Comparative large-scale propagation of retroviruses from old world (mason-pfizer monkey virus) and new world (squirrel monkey virus) primates

Summary A cell culture method is described for the large-scale (50 to 150 1) production of Mason-Pfizer monkey virus and squirrel monkey virus, two primate retroviruses. Virus production was achieved with suspension cultures of chronically infected A204 human rhabdomyosarcoma cells harvested and clarified in the logarithmic stages of cell culture growth. Methods for the subsequent purification and concentration of virus material utilizing zonal centrifugation also are described. Applications of these methodologies resulted in products that afforded biochemical comparisons of these agents in a manner such that host cell-derived variations were minimized. These data indicated that high levels of production and efficient recovery and purification of virus material were achieved. This work was supported by Contract NO1-CO-25423 with the National Cancer Institute, National Institutes of Health.     

Squirrel monkey retrovirus: an endogenous virus of a new world primate.

Squirrel monkey retrovirus (SMRV) was isolated by cocultivation of squirrel monkey lung cells with canine cells. 3H-labeled 60-70S SMRV RNA, isolated from virus grown in canine cells, hybridized to the same extent and to the same Cot1/2 value to the DNA of all tissues of all squirrel monkeys tested; Cot1/2 values show that SMRV proviral sequences are present in the low repetitive range. No SMRV proviral sequences were detected in tissues from a variety of other New World monkeys, Old World monkeys, or apes. Murine, feline, bovine, and canine cells also contain no detectable SMRV proviral sequences. Competitive molecular hybridization studies revealed no detectable sequence homology between the 60-70S RNAs of SMRV and Mason-Pfizer virus (MPV). The virion-associated DNA polymerase of SMRV is similar to that of MPV in that it has a molecular weight of approximately 80,000 and prefers magnesium as a divalent cation using oligo(dG)-poly(rC) as primer-template. The virion-associated DNA polymerase of SMRV can be clearly distinguished from those of MPV and the mouse mammary tumor viruses, however, by its preference for manganese as a divalent cation in the presence of high salt.     

Feline leukemia virus: biochemical and immunological characterization of gag gene-coded structural proteins.

The major non-glycosylated structural proteins of feline leukemia virus have been isolated, and competition immunoassays have been developed for each. These proteins include the 27,000- to 30,000-molecular-weight major internal antigen designated p30, a 15,000-molecular-weight protein (p15), an acidic protein of 12,000 molecular weight (p12), and a highly basic 10,000-molecular-weight protein (p10). Immunologically and biochemically corresponding proteins of feline and murine leukemia viruses have been identified. and, on the basis of analogy to the known sequence of a prototype type C virus of mouse origin, the map order of the gag region of the feline type C viral genome has been tentatively deduced as NH2-p15-p12-p10-COOH. The demonstration of two feline leukemia virus gag gene-coded proteins, p15 and p12, expressed in the form of an uncleaved precursor in a mink cell line nonproductively transformed by feline sarcoma virus provides indirect support for the proposed sequence.     

Reticuloendotheliosis virus: detection of immunological relationship to mammalian type C retroviruses.

Reticuloendotheliosis virus (REV) p30 shares cross-reactive determinants and a common NH2-terminal tripeptide with mammalian type C viral p30's. An interspecies competition radioimmunoassay was developed, using iodinated REV p30 and a broadly reactive antiserum to mammalian virus p30's. The avian leukosis-sarcoma viruses and mammalian non-type C retroviruses did not compete in this assay. Previous data indicating that the REV group is not represented completely in normal avian cell DNA lead us to speculate that this may be the first example of interclass transmission, albeit in the remote past, among the Retroviridae.     

Purification of bovine protoporphyrinogen oxidase: immunological cross-reactivity and structural relationship to ferrochelatase

Protoporphyrinogen oxidase, the penultimate enzyme in the haem biosynthetic pathway has been purified to apparent homogeneity from bovine liver mitochondria, by a published method (Dailey, H.A. and Fleming, J.E., (1983)), with an additional ion-exchange chromatography step, using a Mono Q column on an FPLC-system. This gave a product with a 68% yield and 870-fold purification. Protoporphyrinogen oxidase (EC 1.3.3.4) has an apparent Mr of 57,000 and the Km for protoporphyrinogen IX was 16.6 microM. Activity of the isolated enzyme was increased by 66% in the presence of oleic acid, and evidence was obtained for a FAD prosthetic group. Ferrochelatase (EC 4.99.1.1) was purified and antibodies were raised in rabbits against ferrochelatase and protoporphyrinogen oxidase, respectively. Anti-protoporphyrinogen oxidase IgG showed marked cross-reactivity with ferrochelatase and anti-ferrochelatase IgG cross-reacted with protoporphyrinogen oxidase. In addition, radiolabelled peptides of both enzymes, generated by chymotrypsin, demonstrated common peptides when analysed by two-dimensional chromatography.     

Antigenic cross-reactivity of major outer membrane proteins in enterobacteriaceae species.

The protein constituents in the outer membrane (OM) of several serotypes of Escherichia coli and some other Enterobacteriaceae cross-reacted antigenically. Solubilized OM preparations of these bacteria were applied in interfacial precipitin tests to antisera elicited in rabbits against whole bacterial cells, absorbed with their appropriate lipopolysaccharide before testing. The resulting immunecomplexes were analysed on polyacrylamide gels. Protein profiles of the immunoprecipitates showed a considerable antigenic cross-reactivity of outer membrane proteins between most E. coli serotypes. Cross-reactivity, though substantially lower, was also found with OM from three other Enterobacteriaceae species, but was not detectable with Pseudomonas aeruginosa OM. When OM preparations were solubilized at room temperature, the peptidoglycan-bound proteins in the molecular weight range 37,000 to 41,000 predominated in the protein profiles of the immunecomplexes. In profiles of immunecomplexes obtained with boiled OM preparations, a heat-modifiable protein (mol. wt 33,000) predominated. The major OM proteins of the Gram-negative bacterium may therefore play a role as common surface antigens of the family of Enterobacteriaceae.     

Sequence analysis of major structural proteins of newly isolated mouse hepatitis virus.

We have isolated the virus from a fecal pellet in the colon of a BALB/c mouse with X-linked immunodeficiency (xid) housed in a room in which there has recently been an epidemic due to mouse hepatitis virus (MHV) and designated it as the MHV-TY strain. Sequence analysis of the MHV-TY strain was performed on major structural, spike (S), membrane (M) and nucleocapsid (N), proteins directly from PCR products. The comparison of nucleotide sequences of MHV-TY with other strains investigated so far revealed that all three structural proteins of the TY strain had some unique amino acid sequences among MHV strains which can be used as markers of this strain.     

Pseudotypes of vesicular stomatitis virus with the envelope properties of mammalian and primate retroviruses.

By employing improved techniques it has been possible to produce and characterize a representative spectrum of mammalian and primate retrovirus pseudotypes of vesicular stomatitis virus (VSV). Selection of appropriate cell lines for both the production and subsequent detection of the VSV pseudotypes has been the most important factor in permitting their demonstration. The host range for penetration of these retrovirus pseudotypes of VSV has been defined and found to differ from that reported for the replication of the corresponding retroviruses. Additionally, retroviruses having an identical host range for replication were distinguishable by differences in their host range for penetration, implying that restriction of replication may be occurring by different mechanisms. Studies of the plaque-forming efficiency of retrovirus pseudotypes of VSV in cell lines nonpermissive for replication of the corresponding retroviruses permitted a distinction to be made between the restriction of replication occurring as a consequence of postpenetration events and that occurring as a consequence of a block of penetration itself. The demonstration of primate retrovirus pseudotypes of VSV permits the use of VSV as a probe for the detection of this group of viruses.     

Shared antigenic epitopes of the major core proteins of human and simian immunodeficiency virus isolates.

Antigenic epitopes on the major core (gag) protein of isolates of simian and human immunodeficiency virus (SIV and HIV) were compared using a panel of eleven mouse monoclonal antibodies (Mabs) that recognized nine distinct gag epitopes. Viral isolates used for comparison were HIV-1IIIb, HIV-2ROD, and SIV isolates from macaque (SIVmac), sooty mangabey (SIVsm-UCD), African green monkey (SIVagm), and stump-tailed macaque (SIVstm-UCD). The relatedness of the various HIV and SIV isolates, as determined by Mabs to core protein epitopes, paralleled that ascertained by genetic sequencing.     

BALB/c myeloma retroviruses: peptide mapping and immunological analysis of the pp12 structural protein.

We have compared the pp12 structural protein of the MO-21 and FL-1 BALB/c myeloma retroviruses with the pp12 of several prototype retroviruses. Chymotryptic peptide maps of 125I-labeled, immune-precipitated pp12 proteins revealed that the MO-21 and FL-1 proteins can be distinguished from one another. The MO-21 pp12 most closely resembled the NIH-xenotrophic virus pp12, and the FL-1 pp12 most closely resembled the pp12 of BV-2 and WN 1802 B. Competition radioimmunoassay studies showed that the MO-21 and FL-1 pp12 proteins are also antigenically distinct from one another and that both contain pp12 antigenic determinants of a xenotropic virus. These data support our proposal that these two BALB/c viruses contain a gag gene that was generated by recombination between endogenous eco- and xenotropic viral sequences.     

Sequence homology between the structural proteins of Kilham rat virus.

The capsid proteins of the autonomous parvovirus Kilham rat virus were purified and analyzed for peptide composition. Partial proteolysis mapping and two-dimensional thin-layer chromatography of tryptic peptide digests revealed extensive amino acid sequence homology between the two major Kilham rat virus capsid proteins.     

Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus.

Cell fractionation and protein electrophoresis were used to study the intracellular sites of synthesis and intermediate structures in the assembly of the virion proteins of vesicular stomatitis virus. Each of the three major virion proteins assembled into virions through a separable pathway. The nucleocapsid (N) protein was first a soluble protein and later incorporated into free, cytoplasmic nucleocapsids. A small amount of N protein was bound to membranes at later times, presumably representing either nucleocapsids in the process of budding or completed virions attached to the cell surface. The matrix (M) protein also appeared to be synthesized as a soluble protein, but was then directly incorporated into membranous structures with the same density as whole virus. Very little M protein was ever found in membranes banding at the density of plasma membranes. The M protein entered extracellular virus very quickly, as though it moved directly from a soluble state into budding virus. In contrast, the glycoprotein (G) was always membrane bound; it appeared to be directly inserted into membranes during its synthesis. Glycosylation of the G protein was completed only in smooth membrane fractions, possibly in the Golgi apparatus. After a minimum time of 15 min following its synthesis, G protein was incorporated into the surface plasma membrane, from which it was slowly shed into virions. These multiple processing steps probably account for its delayed appearance in virus. From this work it appears that the three major structural proteins come into the surface budding structure through independent pathways and together they coalesce at the plasma membrane to form the mature virion.     

Comparisons of the immunological properties of two structural polypeptides of type C RNA viruses endogenous to old world monkeys.

Immunologically very closely related type C RNA viruses are endogenous to the domestic cat and to an old world primate, the baboon. In the present studies, radioimmunological techniques have been developed for detection of the 15,000 and 30,000 molecular weight (MW) polypeptides of each virus. The much more pronounced type-specific antigenic determinants of the lower MW polypeptides made it possible to readily differentiate these viruses from each other as well as from a type C virus isolate from a second baboon species. Normal rhesus monkey tissues were partially purified and shown to contain a reactivity with MW and immunological properties similar to that of the baboon virus 30,000 MW polypeptide. Despite a similar degree of purification, antigenic reactivity like that of the baboon virus 15,000 MW polypeptide was undetectable even in the brodest immunological tests available for this polypeptide. The present findings indicate that the immunological properties of two structural polypeptides of closely related viruses endogenous to primate and feline species have undergone different rates of antigenic change in the course of evolution within their respective host cell genome.     

Correlation of structural differences in several bird lysozymes and their loop regions with immunological cross-reactivity

Goat antibodies that were specific, respectively, to hen egg white lysozyme, its loop region (residues 60 to 83) and to regions other than the loop, were reacted with the intact lysozyme or its loop region. The interference with this reaction by several bird lysozymes was tested. Bobwhite quail lysozyme was as efficient as hen lysozyme in the lysozyme-anti-lysozyme system, but much less reactive with anti-loop antibodies. Turkey lysozyme, on the other hand, was similar to hen lysozyme in its behaviour with anti-loop antibodies but different in its reactivity with anti-lysozyme. It is thus concluded that the loop region of hen lysozyme is far more reactive than that of bobwhite quail lysozyme with loop-specific goat antibodies. The large antigenic difference results from replacement of an arginine residue (at position 68) in the hen loop by a lysine residue in the quail loop. By contrast, the loop region of turkey lysozyme is antigenically similar to that of hen lysozyme. Yet the turkey loop also differs from the hen loop by a single lysine-for-arginine replacement (at position 73). To explain why the lysine substitution has a greater antigenic effect at position 68 than at position 73, two hypotheses are considered. First, as arginine 68 is the i + 2 residue of a β-bend (encompassing residues 66 to 69) and as the frequency of occurrence of lysine at the i + 2 position in β-bends is lower than that of arginine, the presence of lysine at position 68 may lower the stability of the β-bend and thereby cause a conformational change in the β-bend region of the loop. Alternatively, arginine 68 may be more exposed than is arginine 73 in hen lysozyme, and hence goat antibodies may more easily recognize the side-chain difference produced by the lysine substitution at position 68.     

Allomothering behaviour of new and old world monkeys

The holding or transferring of newborn infants at less than 1 month old by individuals other than the mothers was studied in 24 species of New and Old World monkeys under captive conditions. The observed monkey species could be divided into two types. Group A included eight species of three families where the mothers were tolerant to ‘infant transfer’ and readily retrieved their infants from other individuals, the frequency of infant transfer being high. The infant transfer of this group was termed allomothering behaviour. Group B included 16 species of two families where infant transfer did not occur at all or its frequency was very low and the mothers were possessive of their infants. Once transfer did occur, the infant could not be reclaimed with ease. The relationships between the two groups and taxonomic status, life forms and social types were evaluated in a total of 45 species from the present study and the literature. Correspondences were found with social type and taxonomic status. That is, species of Group A were seen only in the family or one-male type, except for one species, although none of this group appeared in the Cercopithecinae regardless of social types. The significance of infant transfer is discussed in relation to the participants' responses to it and the correlations between the two groups and social types.     

Antigenic analysis of major structural proteins of avian leukoviruses.

The antigenic determinants of the avian leukovirus proteins p27, p19, p15, and p12 were examined by radioimmunoassay. There was no evidence of antigenic cross-reactivity among these polypeptides, indicating that they are four distinct components. The concentration of the proteins p27, p19, and p15 in the infected cell was measured and found greatly increased, indicating that synthesis of these proteins is induced after virus infection.