Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes

Epstein-Barr virus (EBV), a herpes virus, infects human B lymphocytes in vitro and efficiently immortalizes them. About 10 of the approximately 100 genes of EBV are expressed in recently immortalized B cells and although there is circumstantial evidence that at least three of these may contribute to the process of immortalization, there is no direct evidence that any particular gene is required. We have developed a genetic analysis of EBV that uses a transformation-defective strain of the virus as a helper virus in conjunction with DNA that contains all of the viral cis-acting elements required for replication, cleavage and packaging during the lytic phase of the viral life cycle. This DNA can include viral genes required for immortalization that complement the transformation-defective virus strain. The DNA can be amplified and packaged by the products of the helper virus and the packaged DNA is infectious. We have analysed two viral genes expressed in immortalized cells and find that the gene encoding EBV nuclear antigen-2 is required for immortalization, whereas the gene for the EBV nuclear antigen leader protein is not.     

Endogenous viral genes are non-essential in the chicken.

DNA sequences homologous to the genomes of type C retroviruses are widespread among vertebrates. Ten genetic loci containing endogenous viral DNA sequences have been documented in the white Leghorn chicken alone. Six of these genetic loci are associated with the production of virus or of viral proteins in embryonic fibroblasts (refs 2--4, and S.M.A., L, B. Crittenden and E.G.B., in preparation) and one of the loci may be expressed in the erythroblasts of 5-day-old embryos. The abiquitous presence of endogenous viral genes among vertebrate species and the association of their expression with development of the haematopoietic system in the mouse have led to the proposal that these genes are involved in ontogeny. In addition, the genes may be implicated in oncogenesis as in the case of the AKR mouse in which a high incidence of spontaneous leukaemia is associated with the expression of endogenous murine laukaemia virus genomes. We report here the production of a fertile rooster which lacks avian leukosis virus-related endogenous viral genes and which seems to be completely normal and healthy. Thus, endogenous viral genes are apparently not essential for the normal development of the chicken. An endogenous virus-free state has also been reported for three species of jungle fowl and for the B-type viral genes of the mouse.     

onc sequences (v-fes) of Snyder-Theilen feline sarcoma virus are derived from noncontiguous regions of a cat cellular gene (c-fes)

Type C sarcoma viruses are genetic recombinants containing portions of replication-competent helper viruses linked to sarcoma virus-specific sequences (generically designated onc genes) which are thought to be required for acute fibroblast transformation. The onc elements of different avian and mammalian sarcoma viral isolates are each homologous to subsets of cellular DNA sequences which have no well-defined role in normal cells. Because of the lack of significant homology between helper viral genes and cellular onc sequences, the recombinational mechanisms which facilitate the formation of sarcoma viral genomes remain unclear. In Moloney murine sarcoma virus, viral onc (or v-mos) and cellular onc (or c-mos) sequences exhibit complete and uninterrupted homology as determined by heteroduplex and restriction enzyme analyses of molecularly cloned DNA. By contrast, the cellular counterparts of the onc elements of Rous sarcoma virus (G. Cooper and R. Parker, personal communication), avian erythroblastosis virus (B. Vennstrom, personal communication), Abelson leukaemia virus (D. Baltimore, personal communication), Harvey sarcoma virus (E. Scolnick, personal communication) and simian sarcoma virus (R. Gallo, personal communication) are now known to contain intervening sequences which do not appear in the respective viral genomes. Here we report the use of the Southern blot technique to examine cat cellular DNA sequences (c-fes) homologous to the onc gene (v-fes) of Snyder-Theilen feline sarcoma virus (ST-FeSV). We used cloned DNA 'probes' containing defined portions of the ST-FeSV genome to show that v-fes sequences originate from at least four noncontiguous sequences in cat cellular DNA, separated from each other by intervening sequences.     

Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours

The recent finding of c-myc activation by insertion of woodchuck hepatitis virus DNA in two independent hepatocellular carcinoma has given support to the hypothesis that integration of hepatitis B viruses into the host genome, observed in most human and woodchuck liver tumours, might contribute to oncogenesis. We report here high frequency of woodchuck hepatitis virus DNA integrations in two newly identified N-myc genes: N-myc1, the homologue of known mammalian N-myc genes, and N-myc2, an intronless 'complementary DNA gene' or 'retroposon' that has retained extensive coding and transforming homology with N-myc. N-myc2 is totally silent in normal liver, but is overexpressed without genetic rearrangements in most liver tumours. Moreover, viral integrations occur within either N-myc1 or N-myc2 in about 20% of the tumours, giving rise to chimaeric messenger RNAs in which the 3' untranslated region of N-myc was replaced by woodchuck hepatitis virus sequences encompassing the viral enhancer. Insertion sites were clustered in a short sequence of the third exon that coincides with a retroviral integration hotspot within the murine N-myc gene, recently described in T-cell lymphomas induced by murine leukaemia virus. Thus, comparable mechanisms, leading to deregulated expression of N-myc genes, may operate in the development of tumours induced either by hepatitis virus or by nonacute retroviruses in rodents. Activation of myc genes by insertion of hepadnavirus DNA now emerges as a common event in the genesis of woodchuck hepatocellular carcinoma.     

Bombyx mori bidensovirus: The type species of the new genus Bidensovirus in the new family Bidnaviridae

Bombyx mori bidensovirus （BmBDV）, which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut columnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence sug- gests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies.     

The oncogenic potential of herpes simplex viruses: evidence for a 'hit-and-run' mechanism

Experiments to determine the mechanism of transformation of herpes simplex virus (HSV) have identified fragments of viral DNA which are able to initiate transformation. No set of viral genes seems to be consistently retained or expressed in the transformed cells or in human cervical tumours, suggesting that viral DNA is not needed to maintain the transformed phenotype. In fact there is no conclusive evidence that initiation of neoplasia is mediated by a viral protein. Here we revisit the 'hit-and-run' hypothesis and its implications for HSV-induced tumorigenicity.     

Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus

The 'Spanish' influenza pandemic of 1918-19 was the most devastating outbreak of infectious disease in recorded history. At least 20 million people died from their illness, which was characterized by an unusually severe and rapid clinical course. The complete sequencing of several genes of the 1918 influenza virus has made it possible to study the functions of the proteins encoded by these genes in viruses generated by reverse genetics, a technique that permits the generation of infectious viruses entirely from cloned complementary DNA. Thus, to identify properties of the 1918 pandemic influenza A strain that might be related to its extraordinary virulence, viruses were produced containing the viral haemagglutinin (HA) and neuraminidase (NA) genes of the 1918 strain. The HA of this strain supports the pathogenicity of a mouse-adapted virus in this animal. Here we demonstrate that the HA of the 1918 virus confers enhanced pathogenicity in mice to recent human viruses that are otherwise non-pathogenic in this host. Moreover, these highly virulent recombinant viruses expressing the 1918 viral HA could infect the entire lung and induce high levels of macrophage-derived chemokines and cytokines, which resulted in infiltration of inflammatory cells and severe haemorrhage, hallmarks of the illness produced during the original pandemic.     

JD病毒基因组片段的克隆和序列分析

扩增并克隆了ＪＤ病毒的ｇａｇ 基因片段（位于３００ｎｔ～５６４ｎｔ,编码基质蛋白ＭＡ）和ｐｏｌ基因片段（位于３４６７ｎｔ～３６９１ｎｔ,编码反转录酶ＲＴ）,测定其序列并同ＢＩＶ、ＢＦＶ 和ＢＬＶ 的相应基因进行比较．所建立的方法能够特异性扩增ＪＤＶ序列,适用于ＪＤＶ的检测     

Drosophila RNAi screen identifies host genes important for influenza virus replication

All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.     

Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes

The pathogenic human retrovirus human immunodeficiency virus type 1 (HIV-1) encodes two trans-acting nuclear proteins, tat and rev, whose functional expression is essential for viral replication in vitro. The tat protein greatly enhances the expression of both structural and regulatory genes of HIV-1 (linked to the viral long-terminal-repeat promoter element), whereas the rev gene product (previously termed art or trs) has only been shown to be required for the synthesis of structural proteins. Here, we demonstrate that rev also moderates the expression of regulatory genes of HIV-1. It decreases the expression of messenger RNAs that encode the full-length form of the viral tat gene product or the rev protein itself, and induces the synthesis of a previously unreported, truncated tat protein. These actions of rev are mediated by a dramatic shift in the ratio of spliced to unspliced cytoplasmic HIV-1 mRNA. Therefore rev not only activates the synthesis of the viral structural proteins, but also modulates the level and quality of HIV-1 regulatory gene expression.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

2株甲型肝炎病毒蛋白酶2A核苷酸序列的测定与比较

从甲型肝炎病毒２个不同株感染的ＫＭＢ１７细胞中提取总ＲＮＡ,经ＲＴ－ＰＣＲ特异性扩增出此２毒株病毒蛋白２Ａ基因,将２个２Ａ基因克隆到ｐＧＥＭ－Ｔ载体上,经ＤＮＡ序列测定,得到２个２Ａ基因的核心苷酸序列,序列比较发现它们及与ＨＡＶ野毒株相对应序列有突变存在,Ｈ２株与ＨＭ１７５相比有一个点突变,同源性为９９．７２％;与ＨＭ１７５比较,Ｈ２株和Ｌ８株第３１９７位核苷酸均由Ａ突变为Ｇ,相应的氨基酸由丝氨酸     

Protein Subcellular Localization Prediction and Genomic Polymorphism Analysis of the SARS Coronavirus

Introduction In March 2003, a novel coronavirus (CoV) was dis-covered in association with the outbreak of severe acute respiratory syndrome (SARS)[1-3]. The complete genome sequence of several SARS-CoV isolates was soon determined and characterized[4,5]. Comparison of variant SARS-CoV genome sequences has identified certain genetic signatures that can be used to trace sources of infection[6]. Vaccines are now being devel-oped and molecular modeling has suggested that modi-fied rhinovir…     

Epstein-Barr virus infection and replication in a human epithelial cell system.

Epstein-Barr virus, a human herpesvirus with oncogenic potential, infects two target tissues in vivo: B lymphocytes, where the infection is largely non-productive, and stratified squamous epithelium in which virus replication occurs. The interaction with B cells, initiated through virus binding to the B-cell surface molecule CR2 (ref. 4), has been studied in vitro and the virus 'latent' genes associated with B-cell growth transformation defined. By comparison, viral infection of epithelium remains poorly understood, reflecting the lack of an appropriate cell-culture model. Here we describe the development of such a model using as targets CR2-expressing transfected cells of two independent human epithelial lines. A high proportion of these cells bind virus and become actively infected, expressing the small EBER RNAs (small non-polyadenylated virus-coded RNAs) and the Epstein-Barr nuclear antigen 1 but not other latent proteins; thereafter, under conditions favouring epithelial differentiation, up to 30% of the cells can be induced to enter virus productive cycle with some progressing to full virus replication. We find significant differences between laboratory virus strains in their ability to infect epithelium that do not correlate with their B-cell growth-transforming activity.