Terry D. Copeland

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.

Amino- and carboxyl-terminal sequence of Moloney murine leukemia virus reverse transcriptase.

The NH2-terminal amino acid sequence of Moloney murine leukemia virus reverse transcriptase was determined to be Thr-Leu-Asn-Ile-Glu-Asp-Glu-Tyr-Arg-Leu-His-Glu-. The comparison of the amino acid analysis data obtained after carboxypeptidase Y digestion with the published nucleotide sequence (T. M. Shinnick, R. A. Lerner, and J. G. Sutcliffe, Nature (London) 293, 543-548, 1981) led to the conclusion that the COOH-terminus is Leu coded by CTC in nucleotide positions 4608-4610, and the tentative COOH-terminal sequence is Pro-Asp-Thr-Ser-Thr-Leu-Leu-OH. In light of these and previously reported results the complexity and map order of the pol gene are discussed.

Detection and characterization of the protein encoded by the v-rel oncogene.

To identify the protein encoded by v-rel, the oncogene of reticuloendotheliosis virus (REV-T), antisera have been raised to three synthetic peptides derived from the translation of our previously published v-rel DNA sequence [R.M. Stephens, N.R. Rice, R.R. Hiebsch, H.R. Bose, Jr., and R.V. Gilden, Proc. Natl. Acad. Sci. USA 80, 6229-6233 (1983)]. Sera to all three peptides precipitate a 59,000 Da protein from REV-T-transformed chicken lymphoid cells. This protein is not detectable in uninfected chick embryo fibroblasts, and its observed size is in good agreement with the 56,000 Da predicted by the DNA sequence. We conclude that this protein is the v-rel product and designate it p59rel. To search for evidence of post-translational processing of this protein, cells were grown in the presence of glycosylation inhibitors. These resulted in no detectable difference in the size of p59rel. Nor was its size detectably altered during the course of a pulse-chase experiment. Growth of cells in the presence of [32P] orthophosphate, however, revealed that p59rel is a phosphoprotein. It is also closely associated with a protein kinase activity, for precipitation with one of the peptide antisera (but not the other two) resulted in strong kinase activity in the immune complex pellet. During this reaction, p59rel itself becomes phosphorylated. Kinase activity was retained in the immune complex following detergent and high salt washes, leaving open the possibility that p59rel is itself a kinase.

Purification and chemical and immunological characterization of avian reticuloendotheliosis virus gag-gene-encoded structural proteins.

Five gag-gene-encoded structural proteins, designated p12, pp18, pp20, p30, and p10 were purified from replication-competent avian reticuloendotheliosis-associated virus (REV-A) by high-performance liquid chromatography complemented with chloroform-methanol extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on amino acid composition and NH2- and COOH-terminal sequence analysis p12, pp18, p30, and p10 are distinct from one another, whereas pp20 is likely identical to pp18 in primary structure. The p12 was resistant to Edman degradation and was found to be myristylated at the NH2-terminal amino group. Sequence comparisons among the retrovirus family show that pp18/pp20 and p10 are, respectively, homologs of phospho-proteins and nucleic acid-binding proteins. A comparison of terminal sequences with the nucleotide sequence of spleen necrosis virus (SNV) revealed that the gag genes of SNV and REV-A are highly conserved; together with the identification of REV-A gag-precursor polyprotein, Pr60gag in immunoprecipitates of radiolabeled cell lysates, this comparison also led to the establishment of the organization of Pr60gag, viz., NH2-p12-pp18-p30-p10-OH. Sequence comparisons show that REV-A/SNV is related to mammalian type C viruses: the pp18-p30 region is most homologous to the macaque/colobus group and least to simian sarcoma virus (SSV), whereas both the 5- and 3-gag regions (i.e., p12 and p10) are clostest to SSV. Immunological studies using monospecific antisera and Western-blot analysis showed that antigenic determinants of REV-A p30 are conserved in most of mammalian type C and type D viruses, but those of REV-A p12 are shared only with simian sarcoma-associated virus (SSAV) and endogenous viruses of macaques.

PROCESSING AND STRUCTURE OF MURINE LEUKEMIA VIRUS gag AND env GENE ENCODED POLYPROTEINS

Partial primary structures of the gag and env gene encoded polyproteins of Rauscher and Moloney murine leukemia viruses are presented and a model for virus assembly is proposed. Amino acid sequences of murine and avian nucleic acid binding proteins indicate an evolutionary relationship in addition to functional homology. The processing of glycosylated and unglycosylated gag gene polyproteins is described, carbohydrate attachment sites are identified, and the structural interrelationships between these molecules is discussed. The amino acid sequence of env gene encoded p15(E) indicates discrete topogenic sequences characteristic of integral membrane proteins.

Regulated Proteolytic Processing within Mature Retroviral Capsids

Capsid particles were prepared from equine infectious anemia virus (EIAV) as a model retrovirus for the human immunodeficiency virus (HIV). There is a stepwise cleavage of the nucleocapsid (NC) protein (pll) and integrase (IN) (p32) during incubation of EIAV capsids at 37°C in 10 mM Tris 1 mM EDTA (TE Buffer) at pH 7.6. The viral protease cleaves the NC protein after the first Cys residue of both conserved (C X2 C X4 H X4 C) regions. The p11 → p6 cleavage occurs at the first Cys array. The p6 is then cleaved at the second Cys array, resulting in three main peptide fragments appearing as a 4 KDa band on an SDS gel. The cleavage of a 6 KDa C-terminal fragment from IN starts when all the pll is cleaved to p6, and therefore occurs during the final fragmentation of the NC protein. Capsids from other retroviruses also show NC protein cleavage when incubated under similar conditions. It has been postulated that proteolytic processing of the NC protein occurs in vivo during the early stages of the viral life-cycle and may be required for replication.

Characterization of Immunoaffinity-Purified Human T-Cell Growth Factor from JURKAT Cells

T-cell growth factor (TCGF) is a lymphokine that was first identified in conditioned media of mitogen-activated human T-cell culture (Morgan et al., 1976). TCGF also occurs in other mammalian systems and its biological properties were recently reviewed (Smith, 1983). TCGF proved to be essential for the isolation of human T-cell leukemia virus. For a recent review see Gallo and Wong-Staal (1982).