The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.Retroviral DNA integration is catalyzed by a viral enzyme, integrase (IN), which nicks the two ends of linear viral DNA and splices them into a site in the host DNA (9, 10). This highly orchestrated reaction produces DNA sequence signatures at the virus-host junctions: the loss of usually 2 bp at the ends of the linear viral DNA and duplication of several base pairs of host DNA at the integration site. However, no gross rearrangements or deletions of either the viral or host DNAs are incurred. The integration reaction can be reproduced in vitro using purified IN and viral and target DNA model substrates. Despite the precision of the integration reaction, there are no strict host DNA sequence requirements. Nevertheless, numerous studies indicate that integration site selection is not likely to be entirely random. For example, various features of host chromosomes have been implicated in influencing integration site selection, including primary DNA sequence (7,14,18), DNA structure (16,20,24), nucleosome structure (27-31), chromatin structure (32,36,37,40), and transcriptional activity (23,33,34,41,43). In addition to the passive influences of chromosomal structure, it has been suggested that retroviral integration could be actively targeted by tethering to specific, chromatin-bound host factors (5). Lastly, the IN proteins from different retroviruses produce unique in vitro integration patterns in naked DNA targets (18). These intriguing but disparate observations have not yet led to a unifying model, and the mechanisms that govern integration site selection in vivo remain obscure.In an infected cell, retroviral DNA is organized in a preintegration complex that in...

(TSA) induces GFP activation in GFP(؊) cells and can also increase GFP expression in GFP(؉) cells. In the case of the GFP(؊)populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(؊) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.

Heart failure in children and adults is often the consequence of myocarditis associated with Coxsackievirus (CV) infection. Upon CV infection, enteroviral protease 2A cleaves a small number of host proteins including dystrophin, which links actin filaments to the plasma membrane of muscle fiber cells (sarcolemma). It is unknown whether protease 2A-mediated cleavage of dystrophin and subsequent disruption of the sarcolemma play a role in CV-mediated myocarditis. We generated knockin mice harboring a mutation at the protease 2A cleavage site of the dystrophin gene, which prevents dystrophin cleavage following CV infection. Compared with wild-type mice, we found that mice expressing cleavage-resistant dystrophin had a decrease in sarcolemmal disruption and cardiac virus titer following CV infection. In addition, cleavage-resistant dystrophin inhibited the cardiomyopathy induced by cardiomyocyte-restricted expression of the CV protease 2A transgene. These findings indicate that protease 2A-mediated cleavage of dystrophin is critical for viral propagation, enteroviral-mediated cytopathic effects, and the development of cardiomyopathy. IntroductionCoxsackievirus (CV) is a member of the enteroviral genus of the picornavirus family and is known to be an important cause of myocarditis and heart failure in children and adults. Enteroviral protease 2A cleaves the viral polyprotein and a small number of host cell proteins such as the cytoskeletal protein dystrophin (1) and the eukaryotic translation initiation factors eIF4G1 and eIF4G2 (2-4). Genetic deficiency of dystrophin causes cardiomyopathy in Duchenne muscular dystrophy and increases susceptibility to myocarditis (5, 6). However, the importance of protease 2A-mediated cleavage of dystrophin is not known. We hypothesized that cleavage of dystrophin by protease 2A is important in sarcolemmal membrane disruption, viral propagation, enteroviral-mediated cytopathic effects, and the development of viral myocarditis. In order to address this hypothesis, we knocked in a mutation at the protease 2A cleavage site of the dystrophin gene, thus inhibiting only the cleavage of dystrophin following CVB3 infection. When mice expressing cleavage-resistant dystrophin were infected with CVB3, there was a decrease in the sarcolemmal disruption, cardiac virus titer, and severity of myocarditis compared with control mice expressing cleavable wild-type dystrophin. In addition, the prevention of dystrophin cleavage in protease 2A-expressing transgenic mice (7) markedly inhibited the protease 2A-induced myocytopathic effect and cardiomyopathy. These findings indicate that disruption of the sarcolemma by protease 2A-mediated cleavage of dystrophin can have a critical role in the pathogenesis of viral myocarditis via alterations in viral propagation and enteroviral-mediated cytopathic effects in the intact wild-type heart.

The voltage-gated sodium channel Na v 1.8 is known to function in the transmission of pain signals induced by cold, heat, and mechanical stimuli. Sequence variants of human Na v 1.8 have been linked to altered cardiac conduction. We identified an allele of Scn10a encoding the α-subunit of Na v 1.8 among mice homozygous for N -ethyl- N -nitrosourea-induced mutations. The allele creates a dominant neurobehavioral phenotype termed Possum , characterized by transient whole-body tonic immobility induced by pinching the skin at the back of the neck (“scruffing”). The Possum mutation enhanced Na v 1.8 sodium currents and neuronal excitability and heightened sensitivity of mutants to cold stimuli. Striking electroencephalographic changes were observed concomitant with the scruffing-induced behavioral change. In addition, electrocardiography demonstrated that Possum mice exhibited marked sinus bradycardia and R-R variability upon scruffing, abrogated by infusion of atropine. However, atropine failed to prevent or mitigate the tonic immobility response. Hyperactive sodium conduction via Na v 1.8 thus leads to a complex neurobehavioral phenotype, which resembles catatonia in schizophrenic humans and tonic immobility in other mammals upon application of a discrete stimulus; no other form of mechanosensory stimulus could induce the immobility phenotype. Our data confirm the involvement of Na v 1.8 in transducing pain initiated by cold and additionally implicate Na v 1.8 in previously unknown functions in the central nervous system and heart.

No abstract