the reverse transcriptase enzyme of the HERV-K(HML-2) endogamous retroviral group. Several members of this group are transcriptionally active and encode biologically active proteins. We expressed and cloned amplimers from a broad amplification of betaretroviral endogamous human RT sequences. The resulting RT proteins 4 came from the HML-2 group. Although there were confounding results, some of the cloned proteins had RT and RNAse H acfivity,

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2001. , 65 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1083
Keyword [en]
Medical sciences, Reverse transcriptase, endogenous retrovirus, deoxycytidine kinase, HIV, HERVS
Keyword [sv]
MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
Research subject
Medical Virology
Identifiers
URN: urn:nbn:se:uu:diva-1442ISBN: 91-554-5132-2 (print)OAI: oai:DiVA.org:uu-1442DiVA: diva2:160983
Public defence
2001-10-12, Universitetshuset, sal IV, Uppsala, 13:15 (English)
Opponent
Available from: 2001-10-16 Created: 2001-10-16 Last updated: 2009-05-08Bibliographically approved
List of papers
1. A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation
Open this publication in new window or tab >>A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation
1996 In: Biotechnol Appl Biochem, Vol. 23, no Pt 2, 95-105 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89486 (URN)
Available from: 2001-10-16 Created: 2001-10-16Bibliographically approved
2. Measurement of levels of human immunodeficiency virus type 1 reverse transcriptase (RT) and RT activity-blocking antibody in human serum by a new standardized colorimetric assay
Open this publication in new window or tab >>Measurement of levels of human immunodeficiency virus type 1 reverse transcriptase (RT) and RT activity-blocking antibody in human serum by a new standardized colorimetric assay
Show others...
1997 In: J Clin Microbiol., Vol. 35, no 5, 1080-9 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89487 (URN)
Available from: 2001-10-16 Created: 2001-10-16Bibliographically approved
3. A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: A model for nucleoside kinase action
Open this publication in new window or tab >>A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: A model for nucleoside kinase action
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1999 In: Biochemistry, Vol. 38, no 26, 8555-61 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89488 (URN)
Available from: 2001-10-16 Created: 2001-10-16Bibliographically approved
4. Retroviral RNA related to ERV9/MSRV in a human serum: a new sequence variant
Open this publication in new window or tab >>Retroviral RNA related to ERV9/MSRV in a human serum: a new sequence variant
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1999 (English)In: AIDS Research and Human Retroviruses, ISSN 0889-2229, E-ISSN 1931-8405, Vol. 15, no 6, 591-593 p.Article in journal (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-54223 (URN)10.1089/088922299311114 (DOI)10221536 (PubMedID)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2017-12-04Bibliographically approved
5. Open reading frames and associated enzymatic activities in the polymerase gene of human endogenous retroviral (HERV)-K sequenses
Open this publication in new window or tab >>Open reading frames and associated enzymatic activities in the polymerase gene of human endogenous retroviral (HERV)-K sequenses
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-89490 (URN)
Available from: 2001-10-16 Created: 2001-10-16 Last updated: 2010-01-13Bibliographically approved

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