Analysis of gag proteins from mouse mammary tumor virus.

Structural proteins designated p10gag, p21gag, p8gag, p3gag, p27gag, and p14gag from the C3H strain of mouse mammary tumor virus (MMTV) were purified by reversed-phase high-pressure liquid chromatography. The N- and C-terminal amino acid sequences and amino acid composition of each protein were determined and compared with the amino acids encoded by the proviral DNA sequences for the MMTV gag gene. The results show that each of the purified proteins is a proteolytic cleavage product derived from the predicted primary translational product of the gag gene (Pr77gag) and that their order in Pr77gag is p10-pp21-p8-p3-n-p27-p14 (where n represents 17 predicted residues that were not identified among the purified proteins). Purified p10gag lacks the initiator methionine and has a myristoyl group attached in amide linkage to the N-terminal glycine residue predicted by the second codon of the gag gene. The cleavage products are contiguous in the sequence of Pr77gag, and the C-terminal residue of p14gag is encoded by the last codon of the gag gene. By analogy with other retrovirus, p14gag is the viral nucleocapsid protein, p10gag is the matrix protein, and p27gag is the capsid protein of mature MMTV. Proteolytic cleavage sites in MMTV Pr77gag bear a striking resemblance to cleavage sites in the gag precursors of D-type retroviruses, suggesting that these viral proteases have similar specificities.     

Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide.

The four major internal structural proteins (the group-specific antigens) of avian myeloblastosis virus are formed by sequential cleavage of a precursor polypeptide with M_r = 76,000 (Pr76). The evidence for this conclusion is based on analysis of immune precipitates from lysates of AMV⁴-infected cells treated with a multivalent antiserum directed against these proteins. Sodium dodecyl sulfate gel electrophoresis of such immune precipitates from cells pulse-labeled with [³⁵S]-methionine reveals five metabolically unstable radioactive polypeptides. These polypeptides behave kinetically as precursors to virion proteins. Double-label ion-exchange chromatography of tryptic digests of the unstable polypeptides demonstrates that the largest precursor, Pr76, contains the amino acid sequences of all four virion proteins. It appears not to contain the sequence of the fifth and smallest internal virion protein. The four smaller precursors are intermediate cleavage products of Pr76.The arrangement of the virion proteins in Pr76 was determined by labeling cells shortly after inhibiting polypeptide chain initiation. The relative amounts of radioactivity both in completed virion proteins and in the tryptic peptides of Pr76 implies the same order for three of the four proteins. The exact position of one protein remains uncertain.On the basis of these experiments, we propose a cleavage pathway for the generation of the structural proteins of AMV. We also demonstrate that cleavage of precursors can proceed in crude extracts of AMV-infected cells. This proteolysis, while resistant to several protease inhibitors, is completely blocked by addition of agents that disrupt membranes.     

Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins

Mouse mammary tumor virus (MMTV) derived from the culture medium of GR cells contained seven proteins, identified as gp55, gp33, p25, pp20, p16, p12, and p10. The major viral phosphoprotein was the 20,000-molecular-weight protein, pp20. Immunoprecipitation of cytoplasmic extracts from pulse-labeled GR cells identified three MMTV gag-specific proteins, termed Pr78(gag), Pr110(gag), and Pr180(gag+). These intracellular polyproteins were precipitable from cytoplasmic extracts by antisera to virions p25 and p12 but not by antisera to gp55. The major intracellular gag-specific precursor polyprotein, Pr78(gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, p10, and presumably pp20. This precursor is presumably derived from nascent chain cleavage or rapid posttranslational cleavage of the larger intracellular precursor-like protein, designated Pr110(gag). Pr110(gag) contained all but one of the leucine-containing tryptic peptides of Pr78(gag), plus several additional peptides. In addition to Pr78(gag) and Pr110(gag), monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weight precursor-like protein, designated Pr180(gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78(gag) and Pr110(gag) plus several additional peptides. By analogy to type C viral systems, Pr180(gag+) is presumed to represent a gag-pol common precursor which is the major pathway for synthesis of MMTV polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two env-specific proteins, designated gPr76(env) and gP79(env). The major env precursor, gPr76(env), could be labeled with radioactive glucosamine and was shown to contain antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A minor glycoprotein, gP79(env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79(env) represents fucosylated gPr76(env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.     

Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55gag and p17MA

The human immunodeficiency virus type 1 (HIV-1) Pr55^gag gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55^gag is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55^gag maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55^gag in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55^gag was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55^gag was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55^gag and p17MA in virions and membranes.     

Primate retroviruses: intracistronic mapping of type D viral gag gene by use of nonconditional replication mutants.

Nonconditional replication mutants of squirrel monkey retrovirus (SMRV), an endogenous type D virus of primates, are shown to be defective in post-translational processing of nonglycosylated virus-coded structural proteins. Utilizing such mutants, in combination with sensitive radioimmunological assays, we demonstrate the existence of a 72,000-molecular-weight precursor polyprotein (Pr72gag) encoded by a region of the SMRV genome designated gag. Post-translational cleavage of this precursor polyprotein gives rise to virion structural proteins of 35,000 (p35), 16,000 (p16), 12,000 (p12), and 9,000 (p9) molecular weight. Three of these viral proteins, p35, p16, and p9, are shown to be phosphorylated. Analysis of viral antigen expression in cell lines nonproductively infected with either of two replication-defective SMRV mutants or mink cells productively infected with wild-type SMRV resulted in the detection of several SMRV Pr72gag intermediate cleavage products. Adjacent proteins within such intermediates are identified by use of specific competition immunoassays, and the intracistropic order of individual structural proteins with SMRV Pr72gag was tentatively deduced as NH2-p16-p12-p35-p9-COOH.     

Common Precursor for Rauscher Leukemia Virus gp69/71, p15(E), and p12(E)

Rauscher murine leukemia virus glycoprotein gp69/71 and non-glycosylated p15(E) are synthesized by way of a 90,000-dalton precursor glycoprotein, termed Pr2a+b. Peptide mapping experiments showed that Pr2a+b contains all the tyrosine-containing tryptic peptides of gp69/71. Two additional tyrosine-containing tryptic peptides in Pr2a+b that are not detected in gp69/71 are found in p15(E). Thus, gp69/71 and p15(E) peptide sequences account for all the tyrosine tryptic peptides of Pr2a+b. The gene order of the two proteins was determined by pulse-labeling infected cells in the presence and absence of pactamycin at concentrations of the inhibitor that prevent initiation of translation, but not elongation. The gene order was found to be: ₂HN-gp69/71-p15(E)-COOH. A newly identified major viral protein, termed p12(E), migrates in sodium dodecyl sulfate-polyacrylamide gels in the “p12” region. It is related to p15(E) as determined by tryptic mapping experiments. p15(E) and p12(E) are not phosphorylated, and both can be separated from phosphoprotein p12 by guanidine hydrochloride-agarose chromatography. p12(E) and p15(E) elute in the void volume fraction, whereas phosphoprotein p12 elutes between p15 and p10. The two p12 proteins can also be separated from each other by two-dimensional gel electrophoresis involving isoelectric focusing in the first dimension and sodium dodecyl sulfate-gel electrophoresis in the second dimension.     

P85: a gag-mos polyprotein encoded by ts110 Moloney murine sarcoma virus

Antibody to a synthetic peptide (anti-C3 serum) with the predicted sequence of the C terminus of the Moloney murine sarcoma virus (strain 124) v-mos gene was used in immunoprecipitation experiments with cytoplasmic extracts of a clone of NRK cells infected with ts110 Moloney murine sarcoma virus, termed 6m2 cells. ts110 Moloney murine sarcoma virus codes for two viral proteins of 85,000 and 58,000 M_r, termed P85 and P58, respectively, in nonproducer 6m2 cells maintained at 33°C. Anti-C3 serum specifically recognized [³H]leucine-labeled P85, but not P58, from infected cells maintained at 33°C, whereas antiserum prepared against murine leukemia virus p12 recognized both proteins. Normal serum and anti-C3 serum pretreated with excess C3 peptide did not precipitate P85. Immunoprecipitation experiments after metabolic labeling of 6m2 cells with ³²P_i showed that P85 is phosphorylated. Both anti-C3 and anti-p12 sera specifically detected ³²P-labeled P85. Cell-free translation of ts110 murine sarcoma virus/murine lukemia virus RNA produces P85, P58, and helper virus protein Pr63^gag. Anti-C3 serum specifically precipitated P85 but neither P58 nor Pr63^gag. We conclude from these studies that P85 is a product of both the gag and mos genes of ts110 murine sarcoma virus, and, therefore, it is referred to as P85^gag-mos. We have not detected any other v-mos gene product in ts110-infected cells.     

Further studies on the glycosylated gag gene products of Rauscher murine leukemia virus: identification of an N-terminal 45,000-dalton cleavage product

A glycosylated 45,000-Mr protein containing Rauscher murine leukemia virus p15 and p12 antigenic sites and tryptic peptides was identified in Rauscher murine leukemia virus-infected cells. This glycoprotein, termed gP45gag, was also shown to contain a single tryptic peptide also present in gPr80gag and its unglycosylated apoprotein precursor Pr75gag, but lacking in Pr65gag or Pr40gag. The presence of this peptide only in viral precursor proteins containing the so-called leader (L) sequence strongly suggests that gPr45gag is an N-terminal fragment of larger glycosylated gag polyproteins, composed of L sequences in addition to p15 and p12. The kinetics of appearance of radiolabeled gPr45gag and its disappearance during chase-incubation is suggestive of a precursor-like role for this intermediate gene product. An observed 27,000-Mr glycosylated polypeptide, termed gP27gag and containing p15 but not p12, p30, or p10 antigenic determinants, is a candidate cleavage product derived from gPr45gag. These observations suggest that gPr45gag and its putative cleavage product gP27gag represent an authentic pathway for intracellular processing of glycosylated core proteins.     

Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites.

The structural relationships among the gag polyproteins Pr65gag, Pr75gag, and gPr80gag of Rauscher murine leukemia virus were studied by endoglycosidase H digestion and formic acid cleavage. Fragments were identified by precipitation with specific antisera to constituent virion structural proteins followed by one-dimensional mapping. Endoglycosidase H reduced the size of gPr80gag to that of Pr75gag. By comparing fragments of gPr80gag and the apoprotein Pr75gag, the former was shown to contain two mannose-rich oligosaccharide units. By comparing fragments of Pr65gag and Pr75gag, the latter was shown to differ from Pr65gag at the amino terminus by the presence of a leader peptide approximately 7,000 daltons in size. The internal and carboxyl-terminal peptides of the two unglycosylated polyproteins were not detectably different. The location of the two N-linked carbohydrate chains in gPr80gag has been specified. One occurs in the carboxyl-terminal half of the polyprotein at asparagine177 of the p30 sequence and the other is found in a 23,000-dalton fragment located in the amino-terminal region of gPr80gag and containing the additional amino acid sequences not found in Pr65gag plus a substantial portion of p15.     

Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides.

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.     

Polyproteins related to the major core protein of mouse mammary tumor virus.

The mouse mammary tumor virus (MuMTV) contains several low-molecular-weight proteins which, together with the genomic RNA, constitute the core structure of the virion. The most abundant protein in the core is the 27,000-dalton protein (p27), and, by analogy to the type C viruses, this protein probably forms the core shell. In mouse mammary tumor cell lines (GR and Mm5MT) producing MuMTV the major p57 antigenic specificity resides in a large protein, which migrates in polyacrylamide gels as a doublet of 77,000 and 75,000 daltons (p 77/75). A series of lower-molecular-weight proteins, p61, p48, p38, and p34, is also present in small amounts and is probably derived by proteolytic cleavage of the p 77/75. These proteins have been identified by immunoprecipitation with monospecific antiserum, and their sequence relatedness to p27 has been determined by an analysis of the peptides after trypsin digestion. After a 15-min pulse with [35S]-methionine, all of the p27-related proteins in these cell lines were labelled and, during a subsequent chase, progressively disappeared. The p27 was labeled poorly during the pulse, but the amount of label in this protein increased during the chase. A quantitation of these experiments suggested that the majority of the p27-related proteins were quite rapidly turned over in these cell lines. Hence, if p27 is derived by a progressive proteolytic cleavage mechanism, then the process is inefficient in the GR cells and only moderately efficient in the Mm5MT cells. When MuMTV was isolated from the culture medium of these cells harvested at 5-min intervals, the major p27-related protein was p34. The p27 accounted for only 29% of the anti-p27 serum immunoprecipitable proteins compared to 95% in virus isolated from an 18-h harvest. Incubation of the rapid-harvest virus at 37 degrees C for 2 h resulted in some conversion of p34 to p27. These results suggest that some of the p27 in MuMTV is formed in the virions by proteolytic cleavage of p34.     

Protein-coding potential of mouse mammary tumor virus genome RNA as examined by in vitro translation.

The protein-coding capacity of the mouse mammary tumor virus genome has been examined by in vitro translation of genome length and polyadenylated subgenomic fragments of viral RNA. Intact genome RNA of about 35S programmed synthesis of the Pr77gag, Pr110gag and Pr160gag/pol precursors seen in infected cells in vivo. Polyadenylated RNA fragments of 18 to 28S encoded products whose tryptic peptide maps resembled those of the nonglycosylated precursor to the envelope glycoproteins, confirming the gene order 5'-gag-pol-env-3'. Translation of polyadenylated RNA fragments smaller than 18S yielded a series of related proteins whose peptide maps bore no resemblance to any of the virion structural proteins. Thus, a region of the mouse mammary tumor virus genome distal to the env gene appears to have an open reading frame sufficient to encode at least 36,000 daltons of protein as of yet unknown function.     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330