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1.
Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes. 相似文献
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The entry of the viral genomic DNA of cauliflower mosaic virus into the nucleus is a critical step of viral infection. We have shown by transient expression in plant protoplasts that the viral coat protein (CP), which is processed from the product of open reading frame IV, contains an N-terminal nuclear localization signal (NLS). The NLS is exposed on the surface of the virion and is thus available for interaction with a putative NLS receptor. Phosphorylation of the matured CP did not influence the nuclear localization of the protein but improved protein stability. Mutation of the NLS completely abolished viral infectivity, thus indicating its importance in the virus life cycle. The NLS seems to be regulated by the N terminus of the precapsid, which inhibits its nuclear targeting. This regulation could be important in allowing virus assembly in the cytoplasm. 相似文献
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The ubiquitous virus JCV is the etiologic agent of the human brain disease progressive multifocal leukoencephalopathy. Although infection usually occurs early in life and the virus can remain latent in human tissues, including brain, little information is available regarding its replication. It is known that DNA replication of primate polyomaviruses is dependent upon the synthesis of T antigen and the subsequent interactions of this protein with cellular factors and the viral origin of replication. We constructed chimeric genomes between JCV and SV40, two genetically similar viruses with distinct biologies, in which segments of the T antigen coding region and the replication origin were exchanged. Because the engineering of these genomes created a defect in the structural protein VP1, their DNA replicating activities could be compared without the complication of secondary infection of adjacent cells and amplification of the replication signal. The ability of the JCV-SV40 hybrid T antigens to initiate replication from the two viral origins in primate cells was investigated. A region of the JCV T antigen that includes the DNA binding and zinc finger domains was found to be responsible for the failure of JCV T antigen to interact productively with the SV40 origin. In addition, the ability to replicate in monkey cells was limited to constructs expressing T antigens which contained the carboxy-terminal host range domain of SV40. 相似文献
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The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex. 相似文献
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Simian virus 40 (SV40), a polyomavirus of rhesus macaque origin, was discovered in 1960 as a contaminant of polio vaccines that were distributed to millions of people from 1955 through early 1963. SV40 is a potent DNA tumor virus that induces tumors in rodents and transforms many types of cells in culture, including those of human origin. This virus has been a favored laboratory model for mechanistic studies of molecular processes in eukaryotic cells and of cellular transformation. The viral replication protein, named large T antigen (T-ag), is also the viral oncoprotein. There is a single serotype of SV40, but multiple strains of virus exist that are distinguishable by nucleotide differences in the regulatory region of the viral genome and in the part of the T-ag gene that encodes the protein's carboxyl terminus. Natural infections in monkeys by SV40 are usually benign but may become pathogenic in immunocompromised animals, and multiple tissues can be infected. SV40 can replicate in certain types of simian and human cells. SV40-neutralizing antibodies have been detected in individuals not exposed to contaminated polio vaccines. SV40 DNA has been identified in some normal human tissues, and there are accumulating reports of detection of SV40 DNA and/or T-ag in a variety of human tumors. This review presents aspects of replication and cell transformation by SV40 and considers their implications for human infections and disease pathogenesis by the virus. Critical assessment of virologic and epidemiologic data suggests a probable causative role for SV40 in certain human cancers, but additional studies are necessary to prove etiology. 相似文献
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Production of an infectious herpes simplex virus (HSV) particle requires sequential progression of maturing virions through a series of complex assembly events. Capsids must be constructed in the nucleus, packaged with the viral genome, and transported to the nuclear periphery. They then bud into the nuclear membrane to acquire an envelope, traffic through the cytoplasm, and are released from the cell. Most of these phenomena are very poorly defined, and no suitable model system has previously been available to facilitate molecular analyses of genomic DNA packaging, capsid envelopment, and intracellular virion trafficking. We report the development of such an assay system for HSV type 1 (HSV-1). Using a reversible temperature-sensitive mutation in capsid assembly, we have developed conditions in which an accumulated population of immature capsids can be rapidly, efficiently, and synchronously chased to maturity. By assaying synchronized scaffold cleavage, DNA packaging, and acquisition of infectivity, we have demonstrated the kinetics with which these events occur. Kinetic and morphological features of intranuclear and extranuclear virion trafficking have similarly been examined by indirect immunofluorescence microscopy and electron microscopy. This system should prove a generally useful tool for the molecular dissection of many late events in HSV-1 biogenesis. 相似文献
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Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39- and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofold-higher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA. 相似文献
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The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen. 相似文献
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Purified full and empty virions of minute virus of mice were separated on CsCl gradients, and their polypeptides were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The empty particle contains two polypeptides, A (83,300 daltons) and B (64,300 daltons), which are 15 to 18% and 82 to 85%, respectively, of the virion mass. The full particle contains the single-stranded DNA genome, proteins A and B, and a third polypeptide, C (61,400 daltons). Again A is 15 to 18% of the protein mass, but the amounts of B and C vary inversely in different preparations of full particles. These polypeptides comprise greater than 99.6% of the protein in either virion, and their molecular weights and molar ratios are independent of the species of host cell on which the virus is propagated, They are not found in uninfected cells, and no protein component of uninfected cells copurifies with either virion under our conditions. Pulse-chase experiments show that the three proteins are synthesized only after virus infection and are therefore probably virus coded. Sequential harvesting from the nuclei of cells infected under one cycle growth conditions shows an increase in the proportion of C in full particles as infection progresses, suggesting that C is derived from B in a late maturation step. 相似文献
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Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events. 相似文献
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The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target beta-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTPgammaS. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import. 相似文献
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WS Weichert JS Parker AT Wahid SF Chang E Meier CR Parrish 《Canadian Metallurgical Quarterly》1998,250(1):106-117
The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection. 相似文献
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Epstein-Barr viral nuclear antigen-1 (EBNA-1) is required for the stable replication of plasmids that contain oriP, the origin of DNA synthesis used during the latent phase of the Epstein-Barr virus life cycle. EBNA-1 acts post-synthetically through unknown mechanisms to facilitate the continued synthesis of oriP plasmids in ensuing S phases. In contrast to viral replicons such as that of SV40, DNA synthesis of oriP is restricted to a single round during each cell cycle. Large T-antigen of SV40 is a DNA helicase and activates the synthesis of SV40 DNA by recruiting cellular proteins required for DNA synthesis to the origin of SV40. Using fusion proteins of EBNA-1 and large T-antigen, we tested whether tethering large T-antigen to oriP is sufficient to initiate multiple rounds of DNA synthesis from oriP during each cell cycle. We report here that, although these fusion proteins retain the biological activities of both EBNA-1 and large T-antigen, their constituent proteins do not confer the properties of one on the other. Thus, it is not possible to subvert the cellular controls that restrict DNA synthesis from oriP to a single round per cell cycle. These results also provide insights into architectural constraints at oriP and at the SV40 ori. 相似文献
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HIV-1 gag p17 protein is an attractive target for molecular intervention, because it is involved in the viral replication cycle at both the pre- and postintegration levels. In the present experiments, we targeted p17 by intracellularly expressing a cDNA encoding an Ab to p17. cDNA from a hybridoma-secreting Ab to p17 was cloned, sequenced, reconstructed as a single-chain Ab fragment (scFv), and expressed in the cytoplasm or nucleus with appropriate retention signals. The expressed scFvs had no effect on T cell growth or CD4 expression and bound specifically to HIV-1 p17. Human CD4+ Jurkat T cells that expressed scFvs and were infected with HIV-1 showed a marked reduction in virus replication compared with cells expressing vector alone. The inhibition of virus replication was more pronounced when scFvs were expressed in the cytoplasm rather than the nucleus. From these studies, we conclude that the intracellular expression of a single-chain Ab to p17 inhibits HIV replication; in addition, the degree of inhibition is related to the intracellular targeting site. 相似文献
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Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis. 相似文献
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The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts. 相似文献