gag-Related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms

Abstract

Immunoprecipitation of labeled extracts from murine leukemia virus-infected cells with antisera specific for internal structural (gag) proteins yields three major gag-related polyproteins with molecular weights of 180,000 (Pr180gag"P"), 80,000, and 65,000 (Pr65ag. It has been shown by others that Pr65'ag is the immediate precursor of the internal structural (gag) protein, and that Pr1809'w-l°i s the precursor to reverse transcriptase. In studies reported here, the 80,000dalton gag-related polyprotein from Moloney strain murine leukemia virus (M-MuLV)-infected cells was found to be glycosylated by the following criteria: (i) incorporation of [3H]mannose, (ii) a change in electrophoretic mobility upon digestion with endoglycosidase H, and (iii) a change in electrophoretic mobility when glycosylation was inhibited by treatment of the cells with tunicamycin during labeling. The 80,000-dalton gag polyprotein has therefore been designated GpP80'"9. The unglycosylated form of GpP80'ga was a polypeptide of 75,000 daltons. A comparison of [3H]mannose and [3H]galactose labeling experiments suggested that GpP809ga is further glycosylated to yield a glycopolypeptide of 95,000 daltons. This 95,000-dalton polypeptide is relatively rapidly cleaved to yield two glycopeptides of 55,000 and 40,000 daltons which are released into the cell culture fluid, as soluble proteins. Cell-free translation of M-MuLV genomic RNA resulted in two major gag-related products of 75,000 and 65,000 daltons. The 65,000-dalton gag-related cell-free translation product comigrated with Pr65'qg, and the 75,000-dalton cell-free product comigrated with the unglycosylated form of GpP809'9. Both of the gag-related cell-free translation products could be labeled with [3S]formyl methionine, which is incorporated only as the N-terminal amino acid during translation. Other investigators have shown that GpP809'9 and Pr659'9 differ at their N-termini, and these results combined with those reported here suggest that GpP809(l and Pr65gag are translated from two separate initiation sites in M-MuLV RNA. The virion proteins of RNA tumor viruses are produced in infected cells by cleavage of three polyprotein precursors (reviewed in 4, 7). All of the internal structural proteins, products of the gag gene, are initially present in a single precursor polyprotein which ranges in size from 60,000 to 80,000 daltons, and the individual internal structural proteins are produced by a series of proteolytic cleavages (12, 21, 23, 30, 37, 38). Viral reverse transcriptase, the product of the pol gene, is initially synthesized as part of a large 180,000-dalton polypeptide which also contains most, if not all, of the gag polypeptides (Prl80"g"w°") (12, 15). The envelope proteins, products of the env gene, are initially synthesized from subgenomic length mRNA (10, 11, 31) as a glycosylated polyprotein of approximately 80,000 to 90,000 daltons (6, 18, 22, 30, 39). In murine leukemia virus (MuLV)-infected cells, two major gag-related polyproteins are observed by pulse labeling: a polypept...

A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB

“…4, odd-numbered lanes) was hydrolyzed by endo H to an unglycosylated core polypeptide (Fig. 4, even-numbered lanes) with an increased electrophoretic mobility and an Mr of 63,000 (7), and endo H-resistant gPr8OenV was not detected (Fig. 4, even-numbered lanes).…”

Identification of diverse full-length endogenous betaretroviruses in megabats and microbats

“…This ORF begins within the 5′ LTR and terminates three nucleotides upstream of the Gag start methionine, within the same reading frame. ORFs upstream of Gag may be relevant to Gag expression considering that murine gammaretroviruses encode an alternative N-terminally extended version of Gag, glyco-gag, that has a role in the promotion of viral replication [39,40]. No promoter elements or TATA boxes were predicted to exist upstream of the ORF, however a TATA box is predicted within the ORF coupled with a possible start methionine downstream, encoding a potential 84 amino acid protein.…”

The secretory core protein of human hepatitis B virus is expressed on the cell surface

“…DISCUSSION From the results described above, we conclude that the HBeAg is an unusual secretory protein, since it is not only secreted from HBeAg-producing cells but also incorporated into the outer cell membrane. To our knowledge, to date there is only one other example of a viral nucleocapsid protein which also exists in a membrane-bound and a secreted form, namely, the gag protein of murine leukemia viruses (9,17,24). How the HBeAg reaches the cell membrane is unknown.…”