A DNA polymerase activity is associated with Cauliflower Mosaic Virus

A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa.     

Nuclear Targeting of the Cauliflower Mosaic Virus Coat Protein

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.     

Virus Factories of Cauliflower Mosaic Virus Are Virion Reservoirs That Engage Actively in Vector Transmission

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.     

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) μA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.Membrane trafficking is essential in eukaryotic cells. Cellular membranes serve as a delivery system for newly synthesized proteins such as transporters and receptors exiting the endoplasmic reticulum after proper folding. They then transit through the Golgi complex, reaching the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. Receptors and transporters returning from the PM are either recycled or targeted to the vacuole for degradation. Delivery and recycling sorting pathways overlap in the trans-Golgi network (TGN)/early endosome (EE), an intermediate compartment for both exocytosis and endocytosis (Reyes et al., 2011). In plant systems, the endoplasmic reticulum and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD), cytoplasmic channels that regulate traffic in the symplasm (Maule et al., 2011).The selective transport of macromolecules between different compartments of the endomembrane system is mediated by coat proteins promoting the generation of small cargo-trafficking coated vesicles (Spang, 2008). The recognition and recruitment of cargo proteins are mediated by so-called adaptor complexes (AP complexes [AP-1–AP-4]; Robinson, 2004) one of which, AP-1, is localized on the TGN/EE and endosomes, whereas AP-2 is in the PM. The μ-subunit of AP complexes is devoted to cargo protein selection via a specific and well-characterized interaction with a Tyr-sorting signal, YXXΦ, where Φ is a bulky hydrophobic residue and X is any amino acid (Bonifacino and Dell’Angelica, 1999). YXXΦ motifs are present in the cytoplasmic tail of many proteins integral to the PM and TGN/EE and have been found in the movement proteins (MPs) of some viruses (Laporte et al., 2003; Haupt et al., 2005). Plant viruses are obligate parasites that exploit host components to move within the cell and from cell to cell into the vascular system for systemic invasion of the host. Virus movement, which requires the passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes (Harries et al., 2010). While most MPs act to increase the size exclusion limit of PD to facilitate the passage of the viral nucleoprotein complex, other MPs are assembled in tubules that pass inside highly modified PD and transport encapsidated particles through their lumen.Here, we focus on this second group of tubule-forming MPs and examine the intracellular trafficking of cauliflower mosaic virus (CaMV) MP. The MP encoded by CaMV forms tubules guiding encapsidated virus particle cell-to-cell transport via an indirect MP-virion interaction (Stavolone et al., 2005; Sánchez-Navarro et al., 2010). However, how CaMV MP (and the other tubule-forming MPs) targets the PM and forms tubules remains to be elucidated. Tubule-forming MPs do not require an intact cytoskeleton for PM targeting (Huang et al., 2000; Pouwels et al., 2002) and/or tubule formation (Laporte et al., 2003). However, suppression of tubule formation upon treatment with brefeldin A (BFA), a specific inhibitor of secretion or endocytosis, suggests the involvement of the endomembrane system in correct functioning of some tubule-forming MPs (Huang et al., 2000; Laporte et al., 2003). In this study, we examined the three Tyr-sorting motifs in CaMV MP and show that each of the three domains interacts directly with subunit μ of an Arabidopsis (Arabidopsis thaliana) AP complex. Mutations in these domains revert in the viral context to maintain CaMV viability. MP is found in endosomal compartments labeled by AtRAB-F2b (ARA7) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). The presence of at least one functional YXXΦ domain is essential for the localization of MP to endosomes and for tubule assembly but is not required for MP targeting to the PM. We provide several lines of evidence to show CaMV MP trafficking in the endocytic pathway. Our findings are discussed in the light of the recent demonstration that the TGN/EE functions as a major hub controlling secretory and endocytic pathways in plants.     

Origins and complexes: the initiation of DNA replication

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.     

Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells.

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.     

玉米矮花叶病毒提纯及特性的研究

改进病毒纯化方法,获得了较高纯度、较强侵染活性的提纯病毒制品。提纯病毒的产量为0.7—1.2mg/100g病叶,病毒粒体大小为720—750×14nm,SDS-聚丙烯酰胺凝胶电泳测得的外壳蛋白分子量为36000道尔顿,用提纯病毒免疫家兔得到较高特异性的抗血清,微量沉淀反应的效价为1/2048,间接ELISA法测出的感病叶汁液的最高稀释倍数为3200—6400倍。     

The c-myc DNA-unwinding element-binding protein modulates the assembly of DNA replication complexes in vitro

The presence of DNA-unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both. We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE-B, with a predicted mass of 23.4 kDa. Based on homology to yeast proteins, DUE-B was previously classified as an aminoacyl-tRNA synthetase; however, the human protein is approximately 60 amino acids longer than its orthologs in yeast and worms and is primarily nuclear. In vivo, chromatin-bound DUE-B localized to the c-myc DUE region. DUE-B levels were constant during the cell cycle, although the protein was preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G1 to S phase and induced cell death. DUE-B extracted from HeLa cells or expressed from baculovirus migrated as a dimer during gel filtration and co-purified with ATPase activity. In contrast to endogenous DUE-B, baculovirus-expressed DUE-B efficiently formed high molecular mass complexes in Xenopus egg and HeLa extracts. In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.     

DNA Polymerase Activities in Healthy and Cauliflower Mosaic Virus-infected Turnip (Brassica rapa) Plants

Turnip plants contain three types of DNA polymerase: DNA polymerase-     

DNA replication intermediates synthesized by lysates of dnaB, dnaG and dnaB dnaG mutants in vitro

Summary Isogenic dnaB, dnaG, and dnaB dnaG mutants were constructed and used as extracts in the cellophane-disc in vitro DNA replication system. The increased proportion of 5S DNA characteristic of the dnaB extract and the lack of Okazaki piece synthesis characteristic of the dnaG extract were both apparent in analysis of the dnaB dnaG mutant extract reaction. A hypothetical scheme to explain these results and those of others is presented.     

Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro.

Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP.     

Prereplicative complexes assembled in vitro support origin‐dependent and independent DNA replication

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2‐7 helicase is first loaded into prereplicative complexes (pre‐RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre‐RCs assembled with purified proteins support complete and semi‐conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK). DDK phosphorylation of Mcm2‐7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin‐dependent in this system. These experiments indicate that Mcm2‐7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.     

Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA.

The Ku protein is composed of two polypeptide subunits, p70 and p80, and binds DNA ends in vitro. Previous studies suggested that p70 and p80 are physically associated in vivo, although such an association may have been mediated by DNA. We have now utilized full-length Ku polypeptides synthesized in vitro to examine the association of p70, p80, and linear DNA to form a complex. In gel filtration chromatography, p70 migrates as a 70-kDa structure, whereas p80 migrates at 150 kDa. Co-translation of the two cDNAs yields complexes which migrate at 300 kDa and contain equimolar quantities of the p70 and p80 polypeptides, providing direct evidence that p70 and p80 assemble into a complex in the absence of DNA. To demonstrate that this recombinant protein complex binds DNA, we developed a radiolabeled protein electrophoretic mobility shift assay. When radiolabeled proteins synthesized in vitro were incubated with linear DNA and fractionated in a nonreducing, nondenaturing gel, a band representing a complex of p70, p80, and the DNA was seen. Formation of this Ku-DNA complex required free DNA ends, and binding to DNA ends was not observed with individual p70 or p80 subunits. DNA binding was not reconstituted by mixing the individual subunits together. These studies thus demonstrate that it is the complex of p70 and p80, not individual p70 or p80, which possesses the DNA binding properties previously described for native Ku protein. These results provide new information about the assembly, structure, and DNA binding properties of the Ku protein.     

DNA replication in Physarum polycephalum: characterization of replication products in vivo.

Synchronous plasmodia of Physarum polycephalum in DNA synthesis were pulse-labelled with [oH]- thymidine for time periods of 15 seconds up to 9 minutes, or given a 30 seconds pulse followed by chase periods of 9 minutes up to 6 hours. Sedimentation analysis in alkaline sucrose gradients revealed at least five species of single stranded DNA14 molecules in the pulse experiments. Co-sedimentation of [14C]-labelled phage-DNA gave relative S-values of 5-7, 13-15, 23-25, 30 and 33-35 for these DNA molecules, all of which can be chased into DNA of higher molecular weight.     

Definition and polarity of action of DNA replication terminators in Bacillus subtilis.

The first stage in termination of chromosome replication in Bacillus subtilis involves arrest of the clockwise fork at the inverted repeat region (IRR), comprising the opposed IRI and IRII sequences, adjacent to the upstream region of the rtp gene, which encodes the replication terminator protein RTP. RTP binds to IRI and IRII. The ability of the IRR and its components to function as terminators, in conjunction with RTP, and their polarity of action have now been tested by the use of plasmids replicating in B. subtilis as unidirectional theta structures and into which potential terminator sequences were inserted in alternate orientations relative to fork movement. When the complete IRR was inserted into such plasmids and the new plasmids transferred into a B. subtilis strain overproducing RTP, it was able to block movement of a replication fork approaching from either direction. IRI and IRII were shown to function as polar terminators, each blocking movement of a fork when it approached from one particular direction but not the other. Furthermore, the polarity of action was in accordance with the IRR being able to operate as a replication fork trap. Thus, a fork approaching the IRR would pass through the first terminator encountered (IRI or IRII) and be halted by the second. The previously observed nonfunctioning of a particular orientation of chromosomal IRR as a fork arrest site probably reflects a limiting level of RTP in the cell. Interestingly, a 21 base-pair core sequence spanning a single RTP binding site within IRI (the 47 base-pair IRI contains 2 binding sites) was unable to arrest a fork approaching from either direction in the plasmid system. This suggests that both binding sites within an IR must be filled in order to function as an arrest site. It is possible that co-operative interaction between adjacent dimers within IRI or IRII provides the necessary conformation for causing fork arrest.     

Comparison of in vivo and in vitro translation of Cowpea Mosaic Virus RNAs

The translation products from Cowpea Mosaic Virus (CPMV) RNAs obtained in two different cell-free systems were compared with the viral polypeptides synthesized in CPMV-infected cowpea protoplasts. It was shown that in both the wheat germ system and the rabbit reticulocyte lysate CPMV M component RNA was translated into two polypeptides of 105,000 and 95,000 dalton, which were not detected in CPMV-infected protoplasts. B component RNA however, gave different products depending on the system used. In the reticulocyte system this RNA was translated into a 200,000 dalton polypeptide which was further cleaved to give 170,000 and 32,000 dalton polypeptides. In the wheat germ system this processing step was lacking as only the 200,000 dalton product was formed. Since the 170,000 and 32,000 dalton polypeptides were also found in CPMV-infected protoplasts the two in vitro systems used apparently represent different stages of the expression of the B component RNA, thus providing a tool to study the mechanism of CPMV gene expression in vivo.