Addition of extra DNA sequences to simian virus 40 DNA in vivo

K Yoshike, V Defendi
1977 Journal of Virology  
The possible addition of extra sequences to simian virus 40 (SV40) DNA was analyzed by electron microscopy in two different cell systems, productively infected monkey cells and activated heterokaryons of monkey and transformed mouse 3T3 cells. We found that the closed circular DNA fraction, extracted from monkey cells at 70 h after infection with nondefective SV40 at a multiplicity of infection of 6 PFU/cell, contained oversized molecules (1.1 to 2.0 fractional lengths of SV40 DNA) constituting
more » ... 0 DNA) constituting about 8% of the molecules having lengths equal to or shorter than SV40 dimer DNA. The oversized molecules had the entire SV40 sequences. The added DNA was heterogeneous in length. The sites of addition were not specific with reference to the EcoRI site. These results suggest that recombination between monkey and SV40 DNAs or partial duplication of SV40 DNA occurs at many sites on the SV40 chromosome. The integrated SV40 DNA is excised and replicates in activated heterokaryons. In this system, besides SV40 DNA we found heterogeneous undersized and oversized molecules containing SV40 sequences in the closed circular DNA population. Additions differing in size appeared to be overlapping and to have occurred at a preferential site on the SV40 chromosome. These results support the hypothesis that host DNA can be added to SV40 DNA at the site of integration at the time of excision. Since simian virus 40 (SV40) DNA in transformed cells has been shown to be linked covalently to cell DNA (11), the integration of SV40 DNA into cell DNA, which occurs in both permissive and nonpermissive systems (4, 5), appears to be one of the essential early events for stable transformation of infected cells. The simplest model for the integration would be that the linear insertion of the entire circular SV40 DNA into cell DNA results from the recombination of the two DNAs by a single crossover (1). From this model it is predicted that the SV40 provirus is excised from cell DNA by the reverse process, or circularization of viral DNA at the site of integration. This model is consistent with the fact that the SV40 provirus is induced to initiate production of infectious progeny virus when the transformed cells are fused with SV40-susceptible monkey cells (8, 16). It is also predicted that recombinant molecules containing both virus and cell DNA sequences are occasionally generated, possibly by an abnormal circularization of the provirus at the time of excision. SV40 .DNA from defective virions produced and accumulated during serial undiluted pas-1 Present address: Department of Enteroviruses, National Tngtitute of Health, Kamiosaki, Shinagawa-ku, To-Kyo 141, Japan. sages in monkey cells (14, 17) has been shown to contain host DNA sequences (9). The types of defective DNA that may contain the cell DNA sequences are substitution molecules (12) and insertion molecules having a deletion at a different site (19). Whether both or only one of the two types are recombinant molecules is unclear. Defective DNA molecules isolated from virions do not necessarily represent those initially formed because selection of viral DNA may occur during encapsidation. Thus, it is possible that an abnormal circularization of provirus results in the formation of a variety of substitution and addition molecules, but only those having appropriate lengths can be encapsidated into mature virions and replicate during the subsequent virus propagation. In the present study we examined by electron microscopy the heterogeneity in size and base sequence of free (nonencapsidated) intracellular closed circular DNA molecules produced in two systems, productively infected monkey cells (in which SV40 DNA may be integrated and excised) and heterokaryons produced by the fusion of SV40-transformed and permissive cells (in which SV40 provirus is excised and replicated). We found that extra DNA sequences can be added to SV40 DNA in the two systems. 323 on May 7, 2020 by guest
doi:10.1128/jvi.23.2.323-337.1977 fatcat:mqqrdfxqz5botcjc6emkcnhb5u