The Left-End and Right-End Origins of Minute Virus of Mice DNA Differ in Their Capacity to Direct Episomal Amplification and Integration In Vivo

Corsini, Joe; Cotmore, Susan F.; Tattersall, Peter; Winocour, Ernest

doi:10.1006/viro.2001.1076

Cited by 9 publications

(7 citation statements)

References 45 publications

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“…Similar syntheses and immunoprecipitations were performed by using HeLa cell in vitro replication extracts in place of T7 DNA polymerase (11). In these experiments, the fraction 11 products were somewhat longer, although still shorter than the products of purified genomic DNA.…”

Section: Resultsmentioning

confidence: 77%

Depletion of Virion-Associated Divalent Cations Induces Parvovirus Minute Virus of Mice To Eject Its Genome in a 3′-to-5′ Direction from an Otherwise Intact Viral Particle

Cotmore¹,

Hafenstein

Tattersall

2010

J Virol

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We describe a structural rearrangement that can occur in parvovirus minute virus of mice (MVMp) virions following prolonged exposure to buffers containing 0.5 mM EDTA. Such particles remain stable at 4°C but undergo a conformational shift upon heating to 37°C at pH 7.2 that leads to the ejection of much of the viral genome in a 3-to-5 direction, leaving the DNA tightly associated with the otherwise intact capsid. This rearrangement can be prevented by the addition of 1 mM CaCl 2 or MgCl 2 prior to incubation at 37°C, suggesting that readily accessible divalent cation binding sites in the particle are critical for genome retention. Uncoating was not seen following the incubation of virions at pH 5.5 and 37°C or at pH 7.2 and 37°C in particles with subgenomic DNA, suggesting that pressure exerted by the full-length genome may influence this process. Uncoated genomes support complementary-strand synthesis by T7 DNA polymerase, but synthesis aborts upstream of the right-hand end, which remains capsid associated. We conclude that viral genomes are positioned so that their 3 termini and coding sequences can be released from intact particles at physiological temperatures by a limited conformational rearrangement. In the presence of divalent cations, incremental heating between 45°C and 65°C induces structural transitions that first lead to the extrusion of VP1 N termini, followed by genome exposure. However, in cation-depleted virions, the sequence of these shifts is blurred. Moreover, cation-depleted particles that have been induced to eject their genomes at 37°C continue to sequester their VP1 N termini within the intact capsid, suggesting that these two extrusion events represent separable processes.The capsids of nonenveloped viruses have evolved to protect their genomes from hostile extra-and intracellular environments and to undergo a specific program of conformational shifts in response to cellular cues, which sequentially expose trafficking effector structures required to transport the nucleic acid to the appropriate cellular location, at the correct time, for its successful replication (22,25,31,42,43,46). In many cases, such shifts are possible because the virion is poised in a metastable configuration, prevented from collapsing to an alternate structure by the energy barrier between the two forms. Cellular or environmental triggers effectively compromise this energy barrier during cell entry, inducing the particle to rearrange. Accordingly, the pattern of particle morphogenesis is in part encrypted within the virion so that probing its structure in vitro, using elevated temperatures or biased ionic conditions, provides an avenue for assessing the types of rearrangements that the virion is capable of sustaining (5,12,22,24,29,34,35,46,47,50). In this study we exploit in vitro manipulation to gain insight into the structure and potential rearrangements programmed into the minute virus of mice (MVM) virion.As for all members of the family Parvoviridae, virions of MVM are exceptionally small and structurally sim...

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Section: Resultsmentioning

confidence: 77%

Depletion of Virion-Associated Divalent Cations Induces Parvovirus Minute Virus of Mice To Eject Its Genome in a 3′-to-5′ Direction from an Otherwise Intact Viral Particle

Cotmore¹,

Hafenstein

Tattersall

2010

J Virol

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“…The appearance of large-disperse forms was consistent with the fact that EBV episomes are large double-stranded, supercoiled DNA molecules able to form concatameric structures within the infected cell and able to undergo amplification. 45,46 Similarly, integrated DNA would appear as closely adjacent dots (doublets, or one signal per chromatid copy). As the Namalwa cell line contains only two integrated viral copies and completely lacks episomes, the distinct doublets seen in this cell line could only originate from integrated virus.…”

Section: Discussionmentioning

confidence: 99%

Rapid determination of Epstein–Barr virus latent or lytic infection in single human cells using in situ hybridization

et al. 2004

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Epstein-Barr (EBV) virus is associated with malignancies such as lymphoma and carcinoma. Infection of cells with EBV may result in either lytic infection with production of viral particles, characterized by the presence of linear DNA forms, or latent infection, characterized by either episomal or integrated DNA forms. To examine whether the different lytic and latent EBV DNA forms can reliably be distinguished in single human cells, in situ hybridization was performed in EBV-positive cell lines. Immunocytochemistry and Southern blot analysis were performed supplementary to in situ hybridization. In latent infection, three in situ hybridization patterns were observed: large-disperse (episomal), small-punctate (integrated) and combined (both), signal types 1, 2 and 3 respectively. These were associated with expression of latent membrane protein 1, but not with Z fragment of Epstein-Barr replication activator or viral capsid antigen. In lytic infection, three additional in situ hybridization patterns were observed: nuclear membrane associated, bubble (filling up the nucleus) and spillover (covering the lysed cells) signals types 4, 5 and 6 respectively. Signal types 4 and 5 were associated with expression of latent membrane protein 1 and Z fragment of Epstein-Barr replication activator but not viral capsid antigen, whereas type 6 was associated with expression of viral capsid antigen only. Southern blot analysis confirmed these results; however, low copy numbers of integrated virus were often missed by Southern blot, confirming that in situ hybridization is more sensitive in determining the presence of all types of EBV DNA. In situ hybridization may prove useful in rapidly screening large series of tissue microarrays and other clinical specimens for the presence of lytic or latent EBV.

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“…Based on Southern blot analysis, integration occurred at random locations, and the vector proviruses had terminal deletions. Because the vector did not express NS1, this process is distinct from the NS1-dependent integration of MVM vectors into episomes (7,8) and the related Rep-dependent site-specific integration of AAV vectors at human chromosome 19 (32,49). Instead, MVM vector integration is similar to Rep-independent random integration of AAV vectors (38,48,53), which uses host enzymes in a reaction resembling nonhomologous end joining at double-strand breaks (36).…”

Section: Discussionmentioning

confidence: 99%

“…Viral genomes were not detected in high-molecular-weight DNA during a lytic MVM infection (44) or in cells persistently infected with the immunosuppressive strain of MVM (45). Corsini et al demonstrated that wild-type MVM can integrate into an episome containing the left terminal origin of replication in a reaction that depended on the viral nonstructural (NS) protein NS1 (7,8). This reaction is analogous to AAV integration at viral repeat sequences that bind the AAV Rep proteins (33,52).…”

mentioning

confidence: 99%

Chromosomal Integration and Homologous Gene Targetingby Replication-Incompetent Vectors Based on the Autonomous ParvovirusMinute Virus ofMice

Hendrie

Hirata

Russell

2003

J Virol

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The molecular mechanisms responsible for random integration and gene targeting by recombinant adenoassociated virus (AAV) vectors are largely unknown, and whether vectors derived from autonomous parvoviruses transduce cells by similar pathways has not been investigated. In this report, we constructed vectors based on the autonomous parvovirus minute virus of mice (MVM) that were designed to introduce a neomycin resistance expression cassette (neo) into the X-linked human hypoxanthine phosphoribosyl transferase (HPRT) locus. High-titer, replication-incompetent MVM vector stocks were generated with a two-plasmid transfection system that preserved the wild-type characteristic of packaging only one DNA strand. Vectors with inserts in the forward or reverse orientations packaged noncoding or coding strands, respectively. In human HT-1080 cells, MVM vector random integration frequencies (neo ؉ colonies) were comparable to those obtained with AAV vectors, and no difference was observed for noncoding and coding strands. HPRT gene-targeting frequencies (HPRT mutant colonies) were lower with MVM vectors, and the noncoding strand frequency was threefold greater than that of the coding strand. Random integration and gene-targeting events were confirmed by Southern blot analysis of G418-and 6-thioguanine (6TG)-resistant clones. In separate experiments, correction of an alkaline phosphatase (AP) gene by gene targeting was nine times more effective with a coding strand vector. The data suggest that single-stranded parvoviral vector genomes are substrates for gene targeting and possibly for random integration as well.The Parvoviridae are nonenveloped viruses with an encapsidated, single-stranded, linear DNA genome of approximately 5 kb (3). Autonomous parvoviruses such as minute virus of mouse (MVM) are a diverse subgroup that differs from dependoviruses such as adeno-associated virus (AAV) in their ability to productively infect host cells in the absence of additional helper viruses (9). Unlike AAV, autonomous parvoviruses have not been shown to integrate into host chromosomes. Viral genomes were not detected in high-molecular-weight DNA during a lytic MVM infection (44) or in cells persistently infected with the immunosuppressive strain of MVM (45). Corsini et al. demonstrated that wild-type MVM can integrate into an episome containing the left terminal origin of replication in a reaction that depended on the viral nonstructural (NS) protein NS1 (7,8). This reaction is analogous to AAV integration at viral repeat sequences that bind the AAV Rep proteins (33, 52). While Rep-dependent site-specific integration of AAV can occur at a sequence in human chromosome 19 similar to the viral terminal repeats (32, 49), murine and human genomes do not contain sequences homologous to the MVM binding and nicking sites for NS1 (see Materials and Methods), which may account for the lack of detectable MVM integration.AAV vectors have been extensively developed for gene transfer applications (reviewed in references 6, 18, and 47) and are curre...

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The Left-End and Right-End Origins of Minute Virus of Mice DNA Differ in Their Capacity to Direct Episomal Amplification and Integration In Vivo

Cited by 9 publications

References 45 publications

Depletion of Virion-Associated Divalent Cations Induces Parvovirus Minute Virus of Mice To Eject Its Genome in a 3′-to-5′ Direction from an Otherwise Intact Viral Particle

Depletion of Virion-Associated Divalent Cations Induces Parvovirus Minute Virus of Mice To Eject Its Genome in a 3′-to-5′ Direction from an Otherwise Intact Viral Particle

Rapid determination of Epstein–Barr virus latent or lytic infection in single human cells using in situ hybridization

Chromosomal Integration and Homologous Gene Targetingby Replication-Incompetent Vectors Based on the Autonomous ParvovirusMinute Virus ofMice

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