Intracellular Replication of Mycoplasmavirus MVL51

The intracellular replication of MVL51, a group L1 mycoplasmavirus, was investigated. The single-stranded parental DNA was found to enter the cell and become converted to double-stranded DNA. This replicated to yield additional double-stranded DNA molecules. The parental viral DNA was found to leave the replication complex and become associated with large molecular weight DNA not involved with viral replication. Progeny viral DNA formed from the double-stranded DNA and an intracellular accumulation of virus chromosome size DNA was observed. The interpretation of this data and a suggested model for the viral replication are discussed and compared to viral DNA replication models for other single-stranded DNA viruses.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.     

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.     

A role for single-stranded templates in cell-free adeno-associated virus DNA replication

Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation

The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [(3)H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [(3)H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [(3)H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis.     

Recognition of single-stranded DNA by the bacteriophage T4-induced type II topoisomerase

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.     

Reovirus protein sigmaNS binds in multiple copies to single-stranded RNA and shares properties with single-stranded DNA binding proteins

Reovirus nonstructural protein sigmaNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of sigmaNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of sigmaNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The sigmaNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, sigmaNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, sigmaNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl(2), distinguishing it from a classical ATP-dependent helicase. These properties of sigmaNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for sigmaNS in replication of the reovirus RNA genome.     

Specific Initiation Site for Simian Virus 40 Deoxyribonucleic Acid Replication

Replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) molecules have been isolated under conditions in which the newly synthesized DNA is uniformly labeled with (3)H-thymidine. These newly synthesized strands are released from the replicative intermediate molecules by alkaline treatment, and it has been possible to isolate single-stranded SV40 DNA which varies in size from 157,000 daltons (from molecules that are 10% replicated) to 1,360,000 daltons (85% replicated). The rates of duplex formation of newly synthesized DNA have been used to relate their genetic complexity to the extent of DNA replication. As DNA replication proceeds, the time required to effect 50% renaturation of the newly synthesized DNA increases at a proportional rate. The data establish that DNA replication is not initiated at random, but rather that there is a single specific initiation site for DNA replication.     

Studies on the Mechanism of Replication of Adenovirus DNA II. The Nature of Single-Stranded DNA in Replicative Intermediates

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.