J. Neurochem. (2010) 115, 1520–1529. Abstract Soluble amyloid‐β peptide (Aβ) oligomers, known to accumulate in Alzheimer’s disease brains, target excitatory post‐synaptic terminals. This is thought to trigger synapse deterioration, a mechanism possibly underlying memory loss in early stage Alzheimer’s disease. A major unknown is the identity of the receptor(s) targeted by oligomers at synapses. Because oligomers have been shown to interfere with N‐methyl‐d‐aspartate receptor (NMDAR) function and trafficking, we hypothesized that NMDARs might be required for oligomer binding to synapses. An amplicon vector was used to knock‐down NMDARs in mature hippocampal neurons in culture, yielding 90% reduction in dendritic NMDAR expression and blocking neuronal oxidative stress induced by Aβ oligomers, a pathological response that has been shown to be mediated by NMDARs. Remarkably, NMDAR knock‐down abolished oligomer binding to dendrites, indicating that NMDARs are required for synaptic targeting of oligomers. Nevertheless, oligomers do not appear to bind directly to NMDARs as indicated by the fact that both oligomer‐attacked and non‐attacked neurons exhibit similar surface levels of NMDARs. Furthermore, pre‐treatment of neurons with insulin down‐regulates oligomer‐binding sites in the absence of a parallel reduction in surface levels of NMDARs. Establishing that NMDARs are key components of the synaptic oligomer binding complex may illuminate the development of novel approaches to prevent synapse failure triggered by Aβ oligomers.

Productive infection by herpes simplex virus type 1 (HSV-1), which occurs in the host cell nucleus, is accompanied by dramatic modifications of the nuclear architecture, including profound alterations of nucleolar morphology. Here, we show that the three most abundant nucleolar proteins-nucleolin, B23, and fibrillarin-are redistributed out of the nucleoli as a consequence of HSV-1 infection. We show that the amount of nucleolin increases progressively during the course of infection. We demonstrate for the first time that a nucleolar protein, i.e., nucleolin, colocalizes with ICP8 in the viral replication compartments, at the time when viral replication is effective, suggesting an involvement of nucleolin in the HSV-1 DNA replication process. At later times of infection, a granular form of nucleolin localizes to the cytoplasm, in structures that display the characteristic features of aggresomes, indicating that this form of nucleolin is very probably destined for degradation. The delocalization of nucleolin from the nucleoli requires the viral ICP4 protein or a factor(s) whose expression involves ICP4. Using small interfering RNA technology, we show that viral replication requires a high level of nucleolin expression, demonstrating for the first time a direct role for a nucleolar protein in herpes simplex virus biology.Herpes simplex virus type 1 (HSV-1) is a human herpesvirus consisting of an outer envelope, a tegument, a capsid, and a linear double-stranded DNA. Productive infection consists of a highly ordered program of viral gene expression, DNA replication, and virion assembly that leads to the formation of infectious viral progeny and cell death (48). The viral DNA contains at least 80 genes whose expression is sequentially and temporally regulated by complex regulatory mechanisms. Viral genes can be divided into immediate-early, early, and late genes, according to their kinetics of expression. Proteins encoded by immediate-early genes are involved in the regulation of the synthesis of early and late proteins. Proteins encoded by early genes participate in viral DNA replication, and proteins encoded by late genes are mainly the structural components of the viral particles.Viral transcription, DNA replication, assembly of new capsids, and packaging of HSV-1 DNA occur in the host cell nucleus. As a consequence, HSV-1-infected cells undergo a variety of changes including dramatic modifications of the nuclear architecture. The formation of viral replication compartments (VRC), which are the sites of replication, transcription, and encapsidation of HSV-1 genomes, is accompanied by the marginalization of chromatin and the disruption of the nuclear lamina and of PML bodies, as well as by a profound modification of nucleolar morphology (2,15,33,39). Soon after infection, nucleoli increase in size, localize close to the nuclear membrane, and finally become fragmented in small pieces (39,49). In addition, several viral proteins, including ICP0, ICP4, ICP27, US11, and gamma 34.5, localize at least transiently to nucl...

Rheumatoid arthritis is characterized by erosive inflammation of the joints, new bone proliferation, and ankylosis, leading to severely reduced locomotion and intense chronic pain. In a model of this disease, adjuvant-induced polyarthritis in the rat, neurons involved in pain transmission and control undergo plastic changes, especially at the spinal level. These changes affect notably neurons that contain opioids, such as enkephalins deriving from preproenkephalin A (PA) precursor protein. Using recombinant herpes simplex virus containing rat PA cDNA, we enhanced enkephalin synthesis in sensory neurons of polyarthritic rats. This treatment markedly improved locomotion and reduced hyperalgesia. Furthermore, the progression of bone destruction slowed down, which is the most difficult target to reach in the treatment of patients suffering from arthritis. These data demonstrate the therapeutic efficacy of enkephalin overproduction in a model of systemic inflammatory and painful chronic disorder.

Within the Herpesviridae family, Alphaherpesvirinae is an extensive subfamily which contains numerous mammalian and avian viruses. Given the low rate of herpesvirus nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in alphaherpesviruses is intimately linked to DNA replication. Both viral and cellular proteins participate in this recombination-dependent replication. The presence of inverted repeats in the alphaherpesvirus genomes allows segment inversion as a consequence of specific recombination between repeated sequences during DNA replication. High molecular weight intermediates of replication, called concatemers, are the site of early recombination events. The analysis of concatemers from cells coinfected by two distinguishable alphaherpesviruses provides an efficient tool to study recombination without the bias introduced by invisible or non-viable recombinants, and by dominance of a virus over recombinants. Intraspecific recombination frequently occurs between strains of the same alphaherpesvirus species. Interspecific recombination depends on enough sequence similarity to enable recombination between distinct alphaherpesvirus species. The most important prerequisite for successful recombination is coinfection of the individual host by different virus strains or species. Consequently the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology, virulence and latency. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.

This study describes the physical and functional interactions between ICP0 of herpes simplex virus type 1 and class II histone deacetylases (HDACs) 4, 5, and 7. Class II HDACs are mainly known for their participation in the control of cell differentiation through the regulation of the activity of the transcription factor MEF2 (myocyte enhancer factor 2), implicated in muscle development and neuronal survival. Immunofluorescence experiments performed on transfected cells showed that ICP0 colocalizes with and reorganizes the nuclear distribution of ectopically expressed class I and II HDACs. In addition, endogenous HDAC4 and at least one of its binding partners, the corepressor protein SMRT (for silencing mediator of retinoid and thyroid receptor), undergo changes in their nuclear distribution in ICP0-transfected cells. As a result, during infection endogenous HDAC4 colocalizes with ICP0. Coimmunoprecipitation and glutathione S-transferase pull-down assays confirmed that class II but not class I HDACs specifically interacted with ICP0 through their aminoterminal regions. This region, which is not conserved in class I HDACs but homologous to the MITR (MEF2-interacting transcription repressor) protein, is responsible for the repression, in a deacetylase-independent manner, of MEF2 by sequestering it under an inactive form in the nucleus. Consequently, we show that ICP0 is able to overcome the HDAC5 amino-terminal-and MITR-induced MEF2A repression in gene reporter assays. This is the first report of a viral protein interacting with and controlling the repressor activity of class II HDACs. We discuss the putative consequences of such an interaction for the biology of the virus both during lytic infection and reactivation from latency.The role of acetylation in the transcription pathway has been widely studied and is generally considered a major step toward gene expression (1). Two classes of enzymes with antagonist activities, called histone acetyltransferases (HATs) and deacetylases (HDACs), significantly contribute to the control of expression by the modification of the acetylation state of predominantly, but not exclusively, histone proteins (5, 70). Whereas HAT activity, and thus acetylation of histones, is generally associated with transcription, HDAC activity, and thus deacetylation, is responsible for repression (69). Three groups of HDACs, defined as classes I, II, and III, have been described in higher eukaryotes, according to sequence similarities with yeast RPD3, HDA1 and SIR2 proteins, respectively (reviewed in references 43 and 73). Class II HDACs have been classified as such mainly on the basis of amino acid sequence homologies with the deacetylation domain of members of the class I family. However, for most of them, and unlike class I HDACs, clear evidence concerning their direct implication in deacetylation of substrates is lacking. It is now thought that, with the exception of HDAC6, class II HDACs acquire their deacetylation activity only through the interaction of their carboxy-terminal end with at ...

The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1–induced AAV replication.

Amplicons are promising helper-dependent HSV-1-derived vectors that allow the transfer and expression of very large foreigner DNA into dividing and quiescent cells. We had already described an approach to prepare large amounts of high-titer amplicon vectors, using Cre-loxP site-specific recombination system to delete the packaging ("a") signals of an HSV-1 recombinant helper virus (HSV-1 LaL). Amplicon vectors prepared using such a system showed a level of contamination with helper particles lower than 1%. The residual helper particles generated by this system are, however, replication-competent, thus precluding their use in gene therapy. To avoid such potential spread of residual particles, we present here the development of a defective Cre-loxP-based helper virus (HSV-1 LaL Delta J), deleted of the genes encoding ICP4 and ICP34.5 proteins from the helper genome, in addition to the native "a" signals. HSV-1 LaL Delta J carries a single floxed "a" signal in gC locus. To produce HSV-1 LaL Delta J and to prepare the amplicon vectors, we have constructed two novel cell lines expressing the essential ICP4 protein, either alone or in combination with Cre recombinase. These cell lines were conceived to complement ICP4 while minimizing the probability of generating replication-competent particles. In this paper we present results demonstrating that the novel helper system allows ready production of large amounts of high-titer amplicon vectors. Residual helper particles generated still do not exceed 0.5% of the viral population and can grow only in cells expressing ICP4. Amplicon vectors produced with this method showed no cytotoxicty for infected cells.

Our observations point to an effect of the therapy on either the survival or the localization of PDCs, rather than a direct detrimental effect due to the viral infection during chronic HCV infection.