Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis

Abstract

The small RNAs of Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis. The pattern of small RNAs is significantly different from that of Rous sarcoma virus. A subset of the virion small RNAs is associated with the genome RNA in the 70S complex. One of the associated molecules, a cellular tRNA, is tightly bound to the genome RNA and serves as the major primer for M-MuLV RNA-directed DNA synthesis in vitro. RNA tumor virus particles such as murine leukemia virus or Rous sarcoma virus (RSV) contain two major size classes of RNA, the high-molecular-weight genome RNA and lowmolecular-weight 4 to 7S RNA (1-3, 7, 8, 12, 19, 21, 25, 27). Some of the low-molecular-weight RNAs are associated with the genome RNA in a 70S complex, whereas others are released free in solution after disruption of virus particles (8, 12, 19, 21, 27). One of the 70S-associated 4S RNAs in RSV virions, the host cell tRNA'r , has been shown to be the primer for initiation of RSV RNA-directed DNA synthesis in vitro (6, 11, 14, 22). To determine whether other viruses use tRNA's as primers, we analyzed the small RNAs of Moloney murine leukemia virus (M-MuLV). One ofthe 70S-associated 4S molecules can serve as the major primer for reverse transcription in vitro. This RNA dissociates from the genome RNA at higher temperatures than do other associated species. An identical molecule is found in uninfected mouse, rat, and chicken cells, suggesting that the M-MuLV primer is a host cell RNA. In a later communication, we will show that this molecule has the amino acid acceptor activity and nucleotide sequence of tRNApro. MATERIALS AND METHODS Virus growth and isolation. The cloned line of M-MuLV designated clone 1 was propagated in a 3T3

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“…Cells were routinely propagated in Dulbecco-modified Eagle medium supplemented with 10% tryptose phosphate, 4% fetal calf serum, and 1% dimethyl sulfoxide. M-MuLV was grown in a cloned line of NIH mouse 3T3 producer cells as previously described (9,31). A line of transformed quail cells, d ,signated 16Q, was used as a source of the Bryan high-titer strain RSV (BH-RSV) (28).…”

“…After labeling for 24 h, the medium was collected and replaced with unlabeled medium containing 0.1 mM sodium phosphate for a further 24 h. In some experiments, this labeling regime was repeated, or alternatively cells were grown in medium containing 0.1 mM phosphate and 0.1 mCi of nPO4 per ml for up to four 24-h periods. Virus was harvested from the pooled culture fluids by high-speed centrifugation, and the viral RNA was prepared by standard procedures (31,37). Fractionation of viral RNA on sucrose gradients to prepare the 70S and free 4S RNA components, and subsequently 35S genome RNA and associated 4S RNAs, has been described elsewhere (31).…”

“…Virus was harvested from the pooled culture fluids by high-speed centrifugation, and the viral RNA was prepared by standard procedures (31,37). Fractionation of viral RNA on sucrose gradients to prepare the 70S and free 4S RNA components, and subsequently 35S genome RNA and associated 4S RNAs, has been described elsewhere (31).…”

“…Analysis of low-molecular-weight RNAs. The low-molecular-weight fractions of 3P-labeled RNA were analyzed by electrophoresis in two dimensions in polyacrylamide slab gels (2-D gels) (19,31,37). The first dimension was in 10% acrylamide (from top to bottom in all figures); the second dimension was in 20% acrylamide (from right to left in the figures).…”

“…The initiation of retrovirus DNA synthesis appears to be primed by one of the several cellular tRNA's found associated with the genome in the viral 70S RNA complex (6,10,31). This primer tRNA is bound to the genome RNA by a short region of base complementarity near the 5' end of the genome (5,8,30,42,46).…”