Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.     

Characterization of the feline c-abl proto-oncogene

Analysis of total feline DNA by genomic blot hybridization, using the viral oncogene of Abelson murine leukemia virus as a specific probe, has led to the identification of multiple v-abl homologous genetic sequences in the cat genome. Upon restriction endonuclease BamHI digestion, the combined size of the v-abl homologous DNA fragments was about 31 kbp. To characterize these sequences further, four independent v-abl homologous cosmid clones with overlapping cellular inserts have been isolated from a gene library of cat lung genomic DNA. These inserts represent a contiguous region of cellular DNA sequences of 56 kbp in length. Within this region of the feline genome, the v-abl homologous sequences are discontinuously dispersed over a region of about 34 kbp. They represent the complete feline v-abl cellular homolog and are colinear with the viral v-abl oncogene. Nine regions of highly repetitive DNA sequences have been mapped in close proximity to v-abl homologous sequences. These results establish the presence of only a single c-abl proto-oncogene in the cat genome and present its genetic organization.     

Herpes simplex virus type 1 Angelotti and a defective viral genotype: analysis of genome structures and genetic relatedness by DNA-DNA reassociation kinetics.

DNA-DNA reassociation kinetics of herpes simplex virus type 1 Angelotti DNA and a class of defective viral DNA revealed that the viral standard genome has a total sequence complexity of about 93 X 10(6) daltons and that a portion of 11 X 10(6) daltons occurs twice on the viral genome. These results agree with structural features of herpes simplex virus type 1 DNA derived from electron microscopic studies and restriction enzyme analyses by several investigators. The defective viral DNA (molecular weight, about 97 X 10(6)) displays a sequence complexity of about 11 X 10(6) daltons, suggesting that the molecule is built up by repetitions of standard DNA sequences comprising about 15,000 base pairs. A 2 X 10(6)-dalton portion of these sequences maps in the redundant region and a 9 X 10(6)-dalton portion maps in the unique part of the standard herpes simplex virus type 1 Angelotti DNA, as could be shown by reassociation of viral standard DNA in the presence of defective DNA and vice versa. No cellular DNA sequences could be detected in defective DNA. A 12% molar fraction of the defective DNA consists of highly repetitive sequences of about 350 to 500 base pairs in length.     

Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate

Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5′-triphosphate (5′-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5′-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5′-PPP moiety for RIG-I activation.     

Regions of the terminal repetitions of the herpes simplex virus type 1 genome. Relationship to immunoglobulin switch-like DNA sequences

Several recombinant clones isolated from a mouse genomic library were previously shown to hybridize with a SmaI fragment located in the terminal repetition of the S component of herpes simplex virus DNA. We report here the nucleotide sequence of the related regions in two mouse clones, TGL19 and TGL35, as well as that of the SmaI fragment of HSV-1. The mouse DNA clones have a core of repetitive sequences 80% homologous to a tandem repeat (reiteration II) in the viral fragment. The regions of homology are in turn related to immunoglobulin class-switch sequences, due mostly to the presence of the pentamer TGGG(G), involved in class-switch recombination. These results suggest that the HSV genome has recombination sequences identical to those of the host cell and provide a possible explanation for the high frequency of recombination events observed in this region of the viral genome.     

Genome composition and tandemly repetitive sequence at some centromeres in the lizard Podarcis s. sicula Raf.

A detailed study on the genome of the lizard P. sicula has been carried out using restriction enzyme analysis followed by identification and cloning of a repetitive DNA fraction. The results show that P. sicula generally possesses a quite homogeneous genome composition, with a single tandemly repetitive sequence family that is easily visualized after digestion of genomic DNA with Taq I. The cloned repeating unit of this satellite (260 bp) has been designed pLCSl. In-situ hybridisation shows that this satellite is localized in the centromeric region. Dot blot experiments show that sequences similar to pLCSl are present in other species of the same family of lizards.     

Bcl10 phosphorylation-dependent droplet-like condensation positively regulates DNA virus-induced innate immune signaling

B-cell lymphoma 10 (Bcl10) is a scaffolding protein that functions as an upstream regulator of NF-κB signaling by forming a complex with Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and CARD-coiled coil protein family. This study showed that Bcl10 was involved in type I interferon (IFN) expression in response to DNA virus infection and that Bcl10-deficient mice were more susceptible to Herpes simplex virus 1 (HSV-1) infection than control mice. Mechanistically, DNA virus infection can trigger Bcl10 recruitment to the STING-TBK1 complex, leading to Bcl10 phosphorylation by TBK1. The phosphorylated Bcl10 undergoes droplet-like condensation and forms oligomers, which induce TBK1 phosphorylation and translocation to the perinuclear region. The activated TBK1 phosphorylates IRF3, which induces the expression of type I IFNs. This study elucidates that Bcl10 induces an innate immune response by undergoing droplet-like condensation and participating in signalosome formation downstream of the cGAS-STING pathway.

     

Molecular epidemiology of DNA viruses: applications of restriction endonuclease cleavage site analysis.

Restriction endonucleases which cleave DNA at specific nucleotide sequences can be used to produce a set of DNA fragments of a viral genome which, when separated by gel electrophoresis, gives a characteristic "fingerprint" for that virus genome. This simple technique has been used to identify and classify DNA viruses of the herpes, adeno, and papova virus groups. Small variants within a given type (e.g., herpes simplex type I) are genetically stable and permit study and identification of individual strains of viruses. Such analyses have recently been applied to study the epidemiology of some DNA virus outbreaks. Restriction endonuclease fingerprinting provides a useful addition to methods for virus identification and classification.     

Interferon prevents the generation of spontaneous deletions at the left terminus of vaccinia virus DNA.

In this report we have shown that Friend erythroleukemia cells persistently infected with vaccinia virus maintain the persistent infection even after 1 year of continuous interferon (IFN) treatment. The persistently infected cultures were responsive to IFN as determined by their ability to induce 2-5A synthetase, to increase the intracellular levels of 2-5A, and to cause rRNA cleavage. While large deletions at the left terminus of vaccinia DNA occurred readily in the virus population from untreated cells, IFN completely suppressed the generation of these spontaneous deletions. Removal of IFN from these cultures led to the appearance of similar deletions at the left terminus of the viral genome. The regions deleted contain more than half of the left-end inverted terminal repetition of the vaccinia genome. These findings show that IFN alters specific events associated with the generation of vaccinia DNA deletions.     

Epstein-Barr virus DNA recombines via latent origin of replication with the human genome in the lymphoblastoid cell line RGN1.

We show here that in a lymphoblastoid cell line Epstein-Barr virus DNA recombines with the human genome. The genetic exchange involves the oriP region of the virus. A junction between viral and human DNA from this line has been cloned and sequenced. The results indicate that the integration of Epstein-Barr virus DNA involves a region of the human genome which contains internal short repetition. An 800-bp probe has been isolated from the human part of the junction. This probe has been used to show that the human region exists as a duplication in normal cells.     

Characterizing the citrus cultivar Carrizo genome through 454 shotgun sequencing

The citrus cultivar Carrizo is the single most important rootstock to the US citrus industry and has resistance or tolerance to a number of major citrus diseases, including citrus tristeza virus, foot rot, and Huanglongbing (HLB, citrus greening). A Carrizo genomic sequence database providing approximately 3.5×genome coverage (haploid genome size approximately 367 Mb) was populated through 454 GS FLX shotgun sequencing. Analysis of the repetitive DNA fraction indicated a total interspersed repeat fraction of 36.5%. Assembly and characterization of abundant citrus Ty3/gypsy elements revealed a novel type of element containing open reading frames encoding a viral RNA-silencing suppressor protein (RNA binding protein, rbp) and a plant cytokinin riboside 5′-monophosphate phosphoribohydrolase-related protein (LONELY GUY, log). Similar gypsy elements were identified in the Populus trichocarpa genome. Gene-coding region analysis indicated that 24.4% of the nonrepetitive reads contained genic regions. The depth of genome coverage was sufficient to allow accurate assembly of constituent genes, including a putative phloem-expressed gene. The development of the Carrizo database (http://citrus.pw.usda.gov/) will contribute to characterization of agronomically significant loci and provide a publicly available genomic resource to the citrus research community.     

Influenza virus-induced type I interferon leads to polyclonal B-cell activation but does not break down B-cell tolerance

The link between infection and autoimmunity is not yet well understood. This study was designed to evaluate if an acute viral infection known to induce type I interferon production, like influenza, can by itself be responsible for the breakdown of immune tolerance and for autoimmunity. We first tested the effects of influenza virus on B cells in vitro. We then infected different transgenic mice expressing human rheumatoid factors (RF) in the absence or in the constitutive presence of the autoantigen (human immunoglobulin G [IgG]) and young lupus-prone mice [(NZB x NZW)F(1)] with influenza virus and looked for B-cell activation. In vitro, the virus induces B-cell activation through type I interferon production by non-B cells but does not directly stimulate purified B cells. In vivo, both RF and non-RF B cells were activated in an autoantigen-independent manner. This activation was abortive since IgM and IgM-RF production levels were not increased in infected mice compared to uninfected controls, whether or not anti-influenza virus human IgG was detected and even after viral rechallenge. As in RF transgenic mice, acute viral infection of (NZB x NZW)F(1) mice induced only an abortive activation of B cells and no increase in autoantibody production compared to uninfected animals. Taken together, these experiments show that virus-induced acute type I interferon production is not able by itself to break down B-cell tolerance in both normal and autoimmune genetic backgrounds.