Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA

Abstract

We re-examined the generally accepted concept that replication of Rous sarcoma virus (RSV) requires host DNA synthesis. We used terminally differentiated chicken myotubes as the host because chromosomal DNA replication is completely abolished by the natural differentiation process. Southern blot analysis detected unintegrated viral DNA in both the nucleus and cytoplasm of infected myotubes. This indicated that reverse transcription of the infecting viral RNA and transport of the newly synthesized viral DNA into the nucleus proceeded normally in myotubes. However, restriction enzyme digestion of high Mr DNA prepared from infected myotubes produced none of the fragments specific for RSV, indicating that the viral DNA had failed to integrate into the myotube chromosomal DNA. In these infected myotubes, viral RNA was detected by in situ hybridization. Northern blot analysis showed the presence of all three RSV mRNAs (38S, 28S and 21S). The amount of these viral RNAs in infected myotubes was comparable with that found in infected fibroblasts. We conclude that host DNA synthesis is required for RSV integration, but, in contrast to the generally accepted concept, viral DNA integration is not an absolute requirement for transcription of the RSV genome.

Infection of terminally differentiated myotubes with Rous sarcoma virus: reduced synthesis of env and v-src proteins

“…This finding is somewhat surprising in view of the fact that all species of viral mRNA are present in late-infected myotubes (Tanaka et al, 1992). It is unlikely that these gene products are translated from the parental virion RSV RNA because the amount of viral gag proteins produced in late-infected myotube cultures was comparable with that in infected CEFs.…”

“…Therefore, it is reasonable to regard the radiolabelled viral proteins in our experiments as the result of synthesis rather than a combination of synthesis and degradation. The mechanism of reduced synthesis of env and v-src proteins must involve translational control because a normal ratio of viral mRNAs had been observed in lateinfected myotubes (Tanaka et al, 1992). Examples of such translational control have been reported in muscle cells (Endo & Nadal-Ginard, 1987;Gross & Merrill, 1989) and in maize cells (Skadsen & Scandalios, 1987).…”

“…Early-and late-infected myotube cultures were prepared as described previously (Tanaka et aL, 1992). Three types of ceils were used as controls: conventional CEFs prepared from the same embryos used for myotube culture; myogenic fibroblasts (MFs), which were mononucleated cells recovered from the supernatant of a trypsinized suspension of a 5-day-old myotube culture [without cytosine arabinoside (Ara-C) treatment]; Ara-C-treated MFs, (CMFs), which were prepared similarly to MFs except that the myotube culture was treated with Ara-C for 2 days followed by 4 days of culturing without Ara-C to allow the growth of cells which survived the Ara-C treatment.…”

“…2d, e and f ) because the expression of the v-src protein would have caused a morphological deterioration in myotubes (Holtzer et al, 1975;pathway IV in Fig. 1 of Tanaka et al, 1992). In this experiment, the negative controls were infected CEFs (Fig.…”

“…In the accompanying paper (Tanaka et al, 1992), we have shown that RSV can infect terminally differentiated myotubes ('late-infected' myotubes). These lateinfected myotubes contain as much viral DNA as t Present address: University of California, San Diego, Medical Center, 225 Dickinson Street, H-814-H, San Diego, California, 92103-1990, U.S.A. Present address: Department of Microbiology, Molecular Genetics and Immunology, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas 66103, U.S.A.…”