Translational products encoded by newly acquired sequences of independently derived feline sarcoma virus isolates are structurally related

Polyproteins encoded by several independent isolates of feline sarcoma virus (FeSV) were analyzed with respect to molecular weight, extent of phosphorylation, and tryptic peptide composition. As previously reported, cells nonproductively transformed by the Gardner strain of FeSV express a polyprotein which has a molecular weight of approximately 115,000 and contains feline leukemia virus p15, p12, and minor portion of p30. In addition, a major 72,000-dalton possible cleavage product can be identified. Snyder-Theilen FeSV-transformed cells express a major polyprotein of approximately 115,000 daltons and a second highly related 80,000-dalton protein. The p12 structural component of Gardner FeSV P115, but not Snyder-Theilen FeSV 115, corresponds to feline leukemia virus subgroup A with respect to immunological type specificity, a finding consistent with the independent origin of these viruses. Tryptic peptide analysis revealed five methionine-containing peptides specific to the nonstructural portion of Gardner FeSV 115, three of which were also represented in Snyder-Theilen FeSV P115, three of which were also represented in Snyder-Theilen FeSV P115. None of these [35S]methionine-labeled tryptic peptides were present in translational products representative of the complete feline leukemia virus subgroup A genome, including Pr180gag-pol, Pr65gag, and Pr82env. Similarly phosphorylated tryptic peptides within the structural (p12) and nonstructural components of Gardner FeSV P115 and Snyder-Theilen FeSV P115 Are highly related. These findings support the possibility that acquired sequences of two independently derived isolates of FeSV encode structurally related proteins.     

Differences in mechanisms of transformation by independent feline sarcoma virus isolates.

The Gardner and Snyder-Theilen isolates of feline sarcoma virus (FeSV) have previously been shown to encode high-molecular-weight polyproteins with a transforming function and an associated tyrosine-specific protein kinase activity. Cells transformed by these viruses exhibited morphological alterations, elevated levels of phosphotyrosine, and a reduced capacity for binding epidermal growth factor. In addition, polyproteins encoded by both of these FeSV isolates bound to, and phosphorylated tyrosine acceptor sites within, a 150,000-molecular-weight cellular substrate (P150). McDonough FeSV-transformed cells resembled Gardner and Snyder-Theilen FeSV transformants with respect to morphological changes and a reduced capacity for epidermal growth factor binding. in contrast to the other two FeSV isolates, however, McDonough FeSV encoded as its major translational product a high-molecular-weight polyprotein with probable transforming function but without protein kinase activity detectable under similar assay conditions. Moreover, total cellular levels of phosphotyrosine remained unaltered in McDonough FeSV-transformed cells, and the major McDonough FeSV polyprotein translational product lacked binding affinity for P150. These findings argue for differences in the mechanisms of transformation by these independently derived FeSV isolates.     

Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.     

Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus.

The McDonough (SM), Gardner-Arnstein (GA), and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) code for high-molecular-weight polyproteins that contain varying amounts of the amino-terminal region of the FeLV gag gene-coded precursor protein and a polypeptide(s) of an as yet undetermined nature. The SM-FeSV primary translational product is a 180,000-dalton polyprotein which is immediately processed into a highly unstable 60,000-dalton molecule containing the p15-p12-p30 fragment of the FeLV gag gene-coded precursor protein and a 120,000-dalton FeSV-specific polypeptide. The GA- and ST-FeSV genomes code for polyproteins of 95,000 and 85,000 daltons, respectively, which in addition to the amino-terminal moiety (p15-12 and a portion of p30) of the FeLV gag gene-coded precursor protein also contain FeSV-specific polypeptides. However, the GA- and ST-FeSV polyproteins appear to be relatively stable molecules (half-lives of around 16 h) and are not significantly processed into smaller polypeptides. Immunological and biochemical analysis of each of the above FeSV translational products revealed that the sarcoma-specific regions of the GA- and ST-FeSV polyproteins are antigenically cross-reactive and exhibit common methionine-containing peptides. These findings favor the concept that these sarcoma-specific polypeptides are coded for by the similar subsets of cellular sequences incorporated into the GA- and ST-FeSV genomes during the generation of these transforming agents.     

Monoclonal antibodies to feline sarcoma virus gag and fes gene translational products

A series of hybridomas have been isolated which produce monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strains of FeSV. Within these are representatives of several immunoglobulin classes including IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM. Antibody produced by one hybridoma recognizes immunologic determinants localized within an FeLV gag gene structural component (p15) common to polyproteins encoded by all three FeSV isolates whereas antibody produced by a second is specific for p30 determinants unique to P170^gag-fms. Additional hybridomas secrete antibody directed against v-fes-encoded determinants common to the Gardner and Snyder-Theilen FeSV-encoded polyproteins. GA P110^gag-fes and ST P85^gag-fes immunoprecipitated by antibody directed against p15 exhibit tyrosine-specific protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components.     

The nonstructural component of the Abelson murine leukemia virus polyprotein P120 is encoded by newly acquired genetic sequences.

The Abelson leukemia virus (AbLV) polyprotein P120 is compared to translational products representing the entire Moloney murine leukemia virus (MuLV) genome on the basis of [35S]methionine tryptic peptide composition. Three methionine-containing tryptic peptides present in Moloney Pr65gag are each shown to be present in both Pr75gag and in Pr180gag-pol. Of these, one peptide, corresponding to Moloney MuLV p12, but neither of two p30-specific peptides are present in AbLV P120. Among the 12 remaining methionine-containing peptides present in AbLV P120, many, if not all, are unique to AbLV P120 and not shared by either Moloney MuLV Pr180gag-pol or Pr82gag.     

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.     

Comparative analysis: intracellular precursor polyproteins of baboon endogenous retroviruses and human viral isolate HL23V.

Intracellular precursor polyproteins of three baboon endogenous retrovirus (BaEV) isolates, m7, 455K, and BILN, were compared with the intracellular proteins of the type C human isolated HL23V by radioimmunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide analysis. Human and canine cells infected with m7-BaEV and canine thymus cells infected with BILN-BaEV were characterized by identical precursor polyproteins Pr85gag, Pr70-71gag, Pr65gag, and gPr85env. Canine cells infected with 455K-BaEV consistently showed a slightly different pattern of precursor polyproteins. These included Pr85gag, Pr70gag, Pr67gag, and gPR85env. By tryptic digest mapping of peptides containing [3H]leucine, m7-BaEV and 455K-BaEV were shown to be highly related. By comparison, mapping studies showed that BILN-BaEV was less highly related to m7-BaEV than ws 455K-BaEV. Differences in these related BaEV isolates presumably reflected virus-specific differential cleavage of core protein precursors or alterations in polyprotein primary structure or both. Chase-incubated cells infected with BaEV also contained a stable, p28-related polyprotein termed P72gag. This polyprotein migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than the major core protein precursor Pr70-71gag and appeared to arise by posttranslational modification of Pr70-71gag. Immunoprecipitation of extracts of HL23V-infected cells with antisera to simian sarcoma-simian-associated virus proteins and BaEV proteins confirmed that these cells contained two unrelated viral components, one that was similar to m7-BaEV or BILN-BaEV and a second that was related to simian sarcoma-simian-associated virus. Tryptic digest mapping of BaEV and HL23V prcursor polyproteins suggested that the BaEV-like component of HL23V weas more closely related to m7-BaEV than to 455K-BaEV or BILN-BaEV.     

McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms).

The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P170gag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (P120fms and pp55gag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 +/- 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRI and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Synder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline-leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes.     

Immunological characterization of the gag gene products of bovine immunodeficiency virus.

The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed.     

Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.     

Gene products of McDonough feline sarcoma virus have an in vitro-associated protein kinase that phosphorylates tyrosine residues: lack of detection of this enzymatic activity in vivo.

The primary translational product of the McDonough (SM) strain of feline sarcoma virus (FeSV) is a 180,000-dalton molecule, SM P180, that contains the p15-p12-p30 region of the FeLV gag gene-coded precursor protein and a sarcoma virus-specific polypeptide. In addition, cells transformed by SM-FeSV express a 120,000-dalton molecule, SM P120, that is highly related to the non-helper virus domain of SM P180. Both SM-FeSV gene products were found to be intimately associated with the membrane fraction of SM-FeSV-transformed cells. Immunoprecipitates containing SM P180 and SM P120 exhibited a protein kinase activity capable of phosphorylating tyrosine residues of both viral gene products but not immune immunoglobulin G molecules. By independently immunoprecipitating each of the two SM-FeSV proteins we found that most of the tyrosine-specific phosphorylating activity was associated with the SM P120 molecule. In vivo analysis of 32P-labeled SM P180 and SM P120 revealed their phosphoprotein nature; however, both molecules exhibited low levels of phosphorylation and did not contain phosphotyrosine residues. Finally, we did not detect any significant elevation in the levels of phosphotyrosine in the protein fraction of SM-FeSV transformants. Thus, if SM-FeSV were to induce malignant transformation by a mechanism involving phosphorylation of tyrosine residues, the viral gene products must interact with a small subset of cellular proteins that do not represent a significant fraction of the total cellular protein content.     

Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein.

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.     

Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors.

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.     

Snyder-Theilen feline sarcoma virus P85 contains a single phosphotyrosine acceptor site recognized by its associated protein kinase.

Cells nonproductively transformed by a variant of the Snyder-Theilen strain of feline sarcoma virus (FeSV) expressed an 85,000-dalton polyprotein (P85) with associated tyrosine-specific protein kinase activity. We identified within this polyprotein a single tyrosine acceptor site for its enzyme activity. This acceptor site, as well as two serine phosphorylation sites localized with the p12 structural component of Snyder-Theilen FeSv P85, was phosphorylated in cells nonproductively transformed by Snyder-Theilen FeSv. In contrast, infection by Snyder-Theilen FeSV transformation-defective mutants resulted in phosphorylation only of the two serine acceptor sites, indicating phosphorylation of the tyrosine acceptor site to be transformation specific. In addition, we describe in vitro labeling conditions, using unfractionated cell extracts, which resulted in preferential phosphorylation of the single Snyder-Theilen FeSV tyrosine-specific acceptor site.     

Nucleotide sequences of feline retroviral oncogenes (v-fes) provide evidence for a family of tyrosine-specific protein kinase genes

The nucleotide sequences encoding the transforming polyproteins of the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV) have been determined. These sequences include a viral transforming gene (v-fes), derived from cellular proto-oncogene sequences (c-fes) of domestic cats by recombination with feline leukemia virus (FeLV). The v-fes sequences are predicted to encode a polypeptide domain strikingly similar to that specified by the transforming gene (v-fps) of the avian Fujinami sarcoma virus. In addition, the 3′ 0.8 kilobase pairs of v-fes encode amino acid sequences homologous to the carboxy-terminal portion of pp60^src, the transforming protein encoded by the avian Rous sarcoma virus src gene. Thus different feline and avian retroviral transforming genes, all of which encode functionally related proteins with associated tyrosine-specific kinase activities, must be derived from divergent members of the same protooncogene family.     

Transformation-defective mutants of feline sarcoma virus which express a product of the viral src gene.

Mink cell cultures infected with the Snyder-Theilen strain of feline sarcoma-leukemia virus were cloned from single cells under conditions favoring single virus-single cell interactions. The primary colonies included (i) typical feline sarcoma virus (FeSV)-transformed nonproducer clones, one of which segregated revertants, and (ii) FeSV-infected, phenotypically normal clones, three of which spontaneously converted to the transformed phenotype. The revertants and spontaneous transformants were compared with parental and sister clones expressing the opposite phenotype. Transformed subclones formed colonies in agar, were tumorigenic in nude mice, and failed to bind epidermal growth factor, whereas flat sister subclones were indistinguishable from uninfected mink cells in each of these assays. Sister subclones derived from the same infectious event contained FeSV proviruses integrated at the same molecular site, regardless of which phenotype was expressed. One revertant clone, however, lacked most FeSV proviral DNA sequences but retained terminal portions of the FeSV genome which persisted at the original site of proviral DNA insertion. Two flat subclones expressed viral RNA and the phosphorylated "gag-x" polyprotein (pp78gag-x) encoded by the gag and src sequences of the FeSV genome. Both of these clones were susceptible to retransformation by FeSV. Although unable to induce foci, the viruses rescued from these cells contained as much FeSV RNA as the focus-forming viruses rescued from transformed sister subclones and could be retransmitted to mink cells, again inducing FeSV gene products without signs of morphological transformation. We conclude that these FeSV genomes represent transformation-defective mutants.     

Requirements for incorporation of Pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles.

The roles of the human immunodeficiency virus precursor polyproteins Pr55gag and Pr160gag-pol in viral core assembly were studied in CMT3-COS cells. To do this, the precursors were expressed separately by using a simian virus 40 late replacement vector system described previously. Consistent with previously published data, our results show that the Pr55gag precursor, when expressed alone, was able to form particles which had an immature morphology and that particle formation required the presence of a myristate addition signal at the amino terminus of the precursor. In contrast, the Pr160gag-pol precursor was not able to form particles when expressed alone, although it still underwent proteolytic processing. Coexpression of the two precursor polyproteins from separate vectors in the same cell resulted in processing of the Pr55gag in trans by the protease embedded in Pr160gag-pol and the formation of virus-like particles containing the products of both precursors. Proteolytic processing occurred independently of the presence of a functional myristate addition signal on either precursor. On the other hand, removal of myristate from one or the other precursor had nonreciprocal effects on virus particle formation. Cells expressing Pr55gag lacking myristate and Pr160gag-pol containing it did not produce particles. Cells expressing a myristylated Pr55gag and unmyristylated Pr160gag-pol still produced virus-like particles which contained nearly normal amounts of Pr160gag-pol. The results suggest that the incorporation of Pr160gag-pol into particles is largely determined by intermolecular protein-protein interactions between the two precursor polypeptides.