Identification of the messenger RNAs coding for the gag and env gene products of the murine mammary tumor virus

Abstract

Full-length (35S) genomic RNA from murine mammary tumor virus (MuMTV) was translated in vitro, using a reticulocyte lysate system, into proteins of 105,000, 75,000, 65,000, 35,000, and 27,000 daltons. These proteins were all immunoprecipitable with a monospecific antiserum to the major viral core protein, p27, but not with antiserum to the major viral envelope glycoprotein, gp47. In the case of type C viruses, it has also been established that viral 35S genomic RNA contains information for the synthesis of the major proteins constituting the virus, namely, the core proteins, the glycoproteins, and the reverse transcriptase (5). The avian sarcoma viral RNA also codes for a src protein responsible for the transformation of fibroblasts in vitro and for the induction of sarcoma in vivo. However, when the full-size virion RNA or intracellular viral specific RNA of similar size is translated in an exogenous Xenopus oocyte system or an in vitro cell-free protein-synthesizing system, only the gag proteins and a gag-pol polyprotein are synthesized. It has been reported that virion RNA of smaller size classes can be translated into the src protein product (5). The env proteins are translated only from a virus-specific intracellular mRNA of about 21 S in size. For MuMTV no information is available regarding the identity of the mRNAs for different viral proteins.We report here the translation in vitro of MuMTV virion RNAs. By hybridization with a representative MuMTV cDNA probe, we have identified at least three size classes of MuMTV-specific intracellular RNAs. Intracellular RNA of 35S size class was translated into MuMTV core proteins, while 24S size class RNA was translated into MuMTV envelope proteins. MATERIALS AND METHODSCell Culture. MuMTV-producing GR cells (8) were grown in monolayer culture in Eagle's minimal essential medium supplemented with 10% calf serum. Confluent cultures were treated with dexamethasone at 1 /ig/ml for the production of MuMTV. The cells were grown in the presence of [3H]uridine at 50 ,uCi/ml (1 Ci = 3.7 X 1010 becquerels) for preparing RNA-labeled MuMTV.Virus Purification. Virus was purified from the culture fluid by using the following method. The culture fluid was first clarified by centrifugation at 10,000 X g for 10 min. The supernatant was then layered on a cushion of 60% sucrose in TNE buffer (10 mM Tris-HCI, pH 7.5/100 mM NaCl/1 mM EDTA) and centrifuged at 100,000 X g for 90 min. The virus layer at the top of the cushion was removed and layered onto a preformed linear gradient of 25% to 60% sucrose in TNE buffer. This was centrifuged at 100,000 X g for 3 hr. The virus banding at a density of 1.17 g/ml was removed and stored at 0C.Isolation of Viral and Cellular RNAs. RNA was extracted from the virus by incubation with self-digested Pronase and phenol/chloroform extraction (9). Total cytoplasmic RNA was extracted from cells that had been treated with dexamethasone for 18 hr, following published procedures (10). Poly(A)-containing RNA was selected from this total RNA by two cons...

Effect of hormones on the expression of proviral genes Mtv‐2 and Mtv‐3 in mouse mammary gland

“…The gag proteins are synthesized from a 35s mRNA, initially as a precursor polypeptide of 73,000 dalton (Schochetman et al, 1978). A 24s mRNA instructs synthesis of env proteins, also via a precursor polypeptide of 73,000 dalton (Sen et al, 1979). It is therefore possible that in the Mtv-3 proviral sequence the env genes are absent, or alternatively that they are present but that their expression is blocked or changed either at the transcriptional or at the translational level.…”

Sequence and expression of the mouse mammary tumour virus env gene.

“…The open reading frame (orf] contained within the LTR (Donehower et al, 1981;Dickson and Peters, 1981;Fasel et al, 1982) (Sen et al, 1979;Groner et al, 1979;Dickson and Peters, 1981;Robertson and Varmus, 1981;Dudley and Varmus, 1981). The 24S mRNA is believed to contain 200-300 bases derived from the 5' end of the provirus which is spliced to a coding region located in the 3' half of the provirus (Majors, 1982).…”

“…Circumstantial evidence suggests that the second or third methionine codon is the most likely start. This is partly based on the estimated size of the primary protein products from the three possible initiator methiones, which are 77 132, 73 159 and 71 822 daltons, respectively, in comparison with the size of the in vitro translated product of env mnRNA, estimated on SDS-polyacrylamide gels to be 68 000 -70 000 daltons (Sen et al, 1979;Dudley and Varmus, 1981). This calculation would favour the third methionine.…”

Localization of a gene for expression of mouse mammary tumor virus antigens in the GR/Mtv-2- mouse strain.

“…The core, or gag, proteins are synthesized from a 35S messenger RNA (mRNA), initially as a precursor polypeptide of 73,000 dalton (16,19). A 24S mRNA instructs synthesis of envelope, or env, proteins, also via a precursor polyprotein of 73,000 dalton (20). To measure the expression of individual representative genes of MMTV in GR/Mtv-2-mice, two proteins from the env gene, gp52 and gp36, and two proteins from the gag gene, p27 and p l0, were used as tracer antigens in the radioimmunoassay (RIA).…”