No abstract

A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone.

Tumor necrosis factor (TNF), a monokine initially described as a tumoricidal agent, facilitated the replication of human immunodeficiency virus (HIV) in vitro. The viability of human T-cell line MOLT-4/HIV, chronically infected with HIV, was affected by the addition of a low dose (10 ng/ml) of recombinant TNF-a (rTNF-t), while uninfected MOLT-4 cells were resistant to treatment with rTNF-a at concentrations up to 1,000 ng/mI. A marked increase in the level of HIV-specific RNA was detected in MOLT-4/HIV cells as early as 1 h after exposure to rTNF-ot and reached almost maximum level within 6 h. Production of HIV particles from MOLT-4/HIV was also increased at 6 h after treatment with rTNF-at. Nearly identical phenomena were observed in CCRF-CEM/HIV, Jurkat/HIV, and H9/HIV cells, although the sensitivity of these cell lines to rTNF-a varied. A human T-lymphotropic virus type 1-infected cell line, MT-4, was insensitive to treatment with rTNF-a.

An improved method was developed for purification of the protein termed S-II that specifically stimulates RNA polymerase II of Ehrlich ascites tumor cells. The specific activity of the final preparation was 400 000 units/mg of protein, which is about 30-fold higher than that of the previous preparation [Sekimizu, K., et al. (1976) Biochemistry 15, 5064]. The final preparation gave a single band on both sodium dodecyl sulfate and nondenaturing gel electrophoresis, and the protein extracted from the band on nondenaturing gel had stimulatory activity. S-II is a basic protein with a molecular weight of 40 500. The fundamental characteristics of S-II determined with the previous preparation were confirmed with completely purified S-II. A specific antibody to S-II was prepared. This antibody inhibited only the stimulatory activity of S-II and did not affect the activity of RNA polymerase II itself. Thus, S-II is probably not a component of the multimeric proteins of RNA polymerase II.

Human T cell lines Molt-4, Jurkat, and TLOm-1 were infected by HIV-1 in the presence of various concentrations of r-TNF. The infectivity of HIV-1 was monitored by an indirect immunofluorescence method using anti-HIV-1-positive human serum. We found that r-TNF enhanced the replication of HIV-1. HIV-1-induced giant cell formation between HIV-1 chronically infected Molt-4 cells and HIV-1-uninfected Molt-4 cells was accelerated by r-TNF. The median tissue culture infectious doses (TCID50) of HIV-1 determined in the presence of TNF revealed that TNF apparently accelerated the time of the appearance of CPE but did not affect final titer of HIV-1.