Infectivity of visna virus DNA

Haase, Ashley T.; Traynor, Betty; Ventura, Peter; Alling, David W.

doi:10.1016/0042-6822(76)90236-1

Cited by 32 publications

(11 citation statements)

References 31 publications

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“…The fact that this is higher than that reported for visna virus (Haase et al 1976) and avian tumour viruses (Cooper & Temin,I974) is probably a result of the increased efficiency of the transfection assay due to the DMSO treatment. We are now using this assay to investigate the genome structure of FSFV and the relationship between the proviral DNA and the host cell genome.…”

Section: Short Communications (A)contrasting

confidence: 47%

Infectious DNA from Cells Infected with Feline Syncytium-forming Virus (Spumavirinae)

Chiswell

Pringle

1977

Journal of General Virology

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SUMMARYDNA isolated from cells infected with FSFV (a foamy virus) is infectious when tested on susceptible cells. The virus produced by this infectious DNA is identical to the original infecting virus in terms of plaque and virion morphology and serology.Feline syncytium-forming virus (FSFV) has been isolated from both normal and diseased cats (Riggs et al. 1969; Jarrett, Hay & Laird, 1974). In morphological, biological and biochemical characteristics, FSFV appears to be a typical member of the Spumavirinae (formerly foamy viruses) a sub-family of the Retroviridae (Fenner, I976). DNA was extracted from FEA cells grown at 37 °C, either mock-infected, or infected 4 to 5 days previously with FSFV at a multiplicity of infection of o.I, by a modification of the method of Marmur (I96I). Essentially, when infected monolayers showed approx. 70 to 8o % of cells involved in syncytial c.p.e., the medium was removed and the cells scraped into Dulbecco's phosphate buffered saline (PBS), washed twice with PBS, then resuspended in 0-2 M-NaCI, 0"02 M-tris-HC1, pH 7"4, o.ooi M-EDTA (NTE). Boiled protease (Sigma Chemicals, type VI) and SDS were added to final concentrations of 5o0/zg/rnl and o-5 % respectively. The mixture was heated to 80 °C for IO min, then incubated at 37 °C for 2 h. The digest was then extracted twice by gentle mixing with equal volumes of phenol saturated with NTE and twice with chloroform: iso-amyl alcohol (24: I). The extract was then dialysed overnight against NTE buffer before the addition of RNase A (Sigma Chemicals, boiled for 3 min) to a concentration of 50/zg/ml; incubation was carried out at 37 °C for 3 h. The digest was extracted twice with equal volumes of phenol saturated with NTE, and then chloroform: iso-amyl alcohol extractions performed until protein was no longer observable at the interface (approx. 2 to 4 times). The extract was dialysed against o.I x SSC (SSC-I.5 Msodium chloride, o'15 M-sodium citrate, o"15 M-citric acid, pH 7"5) before being stored at -2o °C. DNA concentrations were estimated using an assay based on the specific binding of mithromycin to double-stranded DNA (Hill & Whateley, I975). Calf thymus DNA was

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Section: Short Communications (A)contrasting

confidence: 47%

Infectious DNA from Cells Infected with Feline Syncytium-forming Virus (Spumavirinae)

Chiswell

Pringle

1977

Journal of General Virology

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“…The data are consistent with an IdUrd-sensitive restriction in the recipient NIH 3T3 cells that revents viral replication from endogenous AKR MuLV DNA Cut does not prevent viral replication from MuLV DNA of productively infected cells. Previous transfection studies have shown that retroviral DNA is infectious either as an unintegrated proviral DNA genome from acutely infected cells (1)(2)(3)(4) or as an integrated proviral DNA genome from chronically infected cells (5)(6)(7)(8)(9)(10). However, DNA from cells known to contain unexpressed but potentially infectious endogenous viral genomes is not infectious under the same transfection conditions (11).…”

Section: Akr Mulv or Rauscher Mulv Is Infectious With Or Withoutmentioning

confidence: 99%

Infectious murine leukemia virus from DNA of virus-negative AKR mouse embryo cells.

Lowy¹

1978

Proc. Natl. Acad. Sci. U.S.A.

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DNA from virus-negative AKR mouse embryo cells is infectious for NIH 3T3 cells if the transfected cells are treated with 5-iododeoxyuridine (IdUrd) either before or after addition of the DNA. The virus isolated after transfection of the AKR DNA is an ecotropic murine leukemia virus (MuLV) Previous transfection studies have shown that retroviral DNA is infectious either as an unintegrated proviral DNA genome from acutely infected cells (1-4) or as an integrated proviral DNA genome from chronically infected cells (5-10). However, DNA from cells known to contain unexpressed but potentially infectious endogenous viral genomes is not infectious under the same transfection conditions (11). Thus, except for the demonstration that DNA from avian cells can complement the reverse transcriptase lesion in a temperature-sensitive mutant of Rous sarcoma virus (12), DNA from normal cells has not yet been shown to be infectious, despite genetic, biologic, and biochemical studies that indicate that endogenous retroviral genomes are present in cells from many species (13,14). The demonstration of complete retroviral expression from the DNA of normal cells would show directly that the cell DNA is potentially infectious. In addition, an infectivity assay for an endogenous viral genome would be useful in attempting to elucidate the cellular and viral mechanisms that restrict the expression of the viral genome in the endogenous state and permit its expression in the productively infected state.Although all inbred mice (Mus musculus) apparently contain endogenous retroviral genomes (15, 16) that are potentially infectious (17), strains vary markedly in their viral expression in vivo and in vitro (18)(19)(20)(21) Chemicals. IdUrd was purchased from Sigma and it was stored at 1 mg/ml in sterile phosphate-buffered saline at -20°C.DNA Isolation. Bulk cellular DNA was isolated as described (30) except that, prior to treatment with RNase, the ethanolprecipitated nucleic acid was wound on a rod instead of being concentrated by centrifugation. This modification was used because the centrifuged DNA was more difficult to redissolve than was the "spooled" DNA. This DNA had an average molecular weight of greater than 5 X 107. It was stored at 150-300 ,g/ml (20 A260 units/,ug DNA) at 4°C in 10 mM NaCl/10 mM Tris, pH 8.0 /0.5 mM EDTA. Stored DNA is still infectious after 1 year.Because it was critical that untreated AKR 2B cells not be producing virus at the time of DNA isolation, at least 2 X 106 AKR cells from the pool of cells used for each AKR DNA isolation were cocultivated with SC-1 cells, passaged at least twice, and found at that time to be negative for MuLV by the XC plaque test (31). IdUrd-treated AKR DNA was obtained after 24-hr treatment of AKR 2B cells with IdUrd at 40,g/ml. Incorporation of IdUrd into DNA was confirmed by CsCl density gradient centrifugation. Infectious center plating onto SC-1 cells Abbreviations: MuLV, murine leukemia virus; IdUrd, 5-iododeoxyuridine.

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“…The mechanism of the block in transcription is unknown but has generally been assumed to be related to a lysogenic state, since visna virus is a retrovirus, and viral DNA is associated with high molecular weight host DNA (5)(6)(7). However, we recently found that transcription in vitro is governed by the extent of early DNA synthesis and suggested that extrachromosomal DNA might be the template for viral RNA (8).…”

mentioning

confidence: 99%

Slow virus visna: reproduction in vitro of virus from extrachromosomal DNA.

Harris¹,

Blum²,

Scott³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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Under permissive conditions of growth in tissue culture, the retrovirus visna multiplies over the course of a few days to high titer and kills the host cell. We show that in this lytic life cycle, viral DNA is tightly associated with, but not covalently linked to, chromosomal DNA. This finding provides explanations for a number of the unusual properties of the lentivirus subfamily of retroviruses, and suggests potential mechanisms for the block in virus gene expression in vivo responsible for the slow infection in nature.Visna virus is the prototype of the subfamily of retroviruses that cause slow infections in sheep characterized by pathological changes in the lungs and central nervous system (1,2). Both the persistence of virus in the face of the immune response mounted by the host and the slow evolution of disease can be explained by restriction in virus gene expression imposed at the transcriptional level (3, 4). Most of the cells that harbor virus genomes have insufficient antigen to be detected and destroyed by immune surveillance, and limitation in synthesis of virus gene products allows the host cell to survive for the extended periods characteristic of slow infections.The mechanism of the block in transcription is unknown but has generally been assumed to be related to a lysogenic state, since visna virus is a retrovirus, and viral DNA is associated with high molecular weight host DNA (5-7). However, we recently found that transcription in vitro is governed by the extent of early DNA synthesis and suggested that extrachromosomal DNA might be the template for viral RNA (8). Moreover, Panganiban and Temin (9) have shown that production of spleen necrosis virus, an avian C-type retrovirus, can occur from unintegrated viral DNA. For these reasons, we reexamined the role of integration in the visna life cycle in vitro and found that in the vast majority of cells unintegrated DNA must serve as the template for transcription and for virus production. MATERIALS AND METHODSInfection of Cells and Isolation of DNA. Confluent cultures of sheep choroid plexus (SCP) cells were infected at a multiplicity of 3 plaque-forming units per cell as described (8,10). At 60-70 hr after infection, cells were removed by trypsinization and either replated under agarose for infectious center assay or separated into nuclear and cytoplasmic fractions (11). High molecular weight DNA was isolated by the Hirt fractionation procedure (12). In some experiments, high molecular weight DNA was purified further by electrophoresis in 0.5% low-gelling temperature agarose; the portion of the gel containing DNA 20 kilobase pairs (kbp) or greater in size was excised and melted, and DNA was isolated by phenol/chloroform extraction.Cloning of Visna DNA. Viral DNA for probes and for reconstruction experiments was obtained by cloning. The relevant restriction enzyme sites in visna DNA (9) are shown in Fig. 1. DNA from the Hirt supernatant fraction from SCP cells infected for 60 hr was digested with Sst I, and the fragments were inserted into ...

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Infectivity of visna virus DNA

Cited by 32 publications

References 31 publications

Infectious DNA from Cells Infected with Feline Syncytium-forming Virus (Spumavirinae)

Infectious DNA from Cells Infected with Feline Syncytium-forming Virus (Spumavirinae)

Infectious murine leukemia virus from DNA of virus-negative AKR mouse embryo cells.

Slow virus visna: reproduction in vitro of virus from extrachromosomal DNA.

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