Unit-length Molecules Research Articles

The cleavage recognition signal is contained within sequences surrounding an a- a junction in herpes simplex virus DNA

Herpesvirus genome maturation involves site-specific cleavage of viral DNA concatemers and encapsidation of unit-length molecules, processes that are apparently coupled. Here, applying a transfection-infection approach, we have investigated the arrangement of the DNA sequence elements involved in cleavage and shown that specific cleavage occurs independently of DNA replication. We show that the cis-acting signal for cleavage is located within a 179-bp fragment from across an a- a junction formed as part of the genome maturation process of herpes simplex virus 1. Plasmids carrying the 179-bp fragment are cleaved at the appropriate site even though they are unable to replicate in HSV-infected cells. When linked to an origin, the same 179-bp a- a fragment will replicate and package into progeny virus as a defective genome. Two highly conserved homologies, pac1 and pac2, that have been observed in all herpesviruses examined, including cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, and herpes simplex virus 2 as well as the herpes simplex virus 1 genome, are contained within the 179-bp fragment. This suggests that a common mechanism is utilized for genome maturation in the herpesvirus group.

Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1.

The linear double-stranded DNA genome of herpesvirus as it is present in infectious virions needs to be circularized after infection of host cells and before DNA replication. Replicative-form genomes have to be cleaved into linear unit-length molecules during virion maturation and are most probably the substrate for inversion of the short segment relative to the long segment of the bovine herpesvirus 1 (BHV-1) genome. Those regions of the BHV-1 genome which are functionally involved in these processes have been analyzed at the molecular level by cloning and sequencing the genomic termini, the fusion of both termini from replicative-form molecules, and the junction between the short and the long genome segment. On the basis of the simple genome arrangement of BHV-1, it was inferable that the cleavage of replicative-form genomes by a hypothetical BHV-1 terminase activity may be specified by a sequence at the left end of UL (An element), which is located proximal to a reiterated beta element that makes up the cleavage site itself. The relationship of those elements in BHV-1 and the comparison to similar regions of other herpesviruses indicate consensus sequence elements which are functionally important for cleavage and isomerization of viral DNA during maturation of virions.

DNA processing in temperature-sensitive morphogenic mutants of HSV-1

The anatomy of DNA synthesized by five HSV-1 mutants previously shown to accumulate predominantly empty capsids at the nonpermissive temperature (NPT) was analyzed with Bg/ll restriction digestion. At the NPT, all five generated DNA lacking termini, indicating that in the absence of packaging, viral DNA is not processed to unit length. One mutant, F18, was able to process DNA made at the NPT to unit length molecules during a 6-hr period after shift to the permissive temperature. The appearance of unit length molecules correlated with the appearance of staphylococcal nuclease-resistant F18 DNA.

Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence.

The sequence necessary for the infectivity of hop stunt viroid cDNA clones

Recombinant plasmids carrying more than two units of the hop stunt viroid (HSV) cDNA sequence linked in tandem were highly infectious. On the other hand, those carrying one unit were not. To determine the length of the HSV cDNA sequence necessary for the infectivity, we constructed series of plasmids carrying more than one but less than two units of the HSV sequence and investigated their infectivities. The results showed that cDNA clones were infectious if about 60 nucleotides of the internal part of the HSV sequence (region A) were undeleted and remained with the same arrangement as in the infectious two-unit clone. The region A can be folded into a stable secondary structure. Viroid replication is thought to involve a site-specific endonucleolytic cleavage of plus strand RNA multimers produced by a rolling circle mechanism to yield unit length molecules. Our results suggest that the region A contains the sequence or structure recognized by the putative endonuclease.

Analysis of replicating vaccinia DNA in interferon-treated, virus-infected cells.

The effect of interferon (IFN) on the replication of vaccinia DNA has been examined. Studies were carried out in infected mouse L cells grown in monolayer cultures. We examined discontinuous synthesis of small DNA fragments, ligation of the small fragments to form intermediate-sized DNA molecules, completion of unit-length DNA molecules, and introduction of crosslinks at each end of the newly replicated DNA molecules late in infection. We measured by pulse-label and pulse-chase experiments the extent of incorporation of 3H-thymidine into DNA and the conversion of small size DNA into mature and cross-linked viral DNA molecules. Interferon inhibited the initiation of viral DNA and this correlated with an overall inhibition of protein synthesis. Interferon inhibited elongation of viral DNA but this did not correlate with an inhibition of protein synthesis. The effect on elongation was not the result of increased degradation of newly synthesized DNA. It is suggested that the effects of IFN on initiation and elongation of vaccinia DNA may be the result of a decrease in the availability of enzymes or factors involved in the regulation of viral DNA synthesis.

"Endless" viral DNA in cells infected with channel catfish virus.

The state of intracellular viral DNA in cells infected with channel catfish virus has been studied by the Hirt selective extraction procedure and by restriction endonuclease digestion. The sedimentation properties and restriction patterns of viral DNA in the Hirt supernatant fraction indicate that the majority, if not all, of the DNA is in the form of linear unit-length (Mr approximately equal to 85 x 10(6)) molecules. However, restriction digests of viral DNA in the pellet fraction lacked two fragments corresponding to the molecular ends of unit-length DNA. In addition, there appeared in HpaI digests of pellet DNA a new restriction fragment interpretable as the product of fusion between the ends of unit-length molecules. The size of the new fragment requires that fusion occur in such a way that one copy of the terminally repeated sequences (Mr approximately equal to 12.3 x 10(6)) of the unit-length DNA is lost in the process. In pulse-chase experiments, radioactivity flowed from the pellet fraction to the supernatant fraction, suggesting a precursor-product relationship for these DNA species. The results are easily understood if unit-length virion DNA is generated by excision from concatemeric structures.

Aclacinomycin A-inhibition of phage .PHI.X174 DNA synthesis in vitro.

Aclacinomycin A inhibited the in vitro conversion of phage phi X174 single-stranded DNA to the replicative form DNA. DNA synthesis was inhibited by 50% in the presence of 15 microM aclacinomycin A. The inhibition was competitive with respect to template DNA (Ki = 13 microM) and was reversed by addition of Escherichia coli cell extracts. Short complementary strands approximately one-third of unit length molecule were synthesized in the presence of 15 microM aclacinomycin A. The data suggest that aclacinomycin A may inhibit the process of phi X174 DNA chain elongation by a direct interaction with the E. coli host enzymes.

Uncoupling of initiation site cleavage from subsequent headful cleavages in bacteriophage T1 DNA packaging.

The packaging of intracellular DNA into heads is a key feature in the morphogenesis of bacteriophage particles. In many phages a performed empty head precursor, the prohead, is filled with DNA from a concatemeric substrate consisting of tandemly repeated genome lengths. The addition of outer shell proteins completes head formation. The DNA molecules released from particles of the coliphage T1 exist as three major permutations of nucleotide sequence. Such limited permutation can be explained by the modification of Streisinger's 'headful' mechanism proposed for phage P22. DNA packaging is initiated at a specific site (the pac site) on the concatemeric precursor. While this site is cleaved, subsequent cleavages (headful cleavages) are dependent only on head-filling and are not defined in terms of nucleotide sequence. Headfuls of DNA, consisting of slightly more than a genome length, are packaged in three successive cycles of head-filling to produce the permuted and terminally redundant molecules characteristic of T1 DNA. To elucidate the regulation of this process, we have studied the DNA metabolism of T1 head mutants. We describe here the properties of a mutant in gene 13.3 which is defective for headful cleavage but remains proficient in pac site cleavage. The observation in this mutant that concatemers are degraded to unit-length molecules by repeated pac site cleavage suggests a model of headful packaging in which pac site initiation and processive head-filling compete for the DNA substrate.

Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA

The herpes simplex virus genome consists of two components, L and S, which invert relative to each other during viral replication. The a sequence is present at the genomic termini in direct orientation and at the L-S junction in inverted orientation. Previously, we showed that insertion of a fragment spanning the L-S junction into the viral genome causes additional inversions. In this study, we determine the nucleotide sequence of the genomic termini and show that insertion of either the free S terminus or the L terminus causes inversions in the viral genome. We conclude that the a sequence is the inversion-specific sequence, that linear unit-length molecules packaged in virions are generated by cleavage between adjacent copies of the a sequence, that cleavage produces 3′ single-base extensions on the genomic termini and that the signal for cleavage is contained within the a sequence.

The mechanism of cytoplasmic orthopoxvirus DNA replication

Orthopoxvirus DNA replication occurs in the cytoplasm of infected cells within discrete foci designated as virosomes. We show that newly synthesized rabbit poxvirus (RPV) virosomal DNA consists predominantly of concatamers wherein unit length molecules are joined by fusion of two left (LL) or right (RR) ends, resulting in genomes aligned in alternating head-to-head and tail-to-tail mirror image arrays. These concatameric molecules serve as the substrates from which unit length DNA molecules are excised during morphogenesis. We propose a mechanism by which internal deletions within these concatameric arrays prior to genome excision and packaging could create inverted terminal repeats and generate gene duplications.

Restriction and modification of bacteriophage SP10 DNA by Bacillus subtilis Marburg 168: stabilization of SP10 DNA in restricting hosts preinfected with a heterologous phage, SP18.

SP10 phage cannot propagate in Bacillus subtilis Marburg 168 containing the wild-type allele of either gene nonA or gene nonB. The latter gene codes for the intrinsic cellular restriction activity. SP10 DNA was degraded in nonB+ derivatives of Marburg 168. The degree of degradation depended upon the previous host in which SP10 was propagated. In the case of SP10 grown in B. subtilis W23 (a nonrestricting, nonmodifying bacterium), 90% of the phage DNA was hydrolyzed to acid solubles, and the residual acid-precipitable material was recovered as 0.5- to 1-megadalton fragments. In contrast, if SP10 was propagated in B. subtilis PS9W7 (a nonA nonB derivative of Marburg 168 that retains modifying activity), 40 to 50% of the input DNA was degraded to acid solubles, and most of the remainder was recovered as 15- to 20-megadalton fragments. In nonA+ nonB cells, SP10 DNA was conserved as unit-length molecules (ca. 80 megadalton). Prior infection of nonB+ cells with SP18 protected superinfecting SP10 DNA, even when rifampin or chloramphenicol was added before the primary infection. The data are discussed in terms of the following conclusions. (i) The nonB gene product of B. subtilis Marburg 168 is required for restriction of SP10 DNA. (ii) Some sites on SP10 DNA are sensitive to both the restricting and modifying activities, whereas other sites are nonmodifiable even though they are sensitive to the restriction enzyme. (iii) In some manner, SP18 antagonizes the action of the nonB gene product.

Identification of some steps in the replication of bacteriophage T1 DNA

The effects of amber mutations in the 25 known genes of phage T1 on phage DNA replication were examined by sedimentation analysis of pulse-labelled DNA extracted from infected nonpermissive cells. Five groups of mutants were identified according to their DNA phenotype. Group I comprises am20 (gene 1) and am5 (gene 2) which are totally defective in DNA synthesis as previously shown by D. Figurski and J. R. Christensen (1974, Virology 59, 397–407). Group IIA also consists of two mutants, am201 (gene 3.5) and am23 (gene 4), both of which fail to make concatemeric DNA. Group II includes am41(gene 3), am15 (gene 5), am18 (gene 6), am35 (gene 7), am32 (gene 8), am13 (gene 9), am19 (gene 10), am29 (gene 11), am304 (gene 11.5), and am37 (gene 12). This group, represented predominantly by genes controlling tail formation, exhibits a wild-type pattern in which concatemerie DNA is synthesized and then processed to unit-length molecules. Group III mutants are all defective in head formation and accumulate concatemeric DNA molecules. This group contains am10 (gene 13), am216 (gene 13.7), am45 (gene 14), am246 (gene 14.5), am11 (gene 15), am4 (gene 16), am7 (gene 17), and am30 (gene 18). Group IV is represented by a single mutant, am283 (gene 13.3), with a sedimentation pattern intermediate between that of Group II and Group III mutants. It is not known if am283 is defective in concatemer formation or concatemer processing.

The mechanism of replication of φX174 DNA: XVI. Evidence that the φX174 viral strand is synthesized discontinuously

Using a new method of stopping DNA synthesis, we have studied the nascent intermediates present during the final stage of φX174 DNA replication when progeny single-stranded circular DNA molecules are synthesized. We find that 40 to 50% of the [ 3H]thymidine incorporated in a brief pulse, stopped by bringing the infected culture rapidly to 100 °C, is present in DNA molecules shorter than unit length. The molecules, which range from very short to unit length, are not generated by the stopping and isolating procedure, since 32P-labeled infecting parental viral strands remain relatively intact. The proportion of pulse label found in short intermediates varies with pulse length, stopping procedure, aeration level of the infected culture, and host strain. There is no significant difference in the abundance of short nascent intermediates in ung and ung + strains, suggesting that the short molecules are not the result of uracil excision by uracil-DNA glycosylase. The 3H-labeled short molecules hybridize to all regions of the φX genome, but preferentially to the region around the origin/terminus of replication. The relative amount of 3H in the unit length molecules that hybridizes to restriction enzyme fragments increases from the origin to the terminus, indicating that these molecules were completed during the pulse interval. Sensitivity to spleen exonuclease after treatment with alkali or RNase suggests that some of the short molecules isolated both during viral strand synthesis and during replicative form DNA replication have at least one ribonucleotide at the 5′ end. We conclude that the major mode of φX viral strand DNA replication is a discontinuous process, not continuous as proposed by the rolling circle model.

Electron microscopic analysis of partially replicated bacteriophage T7 DNA

Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144--6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460--476, 1961; Doermann et al., J. Cell. comp. Physiol. 45[Suppl.]:51--74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome.

Adeno-associated virus DNA replication: Nonunit-length molecules

Nonunit-length adeno-associated virus (AAV) DNA molecules which incorporate radiolabel during a short pulse were isolated from infected cells and characterized with regard to structure and to their potential role as replicative intermediates. Two classes of molecules were observed. (1) Dimer length duplex molecules containing the entire AAV genome were shown to be precursors to mature unit length AAV DNA. About one-third of the single strands from these molecules were dimers, the rest being of unit length. Some of these dimeric duplex molecules contain a hairpinned terminal structure formed by folding over of the terminal repetition from the right end of the AAV genome. (2) Smaller than unit-length molecules contain only the terminal 10–25% of the AAV genome and are not processed to unit-length genomes. They have one natural AAV terminus and a hairpinned internal terminus. Both classes of AAV molecules can be generated from a replication model utilizing the 3′-OH end of a hairpinned palindromic AAV terminal sequence as a primer for daughter-strand synthesis. The short, defective molecules result from a template strand switch to either the complementary strand of the parental duplex or to the newly synthesized complementary strand.

Circular adenovirus DNA-protein complexes from infected HeLa cell nuclei

Adenovirus (Ad) DNA was extracted by the use of Sarkosyl from Ad5-infected HeLa cell nuclei at 15 hr after infection. Mature and replicating viral DNA molecules in the Sarkosyl extract were purified by sucrose gradient centrifugation followed by equilibrium centrifugation in preformed metrizamide gradients. Part of the material was digested with the Hpa I restriction endonuclease and analyzed by electrophoresis through 1% agarose gels. Both terminal fragments D and E did not enter the gel unless first treated with Pronase, suggesting that intracellular viral DNA molecules were linked to proteins by their termini. Examination under the electron microscope of mature intracellular Ad2 or Ad5 DNA molecules extracted with Sarkosyl showed the presence of a majority of circular unit-length molecules. Most of these were attached to a prominent globular structure which disappeared, together with the circularization of the molecule, after treatment with Pronase. We interpret these structures as protein links responsible for the circularization of the molecule. The mature linear DNA molecules observed also showed evidence for linkage structures attached to one or both of their ends. Examination of replicating Ad5 DNA molecules extracted with Sarkosyl revealed the presence of various types of forked, circular molecules, most of which showed evidence for the presence of linkage structures. It is suggested that replicating and newly made Ad DNA molecules are circular inside the cell, perhaps through linkage with proteins, and the possible role of circularization in the replication of viral DNA is discussed.

Capsid transformation during packaging of bacteriophage lambdaDNA.

Assembly pathways of complex viruses might not be simple additions of one protein after another with rigid tertiary structure. It might in fact involve shifts in subunit structure, movement of subunits relative to each other to form new arrangements, transient action of proteins and protein segments, involvement of structure forming 'microenvironments' of the host. Thus morphogenesis of the bacteriophage lambda head starts with the formation of a core-containing DNA-free petit lambda particle. In a first transition, and dependent on a host function, the core is released, minor protein components of the capsid are processed and the particle's structure is altered, as shown by a change of its hydrodynamic properties. The resulting 'prehead' undergoes a second transition triggered by a complex of DNA and recognition protein (A-protein). This transition is more drastic than the first one. The particle doubles its volume without increasing in protein mass, the shell becomes thinner, and the surface structure is changed. Concomitantly with this process, the DNA becomes packaged and the particle becomes able to bind the small 'D-protein' in amounts equimolar to the capsid protein, which it could not do before. The D-protein addition probably causes another shift of the capsid structure. DNA packaging is completed, and the DNA is cut from concatemeric precursors to unit length molecules. Binding sites are created for the tail connector molecules which in turn allow the independently assembled tail to attach. Research on these processes proceeds along several lines: comparison of physical and chemical properties of particles accumulating in mutants; pulse-chase experiments on assembly precursors; morphogenesis in vitro; and model transitions of aberrant lambda polyheads.

Formation of concatemeric DNA in bacteriophage T7-infected bacteria

The formation of concatemers in T7 wild type- and amber 3 mutant-infected bacteria begins soon after the completion of the first rounds of replication. Unreplicated viral DNA is not incorporated into concatemers. Parental DNA strands are recovered as intact unit-length molecules from concatemers in gene 3 mutant-infected cells. In concatemers from wild type-infected cells a considerable fraction of parental DNA sequences is covalently linked to newly synthesized DNA sequences probably due to molecular recombination. No concatemeric DNA is formed in the absence of a functional gene 6 product (exonuclease) although several rounds of DNA replication occur under these conditions. The replicated gene 6 mutant DNA is partially fragmented.