Characterization of novel populations of MVM virions containing covalent DNA-protein complexes

Faust, E A; Brudzynska, K; Morgan, James I.

doi:10.1016/0042-6822(89)90411-x

Cited by 6 publications

(2 citation statements)

References 40 publications

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“…A DNA form similar to band X has been observed previously in crude cell lysates (Cotmore & Tattersall, 1988) and nuclear extracts (Doerig et al, 1986). A similar DNA form has also been reported to be associated with viral capsids (Doerig et al, 1986;Faust et al, 1989). The DNA component of band X is covalently attached to NS1, similar to MVM RF and progeny ssDNA, because its mobility in SDS-agarose gels is slightly increased following proteinase K treatment and it can be immunoprecipitated with anti-NS1 antibody, even after boiling in 0.2% SDS (Cotmore & TattersaU, 1988;G.…”

Section: Analysis Of the Temporal Accumulation Of M V M Rf In Highly mentioning

confidence: 92%

Characterization of the temporal accumulation of minute virus of mice replicative intermediates

Tullis

Schoborg

Pintel

1994

Journal of General Virology

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We have characterized the temporal appearance and accumulation of minute virus of mice (MVM) replicative forms (RF) in highly synchronized single rounds of infection using a combination of restriction endonuclease analysis and two-dimensional agarose gel electrophoresis. Between 4 and 12 h after release of infected cells into the S-phase, both monomer (mRF) and dimer RF (dRF) increased exponentially at similar rates such that the ratio of mRF relative to dRF remained unchanged. These DNA forms accumulated at a faster rate than MVM RNAs, suggesting that the number of DNA templates available for replication is limiting, not the expression of MVM gene products, and that the majority of DNA templates are likely to be destined for DNA amplification rather than transcription and further gene expression. During this exponential DNA amplification phase, approximately 65% of mRF were in a fully extended form, whereas most of the remaining mRF were covalently closed in the left end and extended in the right end. Although MVM replication presumably generates right-hand turn-around mRF, only a low level of this form persists (5 to 10% of total mRF) at all times examined, suggesting that this form must be quickly converted to the extended form. Greater than 90% of dRF, which have right-hand palindromes on both ends of the molecule, were extended on both ends. A significant proportion of dRF and higher concatemers are nicked in the left-hand palindrome, suggesting that resolution of dRF into two mRFs may occur via single-stranded nicks rather than a double-stranded cut. An additional replicative form, previously termed band X, has been identified as an RNA-DNA duplex. This band is formed predominantly intracellularly, before cell lysis but its biological significance remains unclear. Our results provide direct experimental support for many of the predictions of the current models of parvovirus replication and suggest that the kinetic hairpin transfer model should be adjusted to include a strand-transfer of similar mechanism for the resolution of dRF to account adequately for the production of left-end turn-around forms.

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Section: Analysis Of the Temporal Accumulation Of M V M Rf In Highly mentioning

confidence: 92%

Characterization of the temporal accumulation of minute virus of mice replicative intermediates

Tullis

Schoborg

Pintel

1994

Journal of General Virology

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“…subtypes among preparations of these particles that might be caused by occasional overlapping of protein forms in the gels or, more likely, by the fact that several stages of capsid phosphorylation are compatible with the viral DNA encapsidation. Because, as explained below, these more acidic VP-2 protein forms are precursors of VP-3, this heterogeneity would explain the variable VP-2-to-VP-3 ratio normally found in MVMp preparations (23,50,77).…”

Section: Expression In Bacteria and Purification Of 13-gal-ns Fusionmentioning

confidence: 99%

Protein species of the parvovirus minute virus of mice strain MVMp: involvement of phosphorylated VP-2 subtypes in viral morphogenesis

Santarén

Ramírez

Almendral

1993

J Virol

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The pattern of induced protein species of the prototype strain of the parvovirus minute virus of mice was determined in permissive A9 mouse fibroblast cells by high-resolution two-dimensional gel electrophoresis. Identities of the viral proteins in the gels were assigned by probing two-dimensional blots with antisera raised against either purified capsids (recognizing VP-1 and VP-2) or specific coding regions of the nonstructural proteins (NS-1 and NS-2) expressed as ,-galactosidase fusion products in bacteria. All viral proteins showed posttranslational modifications, phosphate being a common substituent. The NS-1 protein migrated as a basic polypeptide in the pI range of 7.4 to 7.8 with multiple stages of modification and as a likely minor but hyperphosphorylated component in the neutral region of the gel. The NS-2 isoforms were resolved at a pl value close to 5.5 as three groups of unevenly phosphorylated polypeptides, each composed of at least two protein species. Both VP-1 and VP-2 structural polypeptides were induced as heterogeneous phosphoproteins. The major VP-2 protein could be resolved in the form of a consistent pattern of three abundant (a to c), two intermediate (d and e), and one meager (f) neutral isoelectric focusing species or subtypes. This posttranslational modification precedes and is uncoupled from viral assembly, and all of the VP-2 subtypes are packaged into empty capsids at the induced stoichiometry. However, intracellular full virions harbored additional phosphorylated subtypes (g to 1) and a subtle rearrangement in the whole VP-2 composition, while mature virions purified from lysed cultures lacked these subtypes, coordinately with the emergence of six neutral VP-3 subtypes. Thus, the virion coat undergoes a chemical transition entailed by genome encapsidation, in which phosphates seem to play a major role, triggering the preferential proteolytic cleavage of the more acidic VP-2 subtypes to VP-3. Parvoviruses, with small coding capacity, may regulate some morphogenetic steps, such as assembly, genome encapsidation, and maturation, by posttranslational modifications of their structural proteins.

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Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice

Yeung

Brown²,

Tam³

et al. 1991

Virology

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Characterization of novel populations of MVM virions containing covalent DNA-protein complexes

Cited by 6 publications

References 40 publications

Characterization of the temporal accumulation of minute virus of mice replicative intermediates

Characterization of the temporal accumulation of minute virus of mice replicative intermediates

Protein species of the parvovirus minute virus of mice strain MVMp: involvement of phosphorylated VP-2 subtypes in viral morphogenesis

Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice

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