VIRAL RNA EXPRESSION IN FRIEND VIRUS-INDUCED ERYTHROLEUKEMIA CELL LINES

Abstract

Friend erythroleukemia virus (FV) consists of a complex of two distinct viruses, both classified as retroviruses because they contain RNA-dependent DNA polymerase (reverse transcriptase). The two viral components of FV are Friend murine leukemia virus (FMuLV) and spleen focus forming virus (SFFV). FMuLV is a replication-competent virus, whereas SFFV is defective for replication and requires a helper virus such as FMuLV to replicate in infected cells. SFFV most probably arose as a recombinant of the FMuLV genome and that of an endogenous xenotropic virus.;Tumors induced in vivo by FV can be adapted to growth in culture. Several cell lines of this sort have been established from tumors induced in H-2-congenic mice. These cell lines invariably produced infectious FV in early passage generations, but eventually ceased FV production upon continuous growth in culture. In one nonproducer cell line even the expression of the viral envelope antigen (VEA, found on the surfaces of tumor cells) was no longer detectable after about two years of continuous passage in culture. The tumorigenicity of these cells upon grafting into syngeneic hosts was not reduced, however. Nonproducer cells still maintained the entire FV genome, as treatment with halogenated pyrimidines induced production of infectious FV.;To study the mechanism of virus shutdown in erythroleukemia cells, complementary DNA (cDNA) probes were generated representing SFFV- and FMuLV-specific sequences as well as one specific for sequences shared by the two viruses. Molecular hybridization with cytoplasmic and nuclear RNA extracted from clones of the H-2-congenic cell lines representing various stages of viral expression showed a direct correlation between the level of transcription of FMuLV-specific sequences and the degree of virus expression. SFFV-encoded sequences, on the other hand, were transcribed in both producer and nonproducer cell clones including those which no longer expressed VEA on their surfaces.;The molecular hybridization experiments described herein revealed or confirmed the following: (1) the restriction of FV expression in erythroleukemia cell lines is primarly related to the cessation of transcription (and not post-transcriptional modification) of FMuLV-specific sequences; (2) most or all of the SFFV genome, including those sequences shared with FMuLV, continues to be transcribed regardless of the level of virus expression; (3) the genes necessary for viral replication are located in the FMuLV-specific region, and those governing maintenance of transformation are located in the SFFV-encoded region; (4) restriction of FMuLV expression occurs through a stepwise inhibition of transcription instead of an abrupt all-or-none phenomenon; and (5) producer cell lines maintain up to thirty times as much viral RNA as nonproducers, most of which may be destined to be packaged into new viral particles.