Type C virions were spontaneously released from cultures of a diploid human cell strain. The varions have properties of known type C RNA tumor viruses and share antigenic determinants with the major interspecies-specific antigen (p30) of simian sarcoma virus. Antiserum to reverse transcriptase of gibbon ape leukemia virus inhibits the reverse transcriptase of the putative human virions and that of simian sarcoma virus, but has no effect on the corresponding enzymes of avian or murine RNA tumor viruses.
Rat embryo cell cultures were synchronized by a double thymidine block. The DNA replication phase (S) was divided into an early, middle, and late period. Cell cultures in the early, middle, or late S phase were pulsed with 0.1 JM 5-bromo[3H]deoxyuridine (BrdU) or equimolar [3HldT. DNA The thymidine analog 5-bromodeoxyuridine (BrdU) induces the synthesis of a latent type C virus in secondary cultures of normal rat embryo cells (1). Release of type C particles into the culture fluids was first detected 2 days after BrdU removal, peaked 8-10 days later, and disappeared 14-16 days after removal of the analog. Density gradient centrifugation and DNA -DNA reassociation experiments revealed extensive and uniform base substitutions throughout the DNA sequences of rat embryo cells at the optimal virogenic BrdU dose (0.1 mM) (2). However, [3H] (2,3). The specific activity of each DNA sample was determined. Aliquots of radiolabeled DNA were centrifuged to equilibrium in gradients of neutral CsCl (2, 3). Gradient fractions were assayed for radioactivity, absorbance, and refractive index as described (2, 3).Renaturation Kinetics. Each DNA sample was made 0.40 M in sodium phosphate buffer, pH 6.8, and 3 mM in EDTA (2,3,7,8). The initial A260 of the reaction mixture was determined, and the sample was heat sealed in 5 ml glass ampoules and immersed completely in boiling water at 1000 for 10-15 min (2, 3, 7). After thermal denaturation, each ampoule was transferred to a 680 water bath and incubated until the product of initial DNA concentration (mol of 1829 Abbreviations: S, DNA replication phase of cell cycle; p30, group-specific antigen of Friend leukemia virus; BrdU, 5-bromodeoxyuridine; Cot, initial DNA concentration time.
Earlier studies from several laboratories showed that interferon-(x (IFN-ax) is present in the sera of a large percentage of patients with systemic lupus erythematosus (SLE). We now report the detection of IFN-ax by indirect immunofluorescence in renal sections of three patients with SLE but not in six control kidneys. The immunofluorescence reaction was mediated by three hyperimmune antisera to IFN-at raised in three different species, but not by any preimmune serum. The reaction was specifically blocked by absorption of the anti-IFN-a sera with purified IFN-a made by recombinant DNA techniques or with IFN-ot isolated from the serum of an SLE patient, but not by bovine serum albumin or human immunoglobulin G. In contrast, antisera to IFN-3 or IFN-y did not mediate immunofluorescence. The pattern of IFN-at deposition resembled that seen with anti-human immunoglobulin G, suggesting association with immune complexes. Immune complexes were then preparatively eluted from the homogenate of an SLE kidney by treatment with buffer at pH 2.8. Biologically active IFN was found in this eluate and was demonstrated to be IFN-cx by specific neutralization with IFN antisera. These results extend the specific association of IFN-ot with SLE.
Kidneys from patients with lupus nephropathy, non-lupus immune-complex glomerulonephritis and other renal diseases were examined by indirect immunofluorescence for antigens related to a C-type virus from human cells (HEL-12 virus). All 11 specimens of lupus nephropathy contained HEL-12 virus antigens deposited in the same pattern as the immune complexes. The intensity of immunofluorescence with anti-HEL-12 virus serum correlated with the extent of immune-complex deposition. In contrast, nine renal lesions other than lupus nephropathy and seven normal tissues did not react with anti-HEL-12 virus serum. Antibody eluted from one kidney with lupus nephropathy reacted by indirect immunofluorescence with human and dog cells infected with HEL-12 virus but not with uninfected control cells. These findings demonstrate a specific association of lupus nephropathy with a C-type viral antigen that is deposited as antigen-antiviral antibody complex.
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