1. The effect of the novel, naturally occurring nucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid Molt 4 cell line was studied. When exposed to this guanine nucleotide. Molt 4 cells exhibited a marked increase in [3H]thymidine incorporation, up to 200-fold at 50 microM c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher than untreated cells. Stimulation of [3H]thymidine incorporation into the cells was time- and concentration-dependent. This effect was specific and was not observed with GMP or cyclic GMP, nor with the unhydrolysable GTP analogues, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate. C-di-GMP entrance into the cells was experimentally verified and occurred without using any means of cell permeabilization. SDS/PAGE analysis of cells exposed to [32P]c-di-GMP, followed by autoradiography, revealed the labelling of three low-molecular-mass proteins at 18-27 kDa. The labelling is highly specific to c-di-GMP and its extent was not affected by other guanine nucleotides. 2. One of the c-di-GMP-binding proteins was found to be the p21ras protein, by immunoprecipitation with the anti-Ras monoclonal antibody Y13-259. The effects described appear to be unique for c-di-GMP and, taken together, raise the possibility that an irreversible binding of this guanine nucleotide to the growth-promoting p21ras protein results in a fixed active conformation of this protein affecting DNA synthesis. Strikingly, although at 48 h of growth markedly high DNA levels were found in Molt 4 cells treated with c-di-GMP, this guanine nucleotide had no effect on cell replication during this period. Thus Molt 4 cells exposed to c-di-GMP enter the S phase uncoordinated with their overall replication rate.

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The effect of the novel, naturally occurring nucleotide cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid CD4+ Jurkat cell line was studied. When exposed to 50 W WM c-di-GMP, Jurkat cells exhibited a markedly elevated expression of the CD4 receptor of up to 6.3-fold over controls. C-di-GMP also causes blockage of the cell cycle at the S-phase, characterized by increased cellular thymidine uptake, reduction in G2/M-phase cells, increase in S-phase cells and decreased cell division. Additionally c-di-GMP naturally enters these cells and binds irreversibly to the P21 r s protein. The effects described appear to be unique for c-di-GMP.z 1999 Federation of European Biochemical Societies.

SUMMARYThe replication of Sindbis virus was studied in primary chick embryo fibroblasts treated with actinomycin D. The cellular site of viral RNA and coat protein synthesis was found to be localized in the cytoplasmic reticulum. Two different species of RNA were identified in association with the reticulum, namely a double-stranded RNA with a sedimentation constant of 20 S and RNA with a sedimentation constant of 25 S. The single-stranded RNA molecules extracted from the 130 S virus particles had a sedimentation coefficient of 4 ° S. The time course of the association of viral RNA and coat proteins demonstrated that both components appear simultaneously in the I3O S viral ribonucleoprotein particles within I5 rain.

Several independent cell lines transformed by simian virus 40 carry a species of viral RNA of 900,000 to 1,000,000 daltons. A viral RNA species of similar size is found early in the lytic cycle. Late in the viral lytic cycle, two prominent viral RNA species of about 600,000 and 900,000 daltons are seen. The larger late species shares nucleotide sequences with, and is less stable than, the smaller. These RNA species are located in the cytoplasm of the infected cell. The regions of the viral genome coding for these RNA species are mapped by hybridization of lytic RNA species to fragments of the genome produced by cleavage with Haemophilus aegyptius endonuclease.