An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

Abstract

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39°C) during the early part of the virus cycle (between 0-to 20-h postinfection) greatly inhibited virus production. This The replication of the RNA genome of retroviruses by reverse transcriptase is a complex process. Linear doublestranded DNA molecules with segmented (+) strand are first synthesized with a redundant sequence at each end, the long terminal repeats (LTR) (for review, see reference 36). LTR are composed of a sequence derived from the 3' end and another one from the 5' end of the RNA genome. The linear DNA molecules mature as nonsegmented, fully doublestranded molecules and appear to be converted in the nucleus into two species of covalently closed circular viral DNAs (20, 32), one shorter species harboring one LTR copy and the larger species harboring two LTR copies (31, 42). Several of these steps seem to be carried out by reverse transcriptase alone or possibly in combination with other virion proteins. Indeed, infectious double-stranded linear viral DNA, as well as circular viral DNA with one LTR copy can be synthesized in vitro by purified virions (1,2,5,6,10). This in vitro reaction, however, does not yield closed circular viral DNA with two LTR copies. Most importantly, the infectivity of viral DNA made in vitro is several orders of magnitude lower than that of viral DNA made early during infection (10,23,28). This suggests that cell factors are required during the reverse transcription process. Other experiments also indicate that the cell provides essential * Corresponding author.factors during some steps of reverse transcription. Serum starvation has been shown to markedly affect the production of unintegrated viral DNA in newly infected avian (7,8,37) or murine (14) cells. Cycloheximide added early after infection prevented the formation of both species of supercoiled viral DNA, suggesting that newly synthesized proteins are required to complete this step (41). The treatment of newly infected murine (4, 13) or avian (16) cells with aphidicolin, an inhibitor of cellular DNA polymerase a (17) and 5 (24), also prevented the formation of supercoiled viral DNA, suggesting that cellular DNA polymerases may be involved at this step of the virus cycle.Although very suggestive, these experiments do not permit one to distinguish between a number of factors possibly affected by the inhibitors. Also, it is not clear whether the effect of these treatments is directly on retroviral DNA synthesis or only indirectly influencing it. It would therefore be helpful to identify the cellular factors required in the reverse transcription process by using cell mutan...

Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes

“…35,[65][66][67][68][69] Tagged ecotropic and amphotropic murine leukemia virus (MLV) and GaLV envelope proteins have been used to demonstrate that retrovirus binding to target cells depends on the receptor density and envelope binding. [65][66][67][68] Analysis of a cell line with an inducible GALVR gene showed that a 4-to 5-fold increase in GALVR mRNA led to a 150-fold increase in the frequency of GALV transduction.…”

Evidence that retroviruses integrate into post-replication host DNA.

“…The Temin, 1982;Hsu and Taylor, 1982;Richter et al, 1984;Springett et al, 1989;Miller et al, 1990), although this is not true of all cells and all retroviruses (Lewis et al, 1992). Several studies have indicated that S phase is critical for integration (Harel et al, 1981;Humphries et al, 1981;Hsu and Taylor, 1982;Springett et al, 1989;Miller et al, 1990), although precisely what takes place during this phase is not clear.…”

Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.

“…Previous studies have shown that cellular DNA replication is required for integration of murine leukemia virus DNA into the host genome of mammalian cells (14,15). However, 97% of purified mouse stem cells are in the Go or G1 phases of the cell cycle (16).…”