Feline oncornavirus-associated cell membrane antigen: evidence for an immunologically crossreactive feline sarcoma virus-coded protein.

The feline oncornavirus-associated cell membrane antigen (FOCMA) acts as a target for natural immuno-surveillance against tumor development in the cat. In the present study, mink and rat cells nonproductively transformed by feline sarcoma virus (FeSV) were shown to express FOCMA as well as 5'-terminal feline leukemia virus (FeLV) gag gene proteins, p15 and p12. In contrast, such cells lack detectable levels of other FeLV gag gene-coded proteins or the env gene product, gp70. FOCMA, p15, and p12 antigen expression is initially in the form of an 80,000-100,000 molecular weight precursor which, upon post-translational cleavage, gives rise to a 65,000 molecular weight component that contains FOCMA and a 25,000 molecular weight component containing p15 and p12. Feline lymphoma cells, including those from several tumors that lacked detectable levels of FeLV structural protein expression, were shown to be FOCMA-positive. These findings strongly suggest that FOCMA represents an FeSV-coded transformation specific protein and provide preliminary information regarding the position within the FeSV genome coding for its synthesis.     

Abelson murine leukemia virus: molecular cloning of infectious integrated proviral DNA.

The integrated proviral genome of Abelson murine leukemia virus (A-MuLV) was cloned in lambda gtWES . lambda B bacteriophage after EcoRI endonuclease digestion and enrichment of proviral sequences by sequential RPC-5 column chromatography and agarose gel electrophoresis. Recombinant DNA clones containing a 7.8-kilobase-pair EcoRI insert were shown to have the entire integrated A-MuLV genome with both 5' and 3' ends flanked by mink cellular DNA sequences. This DNA fragment was shown to induce focus transformation upon transfection of NIH/3T3 mouse cells. Moreover, focus-forming virus could be rescued from transformed nonproducer cells upon superinfection with a type C helper virus. A polyprotein of molecular weight 120,000 (p120) containing murine leukemia virus gag gene determinants was invariably deteced by immunoprecipitation analysis of individual transformants induced by the 7.8-kilobase-pair DNA. Molecularly cloned integrated A-MuLV in its infectious form should be of use in elucidating the mechanisms involved in transformation by this virus.     

Amino- and carboxyl-terminal amino acid sequences of proteins coded by gag gene of murine leukemia virus

The amino- and carboxyl-terminal amino acid sequences of proteins (p10, p12, p15, and p30) coded by the gag gene of Rauscher and AKR murine leukemia viruses were determined. Among these proteins, p15 from both viruses appears to have a blocked amino end. Proline was found to be the common NH(2) terminus of both p30s and both p12s, and alanine of both p10s. The amino-terminal sequences of p30s are identical, as are those of p10s, while the p12 sequences are clearly distinctive but also show substantial homology. The carboxyl-terminal amino acids of both viral p30s and p12s are leucine and phenylalanine, respectively. Rauscher leukemia virus p15 has tyrosine as the carboxyl terminus while AKR virus p15 has phenylalanine in this position. The compositional and sequence data provide definite chemical criteria for the identification of analogous gag gene products and for the comparison of viral proteins isolated in different laboratories. On the basis of amino acid sequences and the previously proposed H-p15-p12-p30-p10-COOH peptide sequence in the precursor polyprotein, a model for cleavage sites involved in the post-translational processing of the precursor coded for by the gag gene is proposed.     

Characterization of a feline sarcoma virus-coded antigen (FOCMA-S) by radioimmunoassay.

A radioimmunoassay has been developed that detects a unique antigen encoded by the genome of the feline sarcoma virus (FeSV). Pseudotype viral particles containing an FeSV-specific polyprotein (p85) were used both as a source of antigen and to prepare specific antisera in rabbits. Because p85 contains antigens related to two structural proteins (p15 and p12) of feline leukemia virus (FeLV), antibodies directed to these were adsorbed with purified FeLV proteins. The adsorbed rabbit antiserum bound to antigenic determinants (designated FOCMA-S) which are also present in p85 and reacted specifically in immunofluorescence tests with rat cells transformed by FeSV and with FOCMA-positive cat lymphoid tumor cells. Competition assays detect FOCMA-S in pseudotype type C viruses rescued from FeSV-transformed mink and rat cells but not in heterologous type C helper viruses or in FeLV. A crossreactive antigen was also detected in pseudotypes of Kirsten sarcoma virus. The assay permits the quantitative measurement of an FeSV-coded protein whose expression is associated with viral transformation.     

Pseudotypes of feline sarcoma virus contain an 85,000-dalton protein with feline oncornavirus-associated cell membrane antigen (FOCMA) activity.

Feline sarcoma virus (FeSV) rescued from transformed nonproducer mink or rat cells contains two FeSV-specific antigens (p15 and p12), and the feline oncornavirus-associated cell membrane antigen (FOCMA). All three antigens are helper virus-independent and are encoded by the FeSV genome, FOCMA, p15, and p12 antigens cochromatograph as phosphorylated molecules of 85,000 molecular weight (pp85), adsorb to immunoadsorbant columns prepared with antibodies to feline leukemia virus (FeLV), and are precipitated with antisera to FeLV or FOCMA. Antibodies to FOCMA can be adsorbed with fractions containing pp85 but not with FeLV proteins, including p15 and p12. Thus, a virus-coded tumor antigen which immunizes cats against tumors induced by feline type C viruses is packaged in FeSV particles and is linked to viral structural protein.     

Maturation of human immunodeficiency virus particles assembled from the gag precursor protein requires in situ processing by gag-pol protease

The vaccinia virus expression system was used to determine the role of human immunodeficiency virus type 1 (HIV-1) protease in viral morphogenesis and maturation. The unprocessed p55 gag precursor polyprotein alone was assembled to form HIV-1 particles which budded from cells. The particles were spherical and immature, containing an electron-dense shell in the particle submembrane; there was no evidence of core formation. Expression of both gag and pol proteins from a recombinant containing the complete gag-pol coding sequences resulted in intracellular processing of gag-pol proteins and the production of mature particles with electron-dense cores characteristic of wild-type HIV virions. To ascertain the role of protein processing in particle maturation, the pol ORF in the gag-pol recombinant was truncated to limit expression of the pol gene to the protease domain. With this recombinant expressing p55 gag and protease, intracellular processing was observed. Some of the resultant particles were partially mature and contained processed gag protein subunits. In contrast, particle maturation was not observed when the HIV-1 protease and p55 gag were coexpressed from separate recombinants, despite evidence of intracellular gag processing. These findings suggest that HIV-1 protease must be an integral component of the full-length gag-pol precursor for optimal processing and virion maturation.     

Defective virus is associated with induction of murine retrovirus-induced immunodeficiency syndrome.

C57BL/6 mice infected with a mixture of murine leukemia viruses (MuLV) develop a syndrome characterized by lymphoproliferation and profound immunodeficiency. Analyses of this viral mixture (LP-BM5 MuLV) showed that it includes replication-competent ecotropic and mink cell focus-inducing MuLV and defective viruses with genome sizes of 3.8-6.5 kilobases. The ecotropic and mink cell focus-inducing MuLV biologically cloned from the mixture did not induce disease, whereas viral preparations containing the ecotropic MuLV and 4.8-kilobase defective virus were active. Cells producing the 4.8-kilobase defective virus expressed an unusual gag-encoded polyprotein of Mr 60,000.     

Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor.

The mature gag and pol proteins of human immunodeficiency virus (HIV) and all retroviruses derive from large gag and gag-pol polyprotein precursors by posttranslational cleavage. A highly specific, virally encoded protease is required for this essential proteolytic processing. In this study, the HIV protease gene product was expressed in Escherichia coli and shown to autocatalyze its maturation from a larger precursor. In addition, this bacterially produced HIV protease specifically processed an HIV p55 gag polyprotein precursor when coexpressed in E. coli. This system will allow detailed structure-function analysis of the HIV protease and provides a simple assay for the development of potential therapeutic agents directed against this critical viral enzyme.     

Cell-free synthesis of the precursor polypeptide for avian myeloblastosis virus DNA polymerase.

High molecular weight RNA (35S) isolated from avian myeloblastosis virus directs the cell-free synthesis of two prominent polypeptides of 180,000 and 76,000 molecular weight. The latter polypeptide has previously been identified as the precursor to the group-specific antigens of the virus ("gag" proteins) [Vogt, V. M., Eisenman, R. & Diggelmann, H. (1975) J. Mol. Biol. 96, 471-493]. Two-dimensional tryptic peptide analyses of the [35S]methionine-labeled peptides demonstrate that the 180,000-dalton product is a polyprotein that can account for all the peptides of the avian myeloblastosis virus DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) and those of the gag viral proteins. This is direct confirmation of the genomic order of the viral structural genes, placing the polymerase gene adjacent to the 5'-proximal gag gene of the virus. Furthermore, our findings suggest that the primary polymerase gene product is the beta subunit of the enzyme. These results are discussed in relation to the proposed structural gene map for the avian retraviruses and suggest a model for the in vivo processing of the viral polymerase.     

Construction and characterization of Moloney murine leukemia virus mutants unable to synthesize glycosylated gag polyprotein.

Murine leukemia virus (MuLV) encodes two independent pathways for expression of the gag gene. One pathway results in processing and cleavage of the precursor Pr65gag to yield the internal capsid proteins of the virion and is analogous to gag polyprotein precursors for all classes of retroviruses. The other pathway, which is not encoded by several other classes of retroviruses, begins with a glycosylated polyprotein gPr80gag . gPr80gag is synthesized independently of Pr65gag; it contains Pr65gag peptides and additional amino-terminal protein. It is modified by further addition of carbohydrate, exported to the cell surface, and released from the cell but does not appear in virus particles. To investigate the role of glycosylated gag in MuLV infection, two mutants of Moloney MuLV (M-MuLV) deficient for synthesis of gPr80gag but able to synthesize Pr65gag were constructed. The mutants were obtained by substitution into a molecular clone of M-MuLV DNA by DNA from two acutely transforming viruses, Ableson MuLV (Ab-MuLV) and Moloney murine sarcoma virus (M-MSV). Both Ab-MuLV and M-MSV are derived from M-MuLV and they express M-MuLV gag sequences, but some strains do not synthesize glycosylated gag protein. For Ab-MuLV, a 177-base-pair Pst I fragment from the P90 strain containing the initiation codon for Pr65gag was substituted for the equivalent fragment in M-MuLV DNA. For M-MSV, 1.5 kilobases at the 5' end of the genome was substituted. Transfection of the recombined DNAs onto NIH-3T3 cells produced infectious M-MuLV, although the infected cells did not produce gPr80gag. Therefore glycosylated gag is not absolutely required for MuLV replication. Deletion of the glycosylated gag pathway did not significantly reduce the level of virus production, although a minor difference in XC plaque morphology was observed.     

Recombinant bacteriophages containing the integrated transforming provirus of Gardner--Arnstein feline sarcoma virus.

The integrated DNA provirus of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) was molecularly cloned in a bacteriophage lambda vector. The cloned DNA fragment is 14.4 kilobase pairs long and contains a 6.7-kilobase provirus flanked by cellular sequences derived from nonproductively transformed mink cells. Transfection of mouse NIH/3T3 cells with the cloned DNA fragment induced foci of transformation at efficiencies of 10(4) focus-forming units/pmol of sarcoma virus DNA. Restriction endonuclease mapping and heteroduplex analyses were used to compare the GA-FeSV provirus with that of Snyder-Theilen (ST)-FeSV, a second strain that contains homologous transformation-specific sequences (v-fes). Both viruses have the general structure 5'-gag-fes-env-c region-3', each having retained portions of the feline leukemia virus (FeLV) gag and env genes. In addition to segments shared by the two sarcoma viruses, GA-FeSV contains 1.7 kilobases of extra sequences not found in ST-FeSV. Of these, at least 400-500 base pairs located near the 5' end of v-fes encode a portion of the GA-FeSV polyprotein; the remaining 1.2 kilobases are derived from the FeLV env gene but do not appear to encode any detectable product related to the FeLV envelope glycoprotein. The close homology of the v-fes sequences shows that GA- and ST-FeSV were formed by recombination of FeLV with similar portions of a cat cellular gene (c-fes).     

Serological characterization of B-tropic viruses of C57BL mice: possible origin by recombination of endogenous N-tropic and xenotropic viruses.

The serological properties of the gag gene products p15 and p12 of N- and B-tropic viruses of C57BL mice have been examined. Although these viruses were serologically identical by competition assays for proteins gp71 and p30, they were readily distinguishable in competition assays for proteins p15 and p12. Two isolates of N-tropic viruses had p12s serologically indistinguishable from AKR murine leukemia virus p12, while two B-tropic isolates had distinctly different p12s. The latter p12s were serologically indistinguishable from the p12 purified from the B-tropic radiation leukemia virus (RadLV)/VL-3. Moreover, this p12 was indistinguishable from the p12 of the endogenous C57BL/Ka xenotropic virus. Similarly, the p15s of the B-tropic viruses were serologically distinct from the AKR murine leukemia virus type of p15, as was the p15 of one C57BL N-tropic virus, whilc another N-tropic isolate had a p15 identical to the AKR murine leukemia virus p15. These results are interprered to suggest that the endogenous N-tropic virus of C57BL mice undergoes recombination with the endogenous, xenotropic virus and that this mechanism is involved in the generation of B-tropic viruses in C57BL mice.     

Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting

Retroviral Gag proteins perform important functions in viral assembly, but are also involved in other steps in the viral life cycle. Conventional mutational analysis has yielded considerable information about domains essential for these functions, yet many regions of gag remain uncharacterized. We used genetic footprinting, a technique that permits the generation and simultaneous analysis of large numbers of mutations, to perform a near-saturation mutagenesis and functional analysis of 639 nucleotides in the gag region of Moloney murine leukemia virus. We report here the resulting functional map defined by eight footprints representing regions of Moloney murine leukemia virus gag, some previously uncharacterized, that are essential for replication. We found that significant portions of matrix and p12 proteins were tolerant of insertions, in contrast to the N-terminal half of capsid, which was not. We analyzed 30 mutants from our library by using conventional methods to validate the footprints. Six of these mutants were characterized in detail, identifying the precise stage at which their replication is blocked. In addition to providing the most comprehensive functional map of a retroviral gag gene, our study demonstrates the abundance of information that can be gleaned by genetic footprinting of viral sequences.     

Deletion of sequences upstream of the proteinase improves the proteolytic processing of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 expresses structural proteins and replicative enzymes within gag and gag-pol precursor polyproteins. Specific proteolytic processing of the precursors by the viral proteinase is essential for maturation of infectious viral particles. We have studied the activity of proteinase in its immature form, as part of a gag-pol fusion protein, in an in vitro expression system. We found that deletion of p6*, the region in pol upstream of proteinase, resulted in improved processing of the precursor. A modified proteinase is released, but it functions less efficiently than wild type. Improved autoprocessing correlates with increased accessibility of the active site region in the polyprotein carrying the p6* deletion. Our results suggest that p6* is involved in the regulation of proteinase activation, perhaps as a region limiting the interaction of the active site and substrate binding domain with the remainder of the polyprotein. Release of p6* inhibition may be an activation step necessary for infectious particle maturation.     

Demonstration of a Cell-Surface Antigen Associated with Murine Sarcoma Virus by Immunoelectron Microscopy

Cells transformed by murine sarcoma virus have been examined for the presence of a new virus-associated cell-surface antigen by immunoelectron microscopy. A common antigen has been detected on the surface of nonproductively transformed cells that were induced by two different strains of murine sarcoma virus, Kirsten and Moloney. This antigen shows crossreaction with cell lines transformed by murine sarcoma virus that were produced in two different mammalian species, rats and mice. Further, this antigen is distinct from previously described antigens on the surfaces of cells infected by murine leukemia virus, on the viral envelope, and on the surfaces of spontaneously transformed cell lines or cell lines transformed by x-irradiation.     

Host restriction of Friend leukemia virus: gag proteins of host range variants.

The host response to murine ecotropic leukemia viruses is mainly controlled by the mouse Fv-1 gene. This locus controls virus replication at an intracellular stage and prevents provirus integration. Biological studies suggest that the Fv-1 effector molecule recognizes at least one virion structural protein. We have produced host range variants of B-tropic Friend murine leukemia virus in order to study the primary structure of potential viral target proteins. Our results show that conversion of B-tropism to NB-tropism is associated with changes in the primary structure of three gag proteins--p15, p12, and p30. These results suggest that host range conversion is due to a recombinational event, presumably between the parental virus and an endogenous murine virus. They also open the possibility that p12 and p30 may be involved in host range restriction.     

Complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses.

We report the complete 8714-nucleotide sequence of the integrated bovine leukemia virus genome and deduce the following genomic organization: 5' LTR-gag-pol-env-pXBL-3' LTR, where LTR represents a long terminal repeat and pXBL represents a region containing unidentified open reading frames. This genomic structure is similar to that of human T-cell leukemia virus. The LTR contains a putative splice donor site in the R region. The gag gene encodes a precursor protein with the form NH2-p15-p24-p12-COOH. The NH2- and COOH-terminal regions of the pol product show stronger homologies with those of avian, rather than murine, type C retrovirus, and its structure is identical to that of avian virus. The env gene encodes a surface glycoprotein (gp51) and a transmembrane protein (gp30). In contrast to the pol product, the gp30 shows stronger sequence homology with a murine, rather than avian homologue, indicating the chimeric nature of the bovine leukemia virus genome. Comparisons of the best conserved pol sequences and overall genomic organizations between several major oncoviruses allow us to propose that bovine leukemia and human T-cell leukemia viruses constitute a group, designated as type "E," of Oncovirinae.     

Conserved immunogenic region of a major core protein (p24) of human and simian immunodeficiency viruses

A murine monoclonal antibody (MoAb), VAK 4, has been known to specifically react with a major core protein (p24) as well as with its precursor (p55-57) and intermediate precursor (p40) of human immunodeficiency virus strain IIIB (HTLV-IIIB). Radioimmunoprecipitation assays revealed that VAK 4 MoAb precipitated a major core protein and its precursors from a variety of strains of HIV and also from simian immunodeficiency virus (SIV), although the molecular weights of the precursor proteins in each viral strain were slightly different. A protein synthesized by transfected Escherichia coli containing amino acid sequences corresponding to residues 121-436 of the HTLV-IIIB gag gene was reactive with VAK 4 MoAb, but the protein carrying only residues 121-309 was not reactive, suggesting that the epitope recognized by VAK 4 MoAb resides at the carboxyl terminus of p24 protein. A competitive enzyme-linked immunosorbent assay showed that patient sera containing anticore protein antibody inhibited the binding of VAK 4 to HTLV-IIIB. These findings suggested that VAK 4 MoAb recognized an immunogenic and conserved epitope belonging to a major core protein of HIV-related viruses.     

Active human immunodeficiency virus protease is required for viral infectivity.

Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.