An unusual transmissible agent--MaTu.

MaTu is an agent, believed to be derived from a human mammary carcinoma, which displayed several extraordinary properties. These were: RIP and PAGE revealed in MaTu-infected cells only a single protein band of Mr 58 k, the gp 58. This gp 58 was immunoprecipitated by antibodies present in some human sera as well as in some sera of rabbits, sheep, and cattle. MaTu had an extremely restricted host range: it was transmissible only to HeLa cells, but not to human embryo fibroblasts, to three human tumour cell lines (T 47 D, T 24, and HMB 2) or to monkey Vero and rabbit SIRC cells. A retrovirus with a broad host range, used as a helper (X-MLV) enabled the transmission of MaTu to human fibroblasts, but not to Vero or SIRC, which are also permissive for X-MLV. These observations, together with our previous reports, support the view that MaTu might either be a novel type of defective virus, or even a non-viral autonomous genetic element.

Identification of the major immunogenic structural proteins of human foamy virus.

We have identified the major immunogenic structural proteins of the human foamy virus (HFV), a distinct member of the foamy virus subfamily of Retroviridae. Radiolabelled viral proteins were immunoprecipitated from HFV-infected cells by foamy virus antisera of human and non-human primate origin. Precipitated viral proteins were in the range of 31K to 170K. Labelling of proteins with [14C]glucosamine or with [35S]methionine in the presence of tunicamycin, as well as endo-beta-N-acetylglycosaminidase H and F treatment of [35S]methionine-labelled proteins, revealed three viral glycoproteins of approximately 170K, 130K and 47K, most likely representing the env gene-encoded precursor, the surface glycoprotein and the transmembrane protein of HFV, respectively.

Gonadotropes in a novel rat pituitary tumor cell line, RC-4B/C. Establishment and partial characterization of the cell line.

An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai X F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cells; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LH beta) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSH beta), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland.

The use of aqueous two-phase systems to concentrate and purify bovine leukemia virus outer envelope protein gp51.

Enzootic bovine leucosis is a chronic lymphoproliferative disease of cattle. The causative agent, bovine leukemia virus (BLV), is related to the human retroviruses HTLV-I and -II. The external env-protein of BLV, a glycoprotein of 51 kDa, carries neutralizing epitopes and should be an essential component in a vaccine against the virus. Problems have been encountered with the concentration and purification of intact virions of BLV and other retroviruses. During centrifugation procedures the external env-proteins are to a great extent detached and consequently poorly recovered with the virion particles. Therefore, other methods are sought to obtain a high yield of the external glycoproteins. The use of two-phase systems based on water soluble polymers is described for the extraction of BLV-gp51 from culture medium. Several polymer systems were tested and the results showed that some were attractive for large scale application. The classical combination dextran-polyethylene glycol gave promising results; a partition coefficient of about 0.02 was obtained for the distribution of the gp51 between the top and combined inter- and bottom phases. In a single extraction step it was possible to obtain 45% of the glycoprotein in a small volume bottom phase and at the same time about 15-fold purified. That should be compared with a recovery of less than 20% with the conventional centrifugation procedures. It is concluded that extraction in phase systems based on water soluble polymers is a methodology well suited for the concentration and purification of BLV-gp51.

Feline immunodeficiency virus infection.

Feline immunodeficiency virus (FIV) (formerly feline T-lymphotropic lentivirus or FTLV) was first isolated from a group of cats in Petaluma, California in 1986. The virus is a typical lentivirus in gross and structural morphology. It replicates preferentially but not exclusively in feline T-lymphoblastoid cells, where it causes a characteristic cytopathic effect. The major structural proteins are 10, 17 (small gag), 28 (major core), 31 (endonuclease?), 41 (transmembrane?), 52 (core precursor polyprotein), 54/62 (reverse transcriptase?), and 110/130 (major envelope) kilodaltons in size. The various proteins are antigenically distinguishable from those of other lentiviruses, although serum from EIAV-infected horses will cross-react with some FIV antigens. Kittens experimentally infected with FIV manifest a transient (several days to 2 weeks) fever and neutropenia beginning 4 to 8 weeks after inoculation. This is associated with a generalized lymphadenopathy that persists for up to 9 months. Most cats recover from this initial phase of the disease and become lifelong carriers of the virus. Complete recovery does not occur to any extent in nature or in the laboratory setting. One experimentally infected cat died from a myeloproliferative disorder several months after infection. The terminal AIDS-like phase of the illness has been seen mainly in naturally infected cats. It appears a year or more following the initial infection in an unknown proportion of infected animals. FIV has been identified in cats from all parts of the world. It is most prevalent in high density populations of free roaming cats (feral and pet), and is very uncommon in closed purebred catteries. Male cats are twice as likely to become infected as females. Older male cats adopted as feral or stray animals are at the highest risk of infection, therefore. The infection rate among freely roaming cats rises throughout life, and reaches levels ranging from less than 1% to 12% or more depending on the area. Clinically affected cats tend to be 5 years or older at the time of hospitalization. Experimental and seroepidemiologic studies suggest that FIV is transmitted mainly by bites. Intimate, non-traumatic contact (mutual grooming, shared use of food, water and litter pans) is inefficient in transmitting the infection. In utero and venereal transmission could not be demonstrated in laboratory settings. There is no statistical linkage between FIV and feline leukemia virus (FeLV) infections in nature. The FeLV infection rate in FIV-infected animals is the same as it is for non-FIV-infected cats.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid purification of squirrel monkey retrovirus-H major gag protein by high performance liquid chromatography.

The major gag protein (p34) of squirrel monkey retrovirus-H was purified in one chromatographic step by anion-exchange high performance liquid chromatography. The virus in a crude fraction was disrupted with Brij 35 in the presence of three kinds of protease inhibitors. The soluble virus lysate was injected into a Polyanion SI column, and p34 was eluted with a linear salt gradient. The recovery of the protein was about 60%. The purified p34 was nearly homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining.

[Syncytia formation in cultures and analysis of the protein composition of various strains of caprine arthritis encephalitis virus (CAEV)]. / Formation de syncytia en culture et analyse de la composition protéique de plusieurs souches de virus de l'arthrite et de l'encéphalite de la chèvre (CAEV).

The syncytia forming activity of 9 caprine arthritis encephalitis virus (CAEV) strains isolated from lesions or normal goat tissue was evaluated in vitro. No discrepancies were seen between the structural proteins of the 9 strains by gel electrophoresis. Goat synovial membrane cells were the most susceptible to the 9 CAEV strains. Goat monocytes in culture were susceptible to all the strains tested. At 39 degrees C, all the strains produced more syncitial lesions. In nasal turbinate cells, 2 strains did not form syncytia; one strain induced lysis of all the goat cells used.

Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne).

A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities.

Sperm-associated retroviruses in the mouse epididymis.

Sperm adsorbed with retrovirus particles were recovered from the epididymis of apparently normal male mice. Epididymal semen from all four mouse strains examined was positive for retrovirus (10(5) to 10(8) particles per microgram of protein) indicating that epididymal fluids and sperm may be important vehicles for murine retrovirus spread. Immunoblot analyses revealed that the banding patterns of electrophoretically separated epididymal viral proteins from the four strains of males were more similar to each other than to either xenotropic New Zealand Black virus or ecotropic Rauscher leukemia virus proteins. The results indicate that retrovirus particles, possibly a unique strain, are commonly expressed at relatively high titers in the reproductive tract of male mice and are sperm-associated.

Biochemical and immunological characterization of structural proteins from retrovirus-D/New England and comparison to Mason-Pfizer monkey virus and permanent human fibroblast virus.

Biochemical and immunological properties of retrovirus-D/New England (here referred as R-D/NE) recently isolated at the New England Regional Primate Research Center, Southborough, Ma, from a rhesus monkey with acquired immune deficiency syndrome were investigated and compared to the prototype type D retroviruses Mason-Pfizer monkey virus (MPMV) and permanent human fibroblast virus (PMFV) isolated from a breast carcinoma of a rhesus monkey and a continuous human cell line, respectively. The polypeptide composition of R-D/NE propagated in a human lymphoid B cell line (Raji cells) has been investigated using SDS-polyacrylamide gel electrophoresis. Staining with Coomassie blue and labelling with 14C amino acids revealed seven viral polypeptides with molecular weights of 4,000, 10,000, 12,000, 15,000, 18,000, 27,000, and 80,000 Da which were also shared by MPMV and PMFV. The 80,000 Da protein was shown to be a glycoprotein by incorporation of 3H glucosamine. The 18,000 Da protein was identified as a phosphoprotein of R-D/NE. p18 structural proteins of MPMV and PMFV represent phosphoproteins of their respective viruses as well. All three phosphorylated proteins contain O-phosphoserine as major phosphoamino acid. The comparison of tryptic peptide maps of the major internal structural proteins of R-D/NE, MPMV, and PMFV revealed a striking similarity among p 10/p 12 and p 15. proteins. A minor difference was detected among the tryptic peptide digests of p 4 and p 27 proteins. Antiserum against p 15 of MPMV showed a significantly weaker binding to R-D/NE than to MPMV and PMFV at high dilutions.

A new human retrovirus isolate of West African origin (SBL-6669) and its relationship to HTLV-IV, LAV-II, and HTLV-IIIB.

A new human retrovirus of West African origin (SBL-6669) has been isolated from a patient with immunological and clinical signs of immunodeficiency. Using radioimmunoprecipitation assays (RIPA) and Western blot (WB) tests with human sera, the new virus isolate has been compared with HTLV-IV, LAV-II, and the HTLV-IIIB prototype strain of the human immunodeficiency virus (HIV). The West African isolates appeared to be members of the same virus group since their glycoproteins were antigenically indistinguishable. West African sera showed no detectable cross reaction with HTLV-IIIB glycoproteins. The external glycoprotein in the different virus strains only showed minor variations in size. The size of the transmembranous protein was not unambiguously defined. In the West African virus isolates a 30-35 kD protein was seen similar to the protein previously described possibly to represent this component. However, in SBL-6669 a distinct 41 kD protein was also identified. There were interstrain variations in the size of several viral proteins among the West African virus isolates. Only minor differences were seen between SBL-6669 and LAV-II. The variations were most pronounced in two core proteins corresponding to the 19 kD and 24 kD proteins of HTLV-IIIB. In addition, West African human retroviruses appear to differ in pathogenicity. LAV-II and SBL-6669 are associated with immunodeficiency, whereas HTLV-IV was isolated from healthy individuals. Since further spread of these viruses to other parts of the world is imminent, it is necessary to consider their antigenic and immunogenic properties in serodiagnosis of HIV infections and in planning for immunoprophylactic interventions.

Isolation of a lentivirus from a macaque with lymphoma: comparison with HTLV-III/LAV and other lentiviruses.

A retrovirus has been isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma in 1982 at the University of Washington primate center. This isolate, designated MnIV (WPRC-1) (M. nemestrina immunodeficiency virus, Washington Primate Research Center) shows the characteristic morphology of a lentivirus and replicates to high titers in various lymphocyte lines of human and primate origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified MnIV revealed multiple bands of structural proteins, including a major viral gag protein of 28 kilodaltons, that did not comigrate with the viral proteins of a human immunodeficiency virus (HIV [FRE-1]) that was also isolated on HuT 78 cells. The relatedness of MnIV to other lentiviruses (HTLV-III/LAV, EIAV, and visna) was examined in radioimmunoassays, by immunoblot techniques, and by N-terminal amino acid sequence analysis of the viral p28 gag protein. The immunoassays revealed cross-reactivity only between MnIV p28 and HTLV-III/LAV p24, and sequence analysis showed that 14 of the 24 N-terminal residues of MnIV p28 and HTLV-III/LAV p24 are identical. These results indicate that MnIV belongs to the same lentivirus family as HTLV-III/LAV but is only partially related to these human acquired immune deficiency syndrome retroviruses.

Structural components of bovine leukemia virus: further biochemical and immunological characterization of major structural proteins and glycoproteins.

Proteins from bovine leukemia virus (BLV) were investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence and absence of 14C amino acids. Virus grown either in fetal lamb kidney (FLK) or bat lung (BL) cells revealed seven major proteins designated p10, p12, p15(1), p15(2), p24, gp30, and gp64. By tryptic peptide analysis, N-terminal amino acid analysis and radioimmunoassay it could be shown that p10, a basic protein with hydrophobic properties, is a cleavage product of p15(2), the acidic and slightly hydrophobic phosphoprotein of BLV. The structural protein p12 was shown to be a phosphorylated protein identifying it as a second major viral protein to contain phosphorous. Investigation of [3H]glucosamine incorporation, N-terminal amino acid analysis and hydrophobic properties by chloroform-methanol extraction confirmed properties of gp30 and gp64 predicted by nucleotide sequence data. The two p15 proteins have been characterized in more detail.

Characterization of the monomeric and complex-associated forms of the gag-onc fusion proteins of three isolates of feline sarcoma virus: phosphorylation, kinase activity, acylation, and kinetics of complex formation.

The gag-onc fusion proteins of three isolates of feline sarcoma virus (ST-FeSV, GA-FeSV, TP1-FeSV) from a stable noncovalent complex with two cellular phosphoproteins, pp90 and pp50. These two phosphoproteins are the same phosphoproteins which have been shown to complex with the transforming proteins of Rous sarcoma virus, Fujinami sarcoma virus, Yamaguchi 73 virus (Lipsich et al., 1982), and PRCII avian sarcoma virus (Adkins et al., 1982). Both the monomeric and complex-associated gag-onc fusion proteins are phosphorylated on serine, threonine, and tyrosine; however, quantitative and/or qualitative differences in phosphorylation of the two species were apparent. Only the monomeric form of the gag-onc proteins was able to undergo tyrosine specific autophosphorylation in an in vitro kinase reaction. Both the monomeric and complex-associated forms of the proteins were acylated, the complex-associated molecules to a greater degree. Pulse-chase experiments indicated that newly synthesized gag-onc molecules become rapidly incorporated into the complex and that a significant amount of these molecules remained associated with the complex for more than 20 hr.

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.