As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione Stransferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.

The coat protein of alfalfa mosaic virus has both structural and regulating functions. The latter is evident from the fact that the genomic RNAs of the virus, although they are of messenger polarity, cannot start an infection cycle in the absence of cost protein. The reason could be that the coat protein is needed for viral RNA synthesis. Indeed, the coat protein has been found in tight association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182, 309-315). To investigate the role of the coat protein, if any, in viral RNA synthesis, we have isolated that viral RNA polymerase (RNA-dependent RNA polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1 and 2, known as P12 plants (P. E. M. Taschner et al., 1991, Virology 181, 687-693), which express the nonstructural proteins P1 and P2. Such an enzyme (called M-RdRp) will contain the viral subunits P1 and P2 but not the coat protein. As a comparison we also isolated the RdRp from virion-inoculated P12 plants (C-RdRp). This enzyme will contain the coat protein. We found that both M-RdRp and C-RdRp could synthesize minus RNA, showing that coat protein is not needed for minus-strand synthesis. In contrast, minus-strand synthesis by both enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by coat protein in the case of C-RdRp, but strongly stimulated by coat protein in the case of M-RdRp. These data might explain why infected cells, which do not produce coat protein, display a very low accumulation of viral plus-strand RNA. They also give a possible explanation for the noninfectious character of the genomes of alfalfa mosaic virus and ilarviruses in the absence of coat protein. The fact that an active enzyme could be isolated from the same membrane fraction in infected and noninfected P12 plants shows that coat protein is not needed for assembly and targeting of the viral RNA polymerase.

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The leukocyte integrin LFA-1 (CD11a/CD18) plays a key role in many adhesive interactions involving cells of the immune system. Recently, it has been shown that LFA-1 is not only involved in cell adhesion, but that stimulation of LFA-1 can also contribute to cell activation. We now demonstrate that triggering of LFA-1 on T lymphocytes by monoclonal antibodies (mAb) against the LFA-1 alpha chain, but not against the LFA-1 beta chain, promotes cell adhesion. Induction of homotypic adhesion was only observed in T cells that had been pre-activated with anti-CD3 and not in resting peripheral blood T lymphocytes. The induced homotypic adhesion is mediated by LFA-itself, because it was inhibited by anti-LFA-1 beta mAb. This notion is supported by the temperature and divalent cation dependence which is characteristic of LFA-1-mediated adhesion. mAb against ICAM-1 (CD54) did not block LFA-1 alpha-induced adhesion. The sensitivity of LFA-1 alpha-induced adhesion to H7, which prevents the activation of protein kinase C and protein kinase A, and to cytochalasin B, which inhibits microfilament formation, suggests that the activation of the LFA-1 pathway through the LFA-1 alpha chain involves cell activation and requires an intact cytoskeleton.