Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA

Abstract

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was terned "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After celLs acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplification to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transfonned, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.Harvey sarcoma virus (HSV) is a retrovirus that induces fibrosarcomas in infected rats and mice and causes morphological transformation of fibroblasts cultured in vitro. HSV originated through genetic recombination between a nonsarcomagenic retrovirus, Moloney murine leukemia virus (M-MLV), and rat cellular sequences (16,24). As Fig. 1 shows, HSV genomic RNA (size, 5.4 kilobases) contains a large internal rat cellular sequence, whereas the RNA 5' and 3' ends are derived from the 5' and 3' ends of genomic M-MLV RNA (5).The HSV genome replicates within an infected cell through DNA intermediates analogous to the DNA intermediates of other retroviruses. Shortly after infection, HSV RNA is reverse transcribed to generate a linear doublestranded DNA which is slightly longer (6.0 kilobase pairs [kbp]) than genomic RNA (11,20). As Fig. 1 shows, the 6-kbp DNA bears a terminal sequence repetition; nucleotide sequences det Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.on July 6, 2020 by guest

Generation of novel, biologically active Harvey sarcoma viruses via apparent illegitimate recombination

“…38, 1981 cells lacks the right end of the HSV provirus (corresponding to the 3' end of the viral RNA), we concluded that the transforming gene of HSV is located in the central or left-end portion of the HSV provirus (corresponding to the middle or 5' end of the viral RNA). We also concluded that the inefficient transmissibility of these deleted viral genomes was probably correlated with the absence of the right end of the provirus (16). Such a conclusion is consistent with the findings of others that indicate the vital role of 3'-proximal RNA sequences in several steps of viral DNA synthesis (13).…”

“…Polyadenylated cytoplasmic RNA was prepared from three transformed cell lines (clones Xl, 16-1, and 16-7), each of which carried a delHSV genome. In addition, as control reagents, we prepared RNAs from two transformants which bore complete HSV proviruses (clones 14 and 16) (16). The RNA samples were subjected to agarose gel electrophoresis in the presence of methylmercuric hydroxide, and the RNA contents of the gel were transferred and affixed to diazo-derivatized filter paper.…”

“…The RNA samples were subjected to agarose gel electrophoresis in the presence of methylmercuric hydroxide, and the RNA contents of the gel were transferred and affixed to diazo-derivatized filter paper. The filter paper was incubated with a 32P-labeled HSV cDNA sequence probe which had been depleted of sequences homologous to the M-MLV genome (16). Figure 1 shows that cells transformed by wildtype HSV contained a single prominent species of HSV-specific polyadenylated cytoplasmic RNA (lanes d and e), which was the same size as HSV 5.4-kilobase (kb) virion RNA (lanes g and h).…”

“…The recombinant pBR322 plasmid bearing the 3.2-kilobase pair (kbp) rat-specific HSV DNA fragment (see Fig. 2E) was derived by methods described in the accompanying article (16).…”

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

“…Thus, the number of new bands seen indicated directly the number of distinguishable copies of the A-MuLV genome introduced into the cells. The very high intensity of hybridization seen suggested that the new DNA was present at an amplified level of 5 to 10 copies per cell, as has been observed for other transfected DNAs (12). Cleavage with the enzyme XbaI suggested that these new A-MuLV DNAs were not the result of conventional viral infection.…”