Two inhibitors of glycosylation, 2-deoxyglucose and tunicamycin, depressed the synthesis of infectious Rous sarcoma virus greater than 100-fold. Under the same conditions only a twoto threefold decrease in the production of virus particles was observed. The noninfectious particles had a lower density (1.145 g/ml) in isopycnic sucrose gradients and lacked the two virion glycoproteins, gp85 and gp37, found on infectious virions. The four internal structural proteins of the virus, p27, p19, p15, and p12, appeared to be assembled normally into the noninfectious virus. Polypeptides related to the Rous sarcoma virus glycoproteins were immunoprecipitated from pulse-labeled Rous sarcoma virus (Prague strain, subgroup B)-transformed cells. pr959P, the polyprotein precursor to gp85 and gp37, was the major protein precipitated from untreated cells. This was absent in both tunicamycinand 2-deoxyglucose-treated cells, and a new polypeptide of molecular weight 57,000 to 58,000 was the major species precipitated. In tunicamycin-treated cells this product was unstable and was degraded during a 2-h chase; in 2-deoxyglucose-treated cells, on the other hand, the polypeptide appeared to be more stable and underwent partial glycosylation. The synthesis and processing of pr76, the polyprotein precursor to the internal structural proteins of the virion, occurred normally in both treated and untreated cells. It is concluded that the unglycosylated env gene product is a polypeptide of molecular weight 57,000 to 58,000. Rous sarcoma virus (RSV) is an enveloped RNA virus which derives its lipid bilayer from the plasma membrane of the host cell. Inserted
The humoral immune response of BALB/c mice to al---)3 dextran (DEX) 1 has been well characterized with respect to isotype and idiotype expression. The serum antibody induced in BALB/c mice by DEX consists almost entirely of the IgM and IgGs heavy chain and lambda light chain isotypes (1, 2). The majority of serum antibody that binds DEX possesses a cross-reactive idiotype (IdX) present on both the J558 and M 104E myeloma proteins and individual idiotypes (IdI) present on either J558 or M104E (3-5). The serum antibody from DEXimmune BALB/c mice had been previously shown to be homogeneous when examined with heterologous antiidiotype antibodies and by isoelectric focusing (4, 6). However, more recent amino acid sequence analysis of anti-DEX hybridoma antibodies has shown that while the majority of these antibodies are closely related, there is a considerable amount of heterogeneity mainly involving differences in only a few VH region amino acid residues (7-9). Several mechanisms have been proposed for the generation of this limited amount of sequence diversity including imprecise joining of V-D-J gene segments, use of a large number of different D region genes and somatic mutation of variable region genes (9-12). It has also been suggested that somatic mutation of V segments is associated with isotype switching during B cell differentiation. This proposal is based on the observed increase in VH sequence heterogeneity of IgG vs. IgM hybridomas that bind phosphorylcholine (13,14).We have used a panel of monoclonal antiidiotype antibodies (MAIDs) with distinct specificities to ask the following questions in relation to the above problems: (a) Do thymus-independent (TI) and thymus-dependent (TD) B cell precursors produce antibodies with differing idiotope profiles? (b) What effects do T cells have on the generation of clones secreting multiple immunoglobulin
Panels of monoclonal anti-idiotype antibodies (MAIDs) specific for individual (IdI) and cross-reactive (IdX) idiotopes were prepared and used to study the expression of these idiotopes on anti-DEX and anti-PC antibodies produced in response to antigenic stimulation in vivo, clonal expression of idiotopes in an in vitro splenic focus assay, and the alterations in the idiotypic profile of these responses after in vivo administration of monoclonal anti-Id antibodies. Using these panels of MAIDs, it was possible to inactivate IdI-bearing B cells both in neonates and adult mice without affecting the responsiveness of IdI- B cells. By contrast, suppression with IdX-specific antibodies resulted in greatly reduced antibody responses. By studying the idiotypic profile of anti-DEX clones in the splenic focus assay, it was shown that IgM, IgG, and IgA antibody Id were diverse and paralleled those expressed in serum. Within some clones there was evidence that idiotope-isotype associations differed, suggesting that V region variants may have been generated within the progeny of a clone following stimulation by dextran. An anti-anti-Id antibody isolated from a BALB/c mouse undergoing a normal immune response to R36A was shown to have a T-cell independent highly idiotope-specific regulatory effect on the T15+ anti-PC response, apparently affecting induction of anti-idiotypic B cells.
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