Differential Regulation of Interferon Synthesis in Lymphoblastoid Cells

Abstract

We have examined the molecular mechanisms involved in the induction and regulation of expression of alpha and beta 1 human interferons (HuIFN) in Namalva cells. Cloned IFN-alpha and -beta 1 cDNAs, and antisera to purified IFN-alpha and -beta 1 were used as specific probes to determine the expression of HuIFN genes both on the RNA and protein levels. The rates of gene transcription were correlated with the relative levels of HuIFN mRNA present in induced cells and with the amounts of HuIFN peptides synthesized by these cells. The comparative rate of transcription of HuIFN-alpha and -beta 1 genes was measured in nuclei isolated from Namalva cells before and after induction. No transcription of HuIFN-alpha and -beta 1 genes was detected in nuclei isolated from the uninduced cells. The correspondence in the rate of HuIFN-alpha and -beta 1 genes transcription after virus infection with the relative levels of HuIFN mRNA in the induced cells indicates that the stimulation of HuIFN synthesis by viral infection results from the activation of the transcription of HuIFN genes. The relative levels of alpha and beta 1 induced transcripts were the same in spite of the differences in the number of copies of HuIFN-alpha and -beta 1 genes indicating that the beta 1 gene is transcribed more efficiently than the alpha genes. The steady-state levels of HuIFN-alpha and -beta 1 mRNAs in induced Namalva cells are comparable, however, the overall amount of HuIFN-beta 1 synthesized (as determined by radioimmunoassay and biological activity) is approximately 10-fold lower than that of IFN-alpha. No evidence has been found that would indicate that HuIFN-beta 1 mRNA induced in Namalva cells is different from that induced in human fibroblasts. The data indicate, however, that in Namalva cells, the IFN-beta 1 polypeptide has a higher turnover rate and slower rate of release into medium than the HuIFN-alpha polypeptides, indicating that the observed difference in the overall amounts of these two types of interferons present in the medium is due to regulation on posttranslational level.

Induction of interferon messenger RNA in human lymphoblastoid cells (Namalva)

“…3). This size is the same range reported for the major species of IFN-amRNA [15,[26][27][28][29]. The absence of IFN-amRNA transcript in the 1.2 kb range in the 0 hour sample confirmed the low level of the IFN-amRNA.…”

Inhibition of human immunodeficiency virus (HIV) replication by HIV-trans-activated alpha 2-interferon.

“…Total RNA was isolated from VN88 or A3N88 cells containing the pBK88 plasmid (A) and from VN89 or A3N89 cells containing the pBK89 plasmid DNA (B) before (lanes mock) and 24 hr after transfection with pllextatIll (lanes tat) as described (11). RNA (10 gg) was analyzed by Northern blot hybridization with a human a2-interferon riboprobe (28). The levels of secreted human a2-interferon, assayed by the cytopathic method (36) in VN88 cells was also markedly reduced yielding only 35% of control cultures.…”

“…The HIV LTR sequences were obtained as the BamHI-HindIII fragment of pU3RIIICAT (HIV LTR-CAT) plasmid DNA, where CAT is chloramphenicol acetyltransferase (21). The human a2-interferon sequence was obtained from the pCR122 plasmid (22,28) containing the genomic clone of the human a2-interferon gene by digestion with Sau I at a site 20 nucleotides downstream of the cap site. After addition of HindIll linker DNA, further digestion with HindIII and EcoRI (at a site 65 nucleotides downstream of the a2-interferon gene polyadenylylation site), generated a 1000-base-pair (bp) fragment that was cloned between the HindIII and EcoRI sites of pU3RIIICAT to yield pHIVa2.…”

Stimulation of Interferon and Cytokine Gene Expression by Imiquimod and Stimulation by Sendai Virus Utilize Similar Signal Transduction Pathways

“…1). Expression of the IFNB gene without detectable secretion of the IFN-␤ protein has previously been found to occur in Sendai virus-induced Namalwa cells (59,70).…”