Jean-Marie Béchet

The cytopathic effect of HIV is associated with apoptosis.

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2S-M2 and a Tet repressor-KRAB fusion protein (tTSKRAB) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter Ptetbi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTSKRAB to Ptetbi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2S-M2 activates Ptetbi-1.

Detection of HHV-6 by the polymerase chain reaction

The polymerase chain reaction (PCR) was used to detect human herpes virus 6 (HHV-6) sequences in tissue culture. A pair of primers was synthesized and used to amplify a conserved region of the genome. Amplified products were detected either by visualization of UV illuminated ethidium bromide stained gel or, by hybridization with a specific radiolabeled oligonucleotide. As little as 5 fg of HHV-6 could be detected in infected cells, making this assay suitable for diagnostic purposes.

Random cleavage of intranuclear herpes simplex virus DNA by micrococcal nuclease

Biochemical and electron microscopic evidence indicates that the basic structure of eukaryotic chromatin consists of repeated subunits, termed nubodies or nucleosomes, containing -200 base pairs of DNA complexed to a histone octamer [ 11. A similar structure has been shown to exist for some DNA viruses replicating in the nuclei of infected cells. Thus, SV40 DNA has been shown to be associated with cellular histones in a structure similar to cellular chromatin [2]. During the replication of larger DNA viruses, belonging to the adenovirus and herpesvirus groups, the synthesis of cellular proteins is inhibited and the virions appear to lack cellular histones [3]. However, analysis of micrococcal nuclease digestion products has shown that adenovirus DNA is in a repeating-unit chromatin-like structure in the nuclei of infected cells and in the virions [4]. Herpesviruses are another group of large DNA viruses replicating in the nucleus. In the present work, we have used micrococcal nuclease as a probe for analysing the structure of the nucleoprotein of herpes simplex virus in the nuclei of infected cells. Advantage was taken of the fact that, owing to the inhibition of cellular DNA synthesis late in infection, radioactive label is preferentially incorporated into viral DNA. Thus, viral DNA-containing structures can be studied in their intranuclear state, by following the fate of radioactive DNA during digestion of nuclei isolated from cells labelled late in infection. 2. Materials and methods

Enhanced replication of herpes simplex virus type 1 in BHK-21 cells transformed by hamster sarcoma virus.

Untransformed and hamster sarcoma virus-transformed BHK-21 cells were compared for their ability to support productive infection with herpes simplex virus type 1. In both types of cells, infection with herpes simplex virus resulted in a lytic, productive cycle. However, inhibition of host cell RNA and DNA synthesis, transcription and replication of viral DNA, and production of infectious virus began earlier and reached higher levels in transformed cells.

Metabolic stability of nuclear self-complementary RNA in cells infected with herpes simplex virus.

Abstract The self-complementary fraction of RNA labeled late in infection with herpes simplex virus was quantitated as a function of labeling and chase times. The labeled RNA that became double-stranded upon self-annealing was a stable fraction of labeled total cell RNA, and was present almost exclusively in the nucleus. The relative amount of complementary sequences in viral RNA was evaluated by hybridization of total and double-stranded RNA to viral DNA, and was found to remain stable with increasing labeling times. The data suggest that post-transcriptional selection of the coding strand occurs during or after transport from the nucleus.