Ethidium bromide inhibits appearance of closed circular viral DNA and integration of virus-specific DNA in duck cells infected by avian sarcoma virus.

The DNA of avian sarcoma virus assumes a closed circular configuration before integration into the host cell chromosomal DNA. Ethidium bromide reduces the formation of superhelical viral DNA and concurrently blocks integration of the viral genome. Inhibition of integration of viral DNA results in the inhibition of virus replication.     

Baculovirus-mediated expression of a Chinese scorpion neurotoxin improves insecticidal efficacy

An Buthus martensii Karsch Insect Toxin （BmK IT ） gene was inserted into the genome of Autographa californica multicapsid nucleopolyhedrovirus （AcMNPV） to construct a recombinant baculovirus, AcMNPV-BmK IT. The expression of BmK IT was confirmed using RT-PCR, dot blot and SDS-PAGE analysis. Dose-lethal time responses to Spodoptera exigua larvae were compared between wild-type baculovirus AcMNPV and recombinant virus AcMNPV-BmK IT. At the concentration of 1 × 10^7 PIBs/mL, the median lethal time of recombinant baculovirus （LT50 = 73.6 h） on third instar S. exigua larvae showed an improvement of 13.2% over the efficacy of wild type virus （LT50 = 84.8 h） during a 192 h infection.     

自主性细小病毒非结构蛋白NS1对转化细胞的毒性及其调控DNA复制和转录的机制

自主性细小病毒具有对转化细胞专一的毒性,它的非结构蛋白ＮＳ１在其中起着重要的作用．在病毒生活周期中ＮＳ１有着多种功能,包括调控ＤＮＡ 的复制和转录．其机制包括ＮＳ１对ＤＮＡ 复制的反式抑制、对基因表达的反式抑制和对转录的反式激活     

A new mechanism for the neutralization of enveloped viruses by antiviral antibody

Despite the considerable research that has been carried out into viral neutralization by antiviral antibody, its mechanisms remain poorly understood. Cases have been reported in which antiviral antibody can inhibit viral replication without inhibiting the binding and uptake of virus by susceptible cells. It has been shown that many enveloped viruses enter their target cells by endocytosis and are subsequently located in cellular compartments of increasing acidity. With several enveloped viruses this acidic pH can catalyse a fusion reaction between the membrane of the virus particle and that of a prelysosomal endosome, thus enabling the viral core to enter the cytosol and replication to commence. We have recently demonstrated that such an endosomal fusion event at mild acidic pH is involved in the entry pathway of the enveloped flavivirus, West Nile virus (WNV), into macrophages. We now show that antiviral antibody can neutralize WNV by inhibiting this intraendosomal acid-catalysed fusion step and we speculate on possible implications for the future design of antiviral vaccines.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

A BAC clone of MDV strain GX0101 with REV-LTR integration retained its pathogenicity

The complete genome of Marek＇s disease virus （MDV） strain GX0101, which was integrated with the LTR sequences of REV, was cloned in Escherichia coli as a bacterial artificial chromosome （BAC）. BAC vector sequences were introduced into the US2 locus of the MDV genome by homologous recombination. The viral DNA containing the BAC vector was used to transform Escherichia coli strain of DH10B. Then the recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast （CEF）. This BAC viral clone was named bac-GX0101. When the reconstituted virus was inoculated into 1-day-old birds, visceral tumors could be detected as early as 62 d post infection. There was no difference in growth ability and pathogenicity to birds between the BAC derived virus and its parental virus. The BAC derived virus maintained its oncogenicity and immunosuppressive effects. In conclusion, the complete genome of GX0101 strain was successfully cloned into BAC and the infectious clone was rescued. With the powerful BAC manipulation system, the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV,     

Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen

The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting alpha-helices to expand/constrict the channel, producing an 'iris' effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.     

Myristic acid is coupled to a structural protein of polyoma virus and SV40

In the lytic cycle of papova viruses, both uncoating of the viral genome after infection and assembly of functional virions take place in the cell nucleus. The mechanisms by which newly internalized virions are targeted to the nucleus and viral DNA encapsidated into particles are poorly understood. Although the major capsid protein VP1 is involved in endocytosis, and largely defines virion structure, the functions of the minor proteins VP2 and VP3 have remained obscure. Here we show that VP2 from both polyoma virus and simian virus 40 (SV40) is covalently linked to myristic acid; this is the first report of a myristylated protein in the nucleus and of a fatty acid being important in the structure of a nonenveloped virus. We consider the implications of this unusual modification on encapsidation and suggest that VP2 may be a scaffolding protein for virion assembly.     

人肿瘤细胞株中细小病毒H—1的DNA扩增与非结构蛋白NS—1基因的表达

研究三株人癌细胞和两株对照细胞对细小病毒Ｈ－１杀伤作用敏感性的分子机制．表明了在感染复数ｍｏｉ（ｍｕｌｔｉｐｉｃｉｔｙｏｆｉｎｆｅｃｔｉｏｎ）为５ｐｆｕ（ｐｌａｑｕｅｆｏｒｍｉｎｇｕｎｉｔ）／细胞的情况下，作为Ｈ—１病毒复制受纳细胞的人肝癌细胞株ＯＧＹ－７７０３和人胃癌细胞株ＳＧＣ—７９０１，能够支持病毒ＤＮＡ扩增和非结构蛋白ＮＳ—１基因的表达，这和作为阳性对照的由ＳＶ４０转化的新生儿肾细胞株ＮＢ—Ｋ一样，但对Ｈ—１病毒感染有抗性的人肾癌细胞株ＯＵＲ—１０和它的对照人胎肾细胞株ＨｕＫ—１，并不支持病毒ＤＮＡ扩增和ＮＳ—１蛋白的表达．本文结果指出，细小病毒Ｈ—１的杀伤作用与细胞中的病毒ＤＮＡ扩增及ＮＳ—１基因表达的程度相关．     

Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex

In mammalian cells, a conserved multiprotein complex of Mre11, Rad50 and NBS1 (also known as nibrin and p95) is important for double-strand break repair, meiotic recombination and telomere maintenance. This complex forms nuclear foci and may be a sensor of double-strand breaks. In the absence of the early region E4, the double-stranded DNA genome of adenovirus is joined into concatemers too large to be packaged. We have investigated the cellular proteins involved in this concatemer formation and how they are inactivated by E4 products during a wild-type infection. Here we show that concatemerization requires functional Mre11 and NBS1, and that these proteins are found at foci adjacent to viral replication centres. Infection with wild-type virus results in both reorganization and degradation of members of the Mre11-Rad50-NBS1 complex. These activities are mediated by three viral oncoproteins that prevent concatemerization. This targeting of cellular proteins involved in genomic stability suggests a mechanism for 'hit-and-run' transformation observed for these viral oncoproteins.     

The transforming gene of Moloney murine sarcoma virus

A cleavage map of the Moloney murine sarcoma viral DNA was constructed and compared with that of a spontaneously occurring deletion mutant. By restriction enzyme analysis, it was shown that a region encompassing over 40% of the viral information was not essential for transformation or rescue of the deletion mutant. The transforming region was further localised by analysis of the transforming activity in tissue culture of isolated restriction fragments of linear duoble-stranded sarcoma viral DNA. In each case, DNA fragments that retained transforming activity preserved the cell-derived insertion sequences of the viral genome. Moreover, such transformants invariably expressed RNA specific to this region. By these two approaches, it was possible to demonstrate that the transforming region of the viral genome begins very near or within the cell-derived insertion sequences. Thus, the transforming gene of this mammalian sarcoma virus originates from within the mouse cell genome.     

Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells.

Replication of DNA occurs at discrete sites in eukaryotic cell nuclei, where replication proteins are clustered into large complexes, or 'replicases'. Similarly, viral DNA replication is a highly structured process, notably in herpes simplex virus type-1 (HSV-1; reviewed in ref. 4) in which large globular 'replication compartments' containing the viral replication machinery exist. Replicating cellular DNA redistributes to these compartments upon HSV-1 infection. We have now used antibodies raised against several cellular proteins to detect changes in their subnuclear localization on HSV-1 infection. We found that various proteins involved in cellular DNA replication move to sites of viral DNA synthesis, whereas a selection of non-replication proteins do not. The retinoblastoma protein and p53 (the products of two putative anti-oncogenes) relocate to the same sites as known DNA replication proteins, suggesting that they may be associated with DNA replication complexes in normal, uninfected cells.     

N-Acetoxy-acetylaminofluorene reacts preferentially with a control region of intracellular SV40 chromosome

Many chemical carcinogens or their metabolites react with DNA; thus it is of interest to determine what effect chromosomal structure has on these reactions. The chromosome of simian virus 40 (SV40) is well suited for such studies; like chromatin of eukaryotic cells, it is organized into nucleosomes. The nucleotide sequence of SV40 is known, together with much about the pattern of viral gene expression and DNA replication, and the structure of the viral chromosome. We have investigated the binding of the ultimate carcinogen, N-acetoxy-acetylaminofluorene (AAAF), to specific regions of the SV40 chromosome in situ in the intact infected cell. The results, reported here, indicate that a region containing regulatory functions on the intracellular SV40 chromosome has unique structural properties which render it more susceptible to attack by AAAF than the rest of the SV40 genome. The preferential binding of AAAF to regulatory regions of chromatin may have implications for the mechanism of action of this and similar carcinogens.     

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.