HSV-1 genome subnuclear positioning and associations with host-cell PML-NBs and centromeres regulate LAT locus transcription during latency in neurons.

Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.). Latent HSV-1 genomes display two major patterns, called "Single" and "Multiple", which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. "Single" and "Multiple" patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the "Multiple" pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction

A novel cell response triggered by interphase centromere structural instability

Interphase centromeres are crucial domains for the proper assembly of kinetochores at the onset of mitosis. However, it is not known whether the centromere structure is under tight control during interphase. This study uses the peculiar property of the infected cell protein 0 of herpes simplex virus type 1 to induce centromeric structural damage, revealing a novel cell response triggered by centromere destabilization. It involves centromeric accumulation of the Cajal body–associated coilin and fibrillarin as well as the survival motor neuron proteins. The response, which we have termed interphase centromere damage response (iCDR), was observed in all tested human and mouse cells, indicative of a conserved mechanism. Knockdown cells for several constitutive centromere proteins have shown that the loss of centromeric protein B provokes the centromeric accumulation of coilin. We propose that the iCDR is part of a novel safeguard mechanism that is dedicated to maintaining interphase centromeres compatible with the correct assembly of kinetochores, microtubule binding, and completion of mitosis

Herpes simplex virus-1 in the brain. The dark side of a sneaky infection

Herpes simplex virus-1 (HSV-1) establishes latency preferentially in sensory neurons of peripheral ganglia. A variety of stresses can induce recurrent reactivations of the virus, which spreads and then actively replicates to the site of primary infection (usually the lips or eyes). Viral particles produced following reactivation can also reach the brain, causing a rare but severe form of diffuse acute infection, namely herpes simplex encephalitis. Most of the time, this infection is clinically asymptomatic. However, it was recently correlated with the production and accumulation of neuropathological biomarkers of Alzheimer's disease. In this review we discuss the different cellular and molecular mechanisms underlying the acute and long-term damage caused by HSV-1 infection in the brain

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells

Herpesvirus Latency: On the Importance of Positioning Oneself

International audienc

The interaction between herpes simplex virus 1 genome and promyelocytic leukemia nuclear bodies (PML-NBs) as a hallmark of the entry in latency

Herpes simplex virus 1 (HSV-1) is a human pathogen that establishes latency in the nucleus of infected neurons in the PNS and the CNS. At the transcriptional level latency is characterized by a quasi-complete silencing of the extrachromosomal viral genome that otherwise expresses more than 80 genes during the lytic cycle. In neurons, latency is anticipated to be the default transcriptional program; however, limited information exists on the molecular mechanisms that force the virus to enter the latent state. Our recent study demonstrates that the interaction of the viral genomes with the nuclear architecture and specifically the promyelocytic leukemia nuclear bodies (PML-NBs) is a major determinant for the entry of HSV-1 into latency (Maroui MA, Callé A et al. (2016). Latency entry of herpes simplex virus 1 is determined by the interaction of its genome with the nuclear environment. PLoS Pathogens 12(9): e1005834.)

Centromeric protein CENP-B proteasomal degradation induced by the viral protein ICP0.

International audienceThe ICP0 protein of herpes simplex virus type 1 (HSV-1) is a nuclear protein that possesses a well-characterized E3 ubiquitin ligase activity. This activity is responsible for the proteasomal-dependent degradation of several cellular proteins. This study shows that ICP0 induces the proteasomal-dependent degradation of the centromeric protein CENP-B in infected as well as ICP0-expressing cells. It is also shown that the ICP0-induced CENP-B degradation occurs as efficiently in human and mouse cells. CENP-B is one of the major proteins of centromeres and its degradation is likely to contribute to the severe damage induced to centromeres by ICP0

Herpes Simplex Virus Type 1 Immediate-Early Protein Vmw110 Inhibits Progression of Cells through Mitosis and from G(1) into S Phase of the Cell Cycle

Herpes simplex virus type 1 (HSV-1) immediate-early protein Vmw110 stimulates the onset of virus infection in a multiplicity-dependent manner and is required for efficient reactivation from latency. Recent work has shown that Vmw110 is able to interact with or modify the stability of several cellular proteins. In this report we analyze the ability of Vmw110 to inhibit the progression of cells through the cell cycle. We show by fluorescence-activated cell sorter and/or confocal microscopy analysis that an enhanced green fluorescent protein-tagged Vmw110 possesses the abilities both to prevent transfected cells moving from G(1) into S phase and to block infected cells at an unusual stage of mitosis defined as pseudo-prometaphase. The latter property correlates with the Vmw110-induced proteasome-dependent degradation of CENP-C, a centromeric protein component of the inner plate of human kinetochores. We also show that whereas Vmw110 is not the only viral product implicated in the block of infected cells at the G(1)/S border, the mitotic block is a specific property of Vmw110 and more particularly of its RING finger domain. These data explain the toxicity of Vmw110 when expressed alone in transfected cells and provide an explanation for the remaining toxicity of replication-defective mutants of HSV-1 expressing Vmw110. In addition to contributing to our understanding of the effects of Vmw110 on the cell, our results demonstrate that Vmw110 expression is incompatible with the proliferation of a dividing cell population. This factor is of obvious importance to the design of gene therapy vectors based on HSV-1

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent <i>In situ</i> Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue