Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication.

The nucleotide sequences of the terminal regions of monomer replicative form DNA, a pivotal intermediate species in the replication of minute virus of mice, were determined. The left (3') terminus had a unique sequence on both strands and in both 3'-hairpin configurations. In contrast, the right (5') terminus was sequence heterogeneous and extended an additional 18 base pairs beyond that expected from the known sequence of the virion DNA. These data unambiguously establish the sequence complexity at the termini of both the single-stranded viral genome and the pool of replicative DNA. A comparison of the combined sequence information leads us to propose a modified rolling hairpin model for the replication of autonomous parvoviruses which is compatible with all available data.     

Nucleoprotein complexes of minute virus of mice have a distinct structure different from that of chromatin.

We studied the structure of viral nucleoprotein complexes extracted from the nuclei of mouse cells infected with the immunosuppressive strain of the minute virus of mice (MVMi). Two types of complex were detected, with sedimentation coefficients of about 110 and 40S. The complexes sedimenting at 110S contained single-stranded MVMi DNA as well as a second form of viral DNA which apparently had a heat-sensitive secondary structure. The 110S peak also contained proteins which coelectrophoresed with the MVMi capsid proteins. Complexes sedimenting at 40S contained the double-stranded replicative form of MVMi DNA. These complexes sedimented faster than did the pure replicative form DNA (15S), but more slowly than cellular chromatin fragments containing DNA of the same length. They incorporated labeled deoxynucleoside triphosphate in vitro into the replicative form DNA. We investigated the structure of MVMi nucleoprotein complexes in the following ways. Nuclei of MVMi-infected cells were digested with staphylococcal nuclease, and the resulting DNA fragments were electrophoresed, transferred to nitrocellulose, and hybridized first with labeled MVMi DNA and then with cellular DNA. A nucleosomal repeat pattern was seen with the cellular DNA probe but not with the MVMi DNA probe. The DNA in MVMi nucleoprotein complexes was cross-linked with psoralen, purified, denatured, and examined with an electron microscope. Bubbles, indicating the presence of proteins, were seen in the MVMi DNA. The length of the DNA in the bubbles was 90 +/- 29 nucleotides. On the other hand, nucleosomes protected 160 base pairs from cross-linking by psoralen. The MVMi nucleoprotein complexes thus have a distinct structure which is different from that of chromatin.     

Development of physical forms of unintegrated retroviral DNA in mouse spinal cord tissue during ts1-induced spongiform encephalomyelopathy: elevated levels of a novel single-stranded form in paralyzed mice.

ts1 is a murine leukemia virus that causes rapidly evolving hindlimb paralysis in susceptible strains of mice. Following perinatal infection, three physical forms of unintegrated viral DNA were detected in the spinal cord by Southern blot hybridization. Linear and supercoiled closed-circle viral double-stranded DNAs were detected in both the central nervous system and non-central nervous system tissues. An elevated level of a novel minus-sense single-stranded form of viral DNA, which had a very high mobility in agarose gels, was correlated with the onset of symptoms of paralysis. As the severity of paralysis progressed, the level of this single-stranded form increased rapidly, with the highest level in the spinal cords of moribund mice. Since the virulence of a number of cytopathic retroviruses has been associated with the presence of increased amounts of unintegrated viral DNA in the tissues of the infected hosts, this novel form of highly mobile unintegrated single-stranded DNA may have a role in the neuropathogenesis of ts1.     

Replication of DNA containing apurinic sites in human and mouse cells probed with parvoviruses MVM and H-1.

We studied the effect of apurinic sites on DNA replication in mouse and human cells, using parvoviruses MVM (minute virus of mice) and H-1 as probes. Although apurinic sites are efficient blocks to the replication of these single-stranded DNA viruses in vivo, depurinated parvoviruses can be reactivated if host cells have been preexposed to a subtoxic dose of UV light. The target of this conditional reactivation process is the conversion of depurinated input DNA into double-stranded replicative forms; the concomitant increase in viral mutagenesis strongly suggests that apurinic sites can be bypassed in mammalian cells.     

Identification and characterization of a protein covalently bound to DNA of minute virus of mice.

We identified a protein which is covalently linked to a fraction of the DNA synthesized in cells infected with minute virus of mice. This protein is specifically bound to the 5' terminus of the extended terminal conformers of the minute virus of mice replicative-form DNA species and of a variable fraction of single-stranded viral DNA. The chemical stability of the protein-DNA linkage is characteristic of a phosphodiester bond between a tyrosine residue in the protein and the 5' end of the DNA. The terminal protein (TP) bound on all DNA forms has a relative molecular weight of 60,000; it is also seen free in extracts from infected cells. Immunologic comparison of the TP with the other known viral proteins suggests that the TP is not related to the capsid proteins or NS-1.     

A genome-linked copy of the NS-1 polypeptide is located on the outside of infectious parvovirus particles.

The 5' ends of all newly synthesized single-stranded (s1) DNA genomes of the autonomous parvovirus minute virus of mice are covalently linked to the major virally coded nonstructural protein NS-1, but later in infection this association is disrupted, giving rise to an abbreviated form of single-stranded DNA designated s2. Both s1 and s2 forms are encapsidated and migrate in velocity gradients as 110S particles, and, as such, both appear to be infectious. Most virions are released from A9 cells as s1 particles, but the NS-1 molecules are located on the outside of the virion where they are accessible to both antibodies and enzymes. These polypeptides are cleaved from the encapsidated DNA by nucleolytic or proteolytic digestion, which can occur either in the culture medium or upon subsequent entry into further host cells. Since the s1 to s2 cleavage can be minimized by blocking viral reentry, it is likely that most of the processing occurs after entry into the host cell. Incoming virus is rapidly converted to the s2 form when it is used to infect new host cells, but in vitro removal of the NS-1 molecules with proteases or nucleases fails to influence the infectivity of s1 particles under normal culture conditions. Limited proteolysis of s1 particles with trypsin demonstrates that NS-1 is linked to the DNA via its amino-terminal domain. Analysis of the 5' ends of s1 and s2 forms indicates that there are approximately 24 externally located nucleotides linking the NS-1 molecules to the 5.1-kilobase nuclease-resistant DNA core of the virion.     

Transcriptional organization of the Drosophila melanogaster ribosomal RNA genes.

Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis.     

Studies on the biological role of DNA methylation. II. Role of phiX174 DNA methylation in the process of viral progeny DNA synthesis.

In vivo inhibition of bacteriophage phiX174 DNA methylation by nicotinamide resulted in the accumulation of replicative intermediates with multiple-genome length single-stranded "tails". These abnormal replicative intermediates could not be chased into viral single-stranded circular DNA. The effect of nicotinamide on phage maturation and accumulation of abnormal replicative intermediates could be reversed by washing out the inhibitor. The results suggest that the single methyl group present in the viral DNA serves as a recognition site for a specific endonuclease, probably the gene A protein product, that is responsible for the excision of the single-stranded one-genome long viral DNA, before final maturation of the virus occurs.     

Tomato Yellow Leaf Curl Virus DNA Forms in the Viral Capside, in Infected Plants and in the Insect Vector

Tomato yellow leaf curl virus (TYLCV) DNA was used as a probe to identify and analyze virus-related DNAs in the viral capside, in infected tomato plants and in the virus vector, the whitefly. In addition to the single-stranded viral genomic DNA, double-stranded virus-related DNA molecules were detected in infected plants. Not all of the virus-related DNA forms are present simultaneously in the infected plant. The double-stranded molecules, which are probably the replicative form of the viral genome, have been purified from an infected tomato plant. In the viruliferous whitefly, only the single-stranded unit-size viral genome was detected.     

Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice.

The linear single-stranded DNA genome of minute virus of mice, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pBR322. The recombinant clones of minute virus of mice were infectious when transfected into monolayers of human 324K cells and produced virus plaques with an efficiency of about 6% that obtained with duplex replicative-form DNA purified from cells infected with minute virus of mice. Southern blot analysis of transfected cells indicated that the cloned minute virus of mice genome requires both termini to be intact for excision and replication as a linear duplex molecule.     

Interaction between parvovirus NS2 protein and nuclear export factor Crm1 is important for viral egress from the nucleus of murine cells

A mutation that disrupts the interaction between the NS2 protein of minute virus of mice and the nuclear export factor Crm1 results in a block to egress of mutant-generated full virions from the nucleus of infected murine cells. These mutants produce wild-type levels of monomer and dimer replicative DNA forms but are impaired in their ability to generate progeny single-stranded DNA in restrictive murine cells in the first round of infection. The NS2-Crm1 interaction mutant can be distinguished phenotypically from an NS2-null mutant and reveals a role for the Crm1-mediated export pathway at a late step in viral infection.     

Interaction of minute virus of mice with differentiated cells: strain-dependent target cell specificity is mediated by intracellular factors

The prototype strain of minute virus of mice and the immunosuppressive strain are unable to grow lytically in each other's murine host cell type. To characterize these strain-dependent virus-host cell interactions further, we have compared the early events of both productive and restrictive infections. Each virus binds to specific receptors on the surface of both productive and restrictive cell types. Competition experiments show that both viruses recognize the same receptor on each cell type. Penetration and uncoating are presumed to be similar in both productive and restrictive infections, since incoming viral genomes are converted to parental replicative form DNA independent of the final outcome of the virus-host cell interaction. In contrast to the majority of other systems studied to date, these differences in minute virus of mice target cell specificity are not mediated at the cell surface, but by the interaction of a strain-specific viral determinant with intracellular host factors that are expressed in particular cell types as a function of differentiation. These cellular factors catalyze a step in viral replication which occurs after the initiation of viral DNA synthesis, but before the detectable expression of the viral capsid polypeptide genes.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.     

In vivo accumulation of cyclin A and cellular replication factors in autonomous parvovirus minute virus of mice-associated replication bodies

Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases alpha and delta in these bodies suggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruitment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement in activating the conversion of virion single-stranded DNA to the duplex replicative form.     

In Vivo Conversion of the Single-Stranded DNA of the Kilham Rat Virus to a Double-Stranded Form

Kilham rat virus (KRV) contains linear, single-stranded DNA in the virion. The fate of radioactive viral DNA was followed after infection of monolayer cells. Within 60 min after infection of cells, 28 to 42% of the parental viral DNA is converted to a new form. This new DNA form is believed to be double stranded and linear on the basis of its sedimentation in neutral and alkaline sucrose gradients, elution from hydroxyapatite columns, its buoyant density in equilibrium CsCl density gradients, and appearance in the electron microscope. The double-stranded linear KRV DNA may be analogous to the replicative form of certain bacteriophages, including phiX174, which contain single-stranded circular genomes.     

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome.

Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.     

Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.     

In vivo resolution of circular plasmids containing concatemer junction fragments from minute virus of mice DNA and their subsequent replication as linear molecules.

During replication of their linear, single-stranded DNA genomes, parvoviruses generate a series of concatemeric duplex intermediates. We have cloned, into Escherichia coli plasmids, junction fragments from these palindromic concatemers of minute virus of mice DNA spanning both the right end-to-right end (viral 5' to 5') and left end-to-left end (viral 3' to 3') fusions. When mouse cells were transfected with these circular plasmids and superinfected with minute virus of mice, the viral junctions were resolved and the plasmids replicated as linear chromosomes with vector DNA in their centers and viral DNA at their termini. Resolution did not occur when the concatemer joint was replaced by a different palindromic sequence or when the transfected cells were not superinfected, indicating the presence of latent origins of replication which could only be activated by a viral trans-acting factor(s). Moreover, the products of resolution and replication from the two termini were characteristically different. Analysis of individual terminal fragments showed that viral 5' (right-end) sequences were resolved predominantly into "extended" structures with covalently associated copies of the virally encoded NS-1 polypeptide, while bridges derived from the 3' (left) end resolved into both NS-1-associated extended termini and lower-molecular-weight "turn-around" forms in which the two DNA strands were covalently continuous. This pattern of resolution exactly coincides with that seen at the two termini of replicative-form intermediates in normal virus infections. These results demonstrate that the bridge structures are authentic substrates for resolution and indicate that the frequency with which extended versus turn-around forms of each terminus are generated is an intrinsic property of the telomere.     

Replication of the Parvovirus MVM II. Isolation and Characterization of Intermediates in the Replication of the Viral Deoxyribonucleic Acid

The time course of the appearance of intracellular viral DNA has been studied in mouse L cells infected with the single-stranded DNA virus MVM (minute virus of mice) by using a selective extraction procedure. Approximately half of this DNA elutes from hydroxyapatite as single-stranded DNA. It is sensitive to Escherichia coli exonuclease I and shows a sedimentation profile similar to DNA from the virus, suggesting that it is progeny viral DNA. The remainder of the selectively extracted DNA elutes from hydroxyapatite in the position of double-stranded DNA and is resistant to exonuclease I. Most of this DNA has a sedimentation coefficient of 14 to 16S, indicating that its molecular weight is twice that of the viral DNA. Denaturation renders the majority of the double-stranded DNA sensitive to exonuclease I, but a significant fraction renatures spontaneously in a monomolecular fashion, indicating that it has a cross-linked or hairpin structure. Chromatography of the double-stranded DNA on benzoylated diethylaminoethyl cellulose resolves two components, one with duplex structure and one which contains single-stranded regions. A short pulse label late in infection predominantly labels the latter class of DNA, suggesting that it contains replicating intermediates. The possible roles of these various forms of DNA in the replication of the viral genome are discussed.     

Pathogenicity of fibroblast- and lymphocyte-specific variants of minute virus of mice.

We tested two strains of the minute virus of mice (MVM) for pathogenic effects and patterns of infection in laboratory mice. The two strains differ in their ability to infect differentiated cultured cells: the prototype virus, MVMp, infects only fibroblasts, while its variant, MVMi, is restricted to lymphocytes. We find that neither strain has any demonstrable effects on the T-cell function of mice infected as adults. In contrast, MVMi, but not MVMp, is able to induce a runting syndrome accompanied by mild immune deficiencies upon the infection of newborn mice. After neonatal infection, MVMi spreads to many organs, and the presence of viral replicative form DNA is evident in nucleic acid hybridization experiments. In contrast, replication of MVMp can be detected only by the seroconversion of infected animals. Newborn mice that grow abnormally as a result of MVMi infection also have low circulating antibody titers to the virus. This phenomenon may be a consequence of the lymphotropism of MVMi.