Synthesis of Virus-Specific High-Mobility DNA after Temperature Upshift of SC-1 Cells Chronically Infected with Moloney Murine Leukemia Virus Mutant ts1

ts1 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (MoMuLV). Temperature sensitivity results from a single Val-25→Ile substitution in gPr80env (39). gPr80env is a precursor polyprotein for the envelope protein, Env. The mature Env protein consists of gp70, a surface protein, and p15E, a transmembrane protein. At the restrictive temperature, gPr80env is inefficiently transported from the rough endoplasmic reticulum to the Golgi apparatus (38, 39). As a result, there is an elevated steady-state level of misfolded gPr80env in the rough endoplasmic reticulum (39), and gp70-deficient virions are released from infected cells (44). Infection is initiated by attachment of the viral Env protein (45) to the host cell receptor protein MCAT-1 (1, 35). Previously, we showed that the tissues of the central nervous system (CNS) of moribund paralyzed FVB/N mice contained unusually high levels of persistent incomplete physical forms of proviral DNA (36, 37). Due to their migration in agarose gels, these physical forms of DNA were designated high-mobility (HM) DNA. The two major persistent physical forms of retroviral DNA that were found in infected tissues of the CNS consisted of class I HM DNA, minus-sense single-stranded DNA that was shorter than one long terminal repeat (LTR), and class II HM DNA, partial DNA duplexes (Fig. ​(Fig.1A).1A). Except for that of unintegrated linear proviral DNA, form III, the functions of extrachromosomal physical forms of retroviral DNA are not clear (for a review, see reference 9). Form III is the physical form of proviral DNA that integrates into the genome of the host cell (14). In DNA-dependent protein kinase (DNA-PK)-deficient murine scid cells, abortive integration of form III is the trigger for cell death by apoptosis (12). HM DNA is physically similar to damaged DNA. It is possible that high levels of HM DNA are toxic to some types of neural cells in the CNS. FIG. 1 Physical forms of ts1 virus-specific DNA in the spinal cord tissues of paralyzed moribund mice (37). (A) Mobilities of physical forms of unintegrated ts1 virus-specific DNA in agarose gels. The minor free plus-sense single-stranded DNA physical form is ... Replication intermediates of the proviral DNA of retroviruses have previously been reported for reactions in detergent-disrupted virions in vitro (17), in membrane vesicles exposed to virions in vitro (34), and for the early stages of cell-free virus infections of cells in cultures (42). In cell cultures and membrane vesicles, the replication intermediates rapidly mature to the full-length proviral DNA form within a few hours (34, 42). The persistent physical forms of DNA that we described for the CNS tissues of moribund paralyzed mice were unique because high levels persisted for weeks after the initial stages of infection. To date, only low levels of persistent replication intermediates of the proviral DNA of MoMuLV have been detected in quiescent cells in vitro by the PCR (18, 28). Some types of quiescent cells contain suboptimal levels of deoxynucleoside triphosphates (dNTPs) for reverse transcription (5, 15, 25). However, high concentrations of exogenous deoxynucleosides in the culture medium of some types of nondividing cells promote the completion of reverse transcription of MoMuLV genomic RNA into full-length proviral DNA (18, 28). In order to better understand the mechanism of HM DNA synthesis in the CNS in vivo, we developed a method for synthesizing high levels of HM DNA in vitro in cultured cells. HM DNA was synthesized in packed confluent monolayers of SC-1 cells that were chronically infected with ts1. When subconfluent monolayers of chronically infected cells were upshifted from 34 to 37°C, persistent levels of HM DNA were synthesized after the monolayers became confluent. Without temperature upshift, the confluent monolayers of chronically infected cells did not synthesize HM DNA. However, when the packed confluent upshifted cells were downshifted from 37 to 34°C, the incomplete physical forms of HM DNA were extended into full-length physical forms of proviral DNA. These in vitro results suggested that the accumulation of HM DNA both in vitro and in vivo may be the result of superinfection.