PubMed Health⌕ Search

Biomedical subjects

C Hohenadl

Publications and source records attributed to C Hohenadl.

24 records · Page 2Linked to original sources
PubMed1994

Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma.

Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA. Specific proteolysis of protein synthesis initiation factor eIF-4 gamma occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs. Cleavage of eIF-4 gamma in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.e., foot-and-mouth disease viruses), the leader proteinase is responsible for this reaction. We describe here the purification to homogeneity of the Lb form of the leader proteinase expressed in Escherichia coli. The primary cleavage products of eIF-4 gamma obtained in vitro with purified leader or 2A proteinase are electrophoretically indistinguishable from those found during infection in vivo. However, additional proteolysis products of eIF-4 gamma are observed with the leader proteinase and the human rhinovirus type 2 2A proteinase in vitro. The cleavage site of the leader proteinase in eIF-4 gamma from rabbit reticulocyte was determined by sequencing the purified C-terminal cleavage product by automated Edman degradation. The cleavage site is between Gly-479 and Arg-480 and thus differs from that of rhinovirus and enterovirus 2A proteinases, which cleave between Arg-486 and Gly-487.

Amino Acid Sequence↗
PubMed1992

Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation.

Coxsackievirus B3-induced myocarditis in different immunocompetent mouse strains was used as a model to investigate interrelationships between virus replication and development of chronic enteroviral heart disease. Using in situ hybridization to detect enteroviral RNA, we show that heart muscle infection is not only detected in acute myocarditis but is also detected during the chronic phase of the disease. Coxsackievirus B3 could evade immunological surveillance in a host-dependent fashion, thus inducing a persistent infection of the myocardium in association with ongoing inflammation. Patterns of acute and persistent myocardial infection were quantitatively assessed in one representative mouse strain (A.CA/SnJ, H-2f) by applying computer-assisted digital image processing; these patterns were then related to the extent of myocardial tissue damage as well as to inflammation. We observed a strong correlation, both spatial and temporal, between viral replication and development of myocardial lesions, indicating that acute and chronic myocardial injuries are a consequence of multifocal organ infection. Analysis of strand-specific in situ hybridization revealed that viral replication in persistent infection is restricted at the level of RNA synthesis. The described procedure for quantitating organ infection provides a powerful tool for evaluating virus-host interactions and will be of particular interest to those studying human enterovirus-induced cardiomyopathies.

Acute Disease↗
PubMed1991

Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization.

In this report we describe the development and application of single-stranded RNA probes for strand-specific detection of enterovirus RNA in infected heart tissue by in situ hybridization. For synthesis of RNA probes a full-length reverse-transcribed, recombinant CVB3 cDNA was inserted into the transcription vector pSPT18. Run-off transcripts of plus-strand and minus-strand orientation were produced using either T7 or SP6 RNA polymerase. Binding specificity and sensitivity of the radioactively labelled RNA probes were determined by slot-blot hybridization. Due to the high degree of genetic identity among enteroviruses, the in vitro transcribed CVB3 RNA probes hybridized with various enterovirus serotypes, including group A and B coxsackieviruses and echoviruses, which are commonly implicated in human viral heart disease. Strand-specific in situ hybridization led to detection of viral plus-strand or minus-strand RNA in infected cell cultures and in myocardial tissue sections of infected mice. In consecutive sections either viral genomic plus-strand RNA or complementary minus-strand RNA were localized in the same infected myocardial cells. In situ hybridization with enterovirus-specific and highly sensitive single-stranded RNA probes is of particular interest for the diagnosis of myocardial infections and for studies concerning viral RNA replication.

Animals↗
PubMedDate not supplied

Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors.

BACKGROUND: A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles. METHODS: In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)-based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3' LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5' LTR of the vector. RESULTS: PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence-activated cell sorting (FACS). After detection of low-level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3' LTR at the R/U5 border to prevent accidental read-through transcription from neighbouring cellular promoters. Virus-containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells. CONCLUSIONS: This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell.

3T3 Cells↗