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Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies
(2011)
Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells
A virus derived from cells of a Iymphoblastoid line originating from the lymph node of a healthy African green monkey was characterized as a typical member of the foamy virus subgroup of rctroviridac by its morphological, physicochemical, biological and biochemical properties (reverse transcriptase actvity). Besides the usual host range of foamy viruses, the isolated strain revealed a remarkable T -lymphotropism, distinguishing it from the prototypes of foamy viruses previously isolated from African green monkeys. Two foamy virus infectious are demonstrated in human contacts of the African green monkey colony, with the animal barbauring the isolate.
In common with most viruses, measles virus (MV) relies on the integrity of the cytoskeleton of its host cells both with regard to efficient replication in these cells, but also retention of their motility which favors viral dissemination. It is, however, the surface interaction of the viral glycoprotein (gp) complex with receptors present on lymphocytes and dendritic cells (DCs), that signals effective initiation of host cell cytoskeletal dynamics. For DCs, these may act to regulate processes as diverse as viral uptake and sorting, but also the ability of these cells to successfully establish and maintain functional immune synapses (IS) with T cells. In T cells, MV signaling causes actin cytoskeletal paralysis associated with a loss of polarization, adhesion and motility, which has been linked to activation of sphingomyelinases and subsequent accumulation of membrane ceramides. MV modulation of both DC and T cell cytoskeletal dynamics may be important for the understanding of MV immunosuppression at the cellular level.
Expression of human foamy virus is differentially regulated during development in transgenic mice
(1992)
Tbe human foamy virus (HFV) is a recently characterized member ofthe spumavirus family. Although no diseases have been unequivocally associated with HFV infection, expression of HFV regulatory genes in transgenie mice induces a characteristic aeute neuro degenerative disease and a myopathy. To better eharaeterize the sequenee of events leading to disease, and to gain a better understanding of the underlying pathogenetic meehanisms, we have analyzed in detail the transgene expression pattern during development. Transcription of a construet containing all regulatory elements and aneillary genes of mv was analyzed by in situ hybridization and was shown to occur in two distinct phases. At midgestation, low but widespread expression was first deteeted in eells of extraembryonie tissues. Later, various tissues originating from embryonie mesoderm, neuroeetoderm, and neural erest transeribed the transgene at moderate levels. However, expression deereased dramatically during late gestation and was suppressed shortly after birth. After a latency period of up to 5 weeks, transeription of the transgene resumed in single eelJs distributed irregularly in the central nervous system and in the skeletal museIe. By the age of 8 weeks, an increasing number of eells displayed much higher expression levels than in embryonie Iife and eventually underwent severe degenerative ehanges. These findings demonstrate that HFV transgene expression is differentially regulated in development and that HFV cytotoxicity may be dose-dependent. Such biphasic pattern of expression differs from that of murine retroviruses and may be explained by the specificity of HFV regulatory elements in combination with cellular faetors. Future studies of this model system should, therefore, provide novel insights in the mechanisms controlling retrovirallatency.
The human foamy virus (HFV) bel-l transactivator protein was expressed in insect cells by a recombinant baculovirus. For the generation of the recombinant baculovirus, Acbel-1, the bel-l gene of an HFV mutant was used, that bears truncations in the bel-l overlapping bel-2 open reading frame. Acbel-1 infected Sf9 cells produced high amounts of recombinant protein of the same electrophoretic mobility (36 kD) as bel-l expressed in mammalian cells. The baculovirus expressed bel-l proteinwas readily identified by a polyclonal rabbit serum directed against bel-1 in immunoblot assay. As in mammalian cells, bel-l was predominantly localized to the nucleus of Acbel-1 infected insect cells. The baculovirus expressed bel-1 proteinwill be of use to determine the action of this novel viral transactivator more precisely.
Human foamy viruspol gene fragments were molecularly cloned into a procaryotic expression vector. The expression pattern of the cloned fragments and nucleotide sequence analysis of the 5' pol gene region revealed that in HFV the protease (PR) is located in the pol open reading frame. Purified recombinant proteins were used to generate antibodies in rats. ln immunoblot assay, using infected cells as antigen, a precursor protein with an apparent molecular mass (M,) of 127K was identified by antibodies directed against the reverse transcriptase (RT), RNaseH, or integrase (IN) domeins of pol. With concentrated virus as antigen, the RT and RNaseH antibodies recognized a protein of 80K, the IN antiserum recognized a protein of 40K, and the PR antiserum detected a protein of approximately 10K.
We have identified the major immunogenic structural proteins of the human foamy virus (HFV), a distinct member of the foamy virus subfamily of Retroviridae. Radiolabelied viral proteins were immunoprecipitated from HFV -infected cells by foamy virus antisera of human and non-human primate origin. Precipitated viral proteins were in the range of 31 K to 170K. Labelling of proteins with [\(^{14}\)C]glucosamine or with [\(^{35}\)S]methionine in the presence oftunicamycin, as well as endo-ß-N-acetylglycosaminidase Hand F treatment of [\(^{35}\)S]methionine-labelled proteins, revealed three viral glycoproteins of approximately 170K, 130K and 47K, most likely representing the env gene-encoded precursor, the surface glycoprotein and the transmembrane protein of HFV, respectively.
In vitro and in vivo infection of rhesus monkey microglial cells by simian immunodeficiency virus
(1993)
The observation that microglial cells in brain tissue are probably a major target for human immunodeficiency virus (HIV) infection has raised interest in the pathogenic role of this cell population for the development of neuro-AIOS. Since it is very difficult to obtain microglia from normal or diseased human brain we studied microglial cells isolated from fresh brain tissue of uninfected and simian immunodeficiency virus (SIV) infected rhesus monkeys (Macacca mulatta) in comparison to peripheral blood macrophages. Besides the characterization of the phenotypes of these two cell populations, we examined the replication of SIV in the cells in addition to the effect of viral infection on the expression of cell surface molecules. We found that microglia and macrophages support replication of the wild-type SIV\(_{mac25}\), strain as well as the infectious clone (SIV\(_239\)). Infectious viruswas produced and a CPE developed. Isolated microglial cells from SIV-infected monkeys were latently infected independent of the presence of neuropathological lesions and produced infectious virus after 20-25 days in culture. In situ hybridization revealed that only a small percentage of isolated microglial cells are productively infected in vivo, yet the majority of these expressed MHC class II molecules. This indicated a state of activation that is acquired in vivo. These findings indicate that microglia are a prime target cell for SIV infection in CNS tissue.
An infectious molecular clone (pHSRV) of the human Spumaretrovirus (HSRV) was constructed using viral DNA and cDNA clones. The infectivity of pHSRV was proven by transfection of cell cultures and subsequent infection of susceptible cultures with cell free transfection derlved virus. pHSRV derived virus produced foamy virus typical cytopathic effects in susceptible cultures. lnfected cells could be stained specifically with foamy virus antisera by means of indirect immunofluorescence. Radiolmmunoprecipltatlon revealed the presence of characteristic HSRV structural proteins in pHSRV infected cultures. By cotransfection of pHSRV and an indicator plasmid it was found that pHSRV is able to transactivate the viral L TR. Viral transcripts were found to be approximately 200 bases Ionger in pHSRV infected cultures compared to wildtype infected cultures. This difference is most likely due to an Insertion of DNA of non-viral origin ln the U3 region of the 3'L TR of the infectious clone.
DNA ofhuman spumaretrovirus (HSRV) was cloned from both cDNA and from viral DNA into phage A and bacterial plasmid vectors. The recombinant plasm.ids harboring viral DNA were characterized by Southern blot hybridization and restriction mapping. Physical maps were constructed from cDNA and found to be colinear with the restriction maps obtained from viral DNA. The recombinant clones isolated contained viral DNA inserts which rangein size from 2.2 kb to 15.4 kb. The recombinant clones allowed to construct a physical map of the complete HSRV provirus of 12.2 kb.
All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear ftuorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Cag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. Tbis motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus.