Retroviral and related nucleic acid processing enzymes expression and detection : Applications on clinical studies of HIV and HERVs

Sammanfattning: 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,