Kinetics of murine type C virus-specific DNA synthesis newly infected cells

We have examined the kinetics of synthesis of minus [(-)]-and plus [(+)]strand viral DNA in melittin-permeabilized avian retrovirus particles. The reaction was biphasic. There was a very rapid initial rate, followed, after approximately 1 h, by a lower rate. Many discrete bands of subgenomic-length (-) strands were produced after 10 and 20 min of synthesis; genome-length (7.7-kilobase [kb]) (-) strands were detected within 30 min. Extension to an 8.0-kb (-)-strand species was evident by 60 min. This extension was inhibited by actinomycin D. Synthesis of (+) strands (which is also inhibited by actinomycin D) began early, before any (-) strands were completed, and continued for more than 4 h beyond the time when synthesis of full-length DNA had terminated. Two distinct species of (+)strand DNA, 0.27 and 0.35 kb, could be observed at the earliest times. Their presence was quickly obscured by subsequent formation of (+)-strand molecules of molecular length between 0.2 and 2.0 kb.

mentioning

confidence: 99%

Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. I. Kinetics of synthesis and size of minus- and plus-strand transcripts

Boone¹,

Skalka²

1981

“…Free virus-specific DNA molecules are generally detectable before integration of provirus into the cell genome in the acute phase of retrovirus infection (2,6,16,17,29). Previous studies on Moloney murine leukemia virus (8,9,24,35), mouse mammary tumor virus (19), and Rous sarcoma virus (12,22,27,28) have demonstrated mainly two physical forms of free viral DNA: double-stranded linear DNA (form III) and double-stranded, covalently closed circular, supercoiled DNA (form I).…”

mentioning

confidence: 99%

Covalently closed circular DNAs of murine type C retrovirus: depressed formation in cells treated with cycloheximide early after infection

Yang

Kiggans

1980

Formation of viral closed circular supercoiled DNA duplexes and production of progeny virus were both inhibited in cultured mouse cells treated with cycloheximide in the first 4 h of type C retrovirus infection. With different doses of cycloheximide to cause different degrees of inhibition, the number of viral supercoiled DNA duplexes detected in the cells at 11 h showed an apparent correlation with the amount of progeny virus produced in the 12- to 22-h period of infection. A slight accumulation of the full-genome linear duplex and an open circular duplex of viral DNA intermediate was observed in the cycloheximide-treated cells. Cycloheximide given to the cells during the time of conversion of viral DNA from linear to supercoiled duplex forms (6 to 11 h after virus inoculation) did not inhibit the conversion. These kinetic data suggest that a cycloheximide-sensitive metabolic process, probably early viral protein synthesis, is required for retrovirus replication and supercoiled viral DNA formation in the cell.

“…For this purpose, we developed an efficient and sensitive procedure for following cytoplasmic viral DNA synthesis by its radioactive labeling. Our procedure differs in this respect from those used by several other investigators, who have preferred to isolate unlabeled viral DNA and to anneal it to radioactive viral probes (4,10,12,13,22,23). Since IF might exert its effect through inhibition of viral protein synthesis (8), we also examined directly the response of viral DNA synthesis to a known protein synthesis inhibitor such as cycloheximide (CH).…”

mentioning

confidence: 99%

“…The tritium counting efficiency, as determined by spotting a known amount of [3H]thymidine onto prewashed filters, is 21%. The dilution of [3H]thymidine in the intracellular pool can be assumed not to exceed two to threefold under the restricted growth conditions used here (11,13). Taking into consideration the counting efficiency, the [3H]thymidine specific activity, and its possible dilution in the intracellular pool, we roughly estimated that the number of viral DNA copies formed at the peak level was only 1 to 3 per cell.…”

mentioning

confidence: 99%

Cytoplasmic viral DNA synthesis in exogenous infection of murine leukemia virus: effect of interferon and cycloheximide

Aboud¹,

Moldovan-Levin²,

Salzberg³

1981

Cytoplasmic viral DNA synthesis can be followed efficiently by [3H]thymidine labeling of cells exogenously infected with Moloney murine leukemia virus. Both the negative and the positive strands of viral DNA reached their maximal level in the cytoplasm at 3.5 h postinfection. Interferon treatment before infection markedly reduced the amount of viral DNA formed during the first 3.5 h, but led to a second major wave of viral DNA synthesis, peaking at 7.5 h postinfection. No such late cytoplasmic DNA synthesis occurred in the untreated control. Inhibition of protein synthesis by cycloheximide, on the other hand, stimulated cytoplasmic viral DNA synthesis during the first 3.5 h.