Alan M. Schultz

Different recombinant murine leukemia viruses use different cell surface receptors.

Retroviruses can be grouped by viral interference measurements into classes which use common cell surface receptors. We previously tested a large number of isolates of mink cell focus-inducing (MCF) murine leukemia viruses (MuLVs), and reported that all of them share a distinct receptor on NIH/3T3 cells (A. Rein, Virology 120, 251, 1982). We now extend this generalization to several additional recombinant isolates, including two (SL3-2 and GPA-V2) which would not be considered MCFs on the basis of host-range data. We note the superiority of interference tests, based on positive, unambiguous data, over host-range tests for virus classification. We also show that in contrast to the MCFs, which are all derived from ecotropic MuLVs, a recombinant derived from wild mouse amphotropic MuLV (S. Rasheed et al., Int. J. Cancer 29, 345, 1982) uses a unique receptor on NIH/3T3 cells. This suggests that (a) mouse cells contain more than one type of endogenous env sequence; and (b) there is some specificity in the generation of recombinants, since ecotropic MuLVs appear to give rise only to MCFs, while amphotropic MuLV has generated a distinct type of recombinant. It also represents a second case (in addition to the MCFs) in which an env gene recombinant is more pathogenic than its exogenous parent. We also show that xenotropic MuLV does not interfere with MCFs in NZB mouse cells; thus, despite the close homology between MCF and xenotropic env sequences, the gp70 of xenotropic MuLV appears to have no detectable affinity for the MCF receptor.

Hydroxylamine-stable covalent linkage of myristic acid in Goα, a guanine nucleotide-binding protein of bovine brain

G0 alpha, a guanine nucleotide-binding protein with a strong homology to the G1 alpha and Gs alpha regulatory proteins of adenylate cyclase, is shown to contain myristic acid. The attachment of myristate to the protein is stable to hydroxylamine treatment, and since the amino-terminal sequence of G0 alpha is typical of proteins with amino-terminal myristate, the inference is strong that G0 alpha is also myristylated at its amino-terminal glycine.

Maturation of murine leukemia virus env proteins in the absence of other viral proteins

A mutant of Akv which produces env but no detectable gag or pol products (A. Rein, D. R. Lowy, B. I. Gerwin, S. K. Ruscetti, and R. H. Bassin, J. Virol. 41, 626-634 (1982] was examined for maturation of env gene protein products. In comparison with wild-type Akv, the mutant AK 71 synthesizes gPr85env and produces gp70 and Prp15E in normal amounts and with normal kinetics. Cell surface gp70 was found alike in the mutant and wild type. However, cleavage of Pr15E to p15E did not occur in the mutant. This final cleavage step of AK 71 Prp15E could be made to occur by superinfecting cells containing the mutant with baboon endogenous virus. Thus, unlike earlier steps, this final step in maturation of the env gene product appears to require gag or pol gene products. It is proposed that the virus-encoded protease is required for this last step.

Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of [3H]myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chromatography to be [3H]myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid (A. Schultz and S. Oroszlan, J. Virol. 46, 355-361). It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins (L. E. Henderson, H. C. Krutzsch, and S. Oroszlan, Proc. Natl. Acad. Sci. USA 80, 339-343). The possible role of myristylation of transforming proteins is discussed.

Inhibitors of glycosylation reverse retroviral interference

Abstract Cells which are productively infected with retroviruses are extremely resistant to superinfection with the homologous virus. We found that in certain cases, this resistance is largely or entirely eliminated by overnight treatment of the cells with either of two inhibitors of glycosylation (2-deoxyglucose or tunicamycin). This loss of viral interference was accompanied by a loss of gp70 from the cell surface, as demonstrated by surface labeling with 125 I and radioimmunoprecipitation. In other cell-virus combinations, these inhibitors gave much smaller reductions in viral interference and in the level of gp70 at the cell surface. The most likely explanation for these phenomena is that inhibition of glycosylation of the env precursor polyprotein prevents its subsequent processing into gp70 and transport to the cell surface; to the extent that the cellular pool of gp70 is depleted during an overnight treatment, cellular receptors become available for interaction with superinfecting virus particles.

Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera☆

The post-translational modifications of the gag-raf fusion proteins of the 3611 murine sarcoma virus (MSV) have been examined by inhibiting glycosylation with tunicamycin and by in vivo labeling with [3H]myristic acid. The results show that P75gag-raf is myristylated but not glycosylated and that P90gag-raf is glycosylated but not myristylated (and is now termed gP90gag-raf). gP90gag-raf expression appeared to become lost during passage of the transformed cells, and consequently does not appear to be necessary for the maintenance of transformation. raf-specific sera for detecting gag-raf fusion proteins have been obtained from synthetic peptides made from different regions of the predicted v-raf sequence. Immunoprecipitation of P75gag-raf with raf-specific sera directly confirmed the deduced v-raf sequence. The fact that P75gag-raf is both myristylated and precipitated by antiserum to a predicted carboxyl-terminal peptide of the v-raf gene established that the mature protein represents the entire coding region. The gP90gag-raf thus appears to be a glycosylated form of P75gag-raf specified by the gag sequences of the fusion protein, in analogy with Pr65gag and gPr80gag of murine leukemia viruses. Antiserum to the carboxyl-terminal P75gag-raf peptide was the most efficient in immunoprecipitation, and will be useful for detecting the product of the c-raf gene.

Tunicamycin inhibits glycosylation of precursor polyprotein encoded by env gene of Rauscher murine leukemia virus

Abstract The molecular weight of the precursor polyprotein to the envelope proteins of Rauscher murine leukemia virus is reduced from 85,000 to 68,000 daltons when synthesized in the presence of tunicamycin, a specific inhibitor of the synthesis of oligosaccharides that attach to glycoproteins via asparagine residues. The unglycosylated precursor protein (Pr68 env ) is synthesized at a rate comparable to that of the normal carbohydrate-containing envelope precursor (gPr85 env ). Pr68 env is not proteolytically processed and remains undegraded in the cell. Thus, most if not all of the carbohydrate content of gPr85 env is N-linked, and glycosylation appears to be necessary for normal processing of precursor proteins into viral particles.

Murine leukemia virus gag polyproteins: The peptide chain unique to Pr80 is located at the amino terminus

Abstract The gag gene-encoded precursor polyproteins Pr80 gag and Pr65 gag of Rauscher leukemia virus were analyzed by chemical fragmentation followed by immune precipitation with antisera specific to viral structural proteins and one-dimensional peptide mapping. Peptides of similar antigenic determinants and size were obtained from the carboxyl-terminal region of the two polyproteins. Precipitation of cleavage products with antiserum known to react with the amino-terminal region of Pr65 gag resulted in the appearance of two distinct fragments: an ∼33,000-dalton peptide generated from Pr80 gag and an ∼18− to 20,000-dalton fragment generated from Pr65 gag . The data provide evidence that the unique peptide chain (13–15,000 daltons) which distinguishes Pr80 gag is located at the amino terminus and that the carboxyl ends of these two polyproteins may be identical.

Preparation of raf-oncogene-specific antiserum with raf protein produced in E. coli

In this report we describe the expression of v-raf protein in E. coli using a tryptophan-promoter-driven expression vector and its immunological characterization by anti-peptide sera. The purified recombinant protein was used to produce raf-specific antibodies which are suitable for studying v-raf and c-raf proteins in vitro and in vivo in a variety of species ranging from mouse to man.

The env-gene of the spleen focus-forming virus lacks expression of p15(E) determinants☆

Abstract Experiments utilizing NRK cells nonproductively infected with the spleen focus-forming virus (SFFV) and immunoprecipitation of the metabolically radiolabeled proteins followed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis clearly established that the gp52 enr of SFFV, which shares antigenic determinants of gp70, lacks all major antigenic determinants of p15(E) of helper-independent viruses. In the presence of tunicamycin, a nonglycosylated apoprotein of approximately 46,000 daltons was synthesized. An apparently unglycosylated protein of similar size was also obtained after removal of carbohydrate from gp52 env with endoglycosidase H. These results suggest that gp52 env is glycosylated to a lesser extent than gp70 of helper-independent mouse retroviruses.