Search

A replicon of phage JHJ-1 (and JHJ-3) was cloned. The autonomously replicating phage element was maintained as a medium-copy-number shuttle plasmid in many actinomycetes, and was efficiently transmitted to spores without antibiotic selection. One gene was shown to be expressed in a vector containing the JHJ-3 replicon.

Using the broad replicating range JHJ-3 phage replicon, a shuttle vector for Escherichia coli and actinomycetes has been constructed. The vector, pOJ31, bears the lacZ alpha fragment allowing a blue/white gene cloning system. pOJ31 also contains a polylinker of 15 unique cloning sites and the phage T7 promoter. The vector has been used to stably express the mel gene from plasmid pIJ702 in Streptomyces lividans.

Phages JHJ-2 and JHJ-3 were isolated from Saccharopolyspora hirsuta 367 UC 8106 following induction with mitomycin C and amplified on S. hirsuta NRRL B-5792. Their properties were compared with those of phage JHJ-1, isolated previously from S. hirsuta 367 NRRL 12045. The DNA restriction patterns appeared to be identical. One-step growth experiments showed no differences between the replication cycles. Burst sizes ranged from 100 to 110 p.f.u. per cell. However, the three phages showed some differences in their behaviour in different hosts. The host range of phage JHJ-1, on non-lysogenic strains, was emended to include all of the Saccharopolyspora strains tested; the host range of phage JHJ-2 was shown to be identical to JHJ-1. Phage JHJ-3 did not form detectable plaques on strains of S. rectivirgula or S. erythraea except S. erythraea NRRL 2359. Neither phage JHJ-2 nor JHJ-3 formed plaques on any lysogenic strains, while JHJ-1 formed plaques on all such strains except S. hirsuta 367 UC8106. Phage JHJ-3 was characterized as a temperate bacteriophage because it formed turbid, self-limiting plaques and lysogenized S. hirsuta NRRL B-5792. It was spontaneously released from UC8106. Both JHJ-1 and JHJ-2 formed clear and invasive (Inv+ phenotype: the property to grow on old mycelium) plaques on some Saccharopolyspora strains but clear and self-limiting plaques on others. Thus, the expression of the Inv+ phenotype encoded by JHJ-1 and JHJ-2 appears to be modulated by the host cell.

Bacteriophage JHJ-1 was isolated from Saccharopolyspora hirsuta strain 367 NRRL 12045 as an endogenous but virulent phage. The plaque size was not self-limiting, since a few p.f.u. could completely lyse a lawn. Electron microscopy showed that this phage belonged to group B of Bradley's morphological classification. The JHJ-1 genome is a linear DNA molecule of 41.1 kbp with cohesive ends and a G + C content of 68.8-70.0 mol%. The DNA cleavage map was established for 12 restriction endonucleases. The host range is apparently very narrow, being limited to two strains of S. hirsuta (NRRL 12045 and NRRL B-5792). However, JHJ-1 did not lytically infect S. hirsuta strain 367 UC 8106. Phage JHJ-1 was shown, by Southern blot analysis, to lysogenize both S. hirsuta NRRL 12045 and UC 8106. It thus appears to behave as a virulent mutant of a temperate phage on one, but not on the other, JHJ-1 lysogen.

Two in vitro proteinase assay systems were developed and used to study the peptide bond specificity and substrate specificity of the adenovirus endoproteinase. Five adenovirus precursor proteins (PVI, PVII, PVIII, 87K, 11K), all found in the virion of the ts1 mutant grown at the nonpermissive temperature, were digested by the proteinase. All, except 11K, were cleaved to their mature counterparts. Some of the proteins, particularly the 87K terminal protein, were processed via cleavage intermediates similar to those found in vivo. The data suggest that the proteinase specifically hydrolyses Gly-Ala bonds. The high specificity for the natural substrates and the failure to cleave foreign proteins suggest that cleavage activity is determined not only by primary sequence but also by other physical features of the substrate. Enzyme activity was inhibited by diisopropylfluorophosphate, showing that it is a serine proteinase.

The structure of adenovirus chromatin in infected cells was studied by micrococcal nuclease digestion and hybridization with virus-specific probes. In the early phase of infection (5 h) a significant proportion of viral molecules was organized like actively transcribed cellular chromatin. As expected for a transcriptionally active population of molecules, even at high multiplicity of infection the nucleosomal repeating pattern was less distinct than in a transformed cell which contained the corresponding but less active genomic region. The observed repeating pattern in infected cells was unlikely to be due to integrated molecules since less than 0.07% of input genomes became associated with cellular DNA. After the onset of viral DNA replication, the pool of viral chromatin organized like cellular chromatin rapidly increased. In addition, newly replicated molecules also maintained the cellular chromatin-like organization as measured by [3H]thymidine incorporation after the cessation of cellular DNA synthesis. These data suggest that newly replicated viral molecules are organized by histones into cell-like chromatin throughout the infection cycle. Coincident with the peak of viral DNA and core protein synthesis, and the decline of histone synthesis, the late, core-like non-repeating viral chromatin became dominant, increasingly obscuring the underlying repeating pattern. Experiments suggest that this late chromatin is destined for encapsidation, that the early chromatin persists and that viral core proteins do not displace histones on viral DNA. A model is proposed suggesting that transcription and type I replication occur on histone-condensed templates, while type II replication products late in infection are condensed by core proteins and are destined for encapsidation.

We have investigated the role of poly(ADP)-ribosylation of adenoviral proteins in virus infection. Viral core proteins V and the precursor to protein VII were shown to be in vivo and in vitro acceptors of ADP-ribose. In vivo ADP-ribosylation was restricted to viral proteins as the histones were not labeled during the late phase of infection. The ADP-ribosylated core proteins were assembled into mature virus particles. In vitro ADP-ribosylation of adenoviral core proteins performed with purified poly(ADP-ribose) polymerase led to relaxation of the chromatin structure of both ts1 and wild type pyridine cores and pentonless particles and triggered the complete dissociation of wild type particles. A critical role for poly(ADP)-ribosylation in virus infection was confirmed by measuring the effect of the inhibitors 3-aminobenzamide and nicotinamide on virus particle yield and infectivity. Both inhibitors depressed particle yield by up to 9-fold, but infectivity was reduced by up to 10(4)-fold. These results suggest that ADP-ribosylation of adenovirus core proteins may have a role in virus decapsidation.

The nuclear matrix has been implicated in several important cellular processes. In this paper, we investigate the role of the nuclear matrix in adenovirus type 2 assembly. Electron microscopic examination of nuclear matrices isolated from adenovirus infected Hep-2 cells clearly reveals that late in the lytic cycle, adenovirus capsids are intimately associated with the nuclear matrix. SDS-PAGE analysis showed that the viral core polypeptides V, PVII and 11 kDa were enriched in the nuclear matrix fraction. After a 3 h chase period a constant high ratio of PVII to VII prevailed in the nuclear matrix suggesting that mostly young virions and viral cores are bound to this structure. Most of the virus maturation endoproteinase activity co-purified with the nuclear matrix and the data suggest that the enzyme may be released from fragile young virions or assembly intermediates. Together these experiments suggest that the nuclear matrix is the site of adenovirus assembly and that mature virions may be released from the matrix by the viral endoproteinase.

We compared some of the biological and structural features of an adenovirus type 2 temperature-sensitive mutant (ts1) defective for maturation cleavages and uncoating with wild-type (WT) virus. The cleavage defect caused ts1 to produce virions at 39 degrees that contained five precursor proteins (pTP, 11K, PVI, PVII, PVIII). Coinfection of cells with such ts1 virions and a variety of mutants or WT virus not only failed to complement ts1 but actually depressed the infection by the second virus. The uncoating defect could only be overcome by multiplicity-dependent leakiness. The structure of the ts1 virion was compared with that of WT virus by iodination with chloramine-T, chloroglycoluril and lactoperoxidase, by cross-linking, and by digestion with proteases. Aside from the presence of precursor proteins and the greater stability of ts1 virions, no other differences were found that could account for the uncoating defect. Therefore, we postulate that this defect was caused by the greater stability imparted to the virion by precursor proteins PVI, PVII and PVIII.

SE-3 is a virulent bacteriophage isolated from a large-scale culture of Saccharopolyspora erythraea, an erythromycin producer. The host range of the phage is narrow, limited to some strains of this species. Another strain of Sac. erythraea, and a strain of Sac. hirsuta, are able to adsorb phage particles but do not sustain their complete multiplication. SE-3 is closely related to the phage SE-5 as shown by DNA restriction mapping. The differences between SE-3 and SE-5 genomes are apparently limited to two DNA segments flanked by short inverted repeats, visualized by electron microscopy.

The adenovirus major late promoter (MLP) is normally not active in transformed cells. We investigated if it could be activated with 5-azacytidine. Three days of treatment with 10 microM 5-azacytidine induced transient activation of the MLP as shown by hybridization with an L1 r-strand-specific probe. The po/III-transcribed VA-RNAs were not activated. L1 activation was not accompanied by detectable changes in methylation of HpaII sites at the promoter or in the body of the transcript. Stably activated cell clones could be obtained at 20% frequency after long-term drug treatment.

The relationship between viral DNA synthesis and virion assembly was studied in adenovirus type 2 infected HEp2 cells. When cells were infected at the restrictive temperature with ts3, an assembly-negative mutant which permits normal viral DNA and protein synthesis, labeled and shifted to the permissive temperature, only de novo synthesized nonradioactive viral DNA was encapsidated. This suggested that only concurrently synthesized DNA is encapsidated. Blocking protein or DNA synthesis with cycloheximide or hydroxyurea after the temperature shift inhibited virus assembly. Therefore efficient virus assembly requires both concurrent protein and DNA synthesis. When DNA synthesis was arrested by shifting ts125 infected cells to the restrictive temperature, protein synthesis continued but assembly was completely blocked. Sucrose gradient sedimentation analysis of nuclear extracts of wt and ts3 infected cells provided evidence in support of a physical coupling between replication complexes and virus assembly complexes. Further evidence of coupling was also shown by preferential pulse labeling of the molecular right end of the genome isolated from reversibly cross-linked assembly intermediate particles. While DNA replication is not dependent on concurrent virion assembly, at least some significant proportion of replication complexes appear to be coupled to and are prerequisite for virion assembly.