Influenza virus polymerase complexes that were expressed in the absence of genomic viral RNA and nucleoprotein were examined for endonuclease activity and transcriptase ability in vitro. Nuclear extracts of cells that express influenza virus polymerase through recombinant vaccinia virus infection did not display specific endonuclease activity in vitro. This polymerase presumably represents an early form of enzyme present in infected cells prior to ribonucleoprotein assembly. Upon addition of a virus-like model RNA template, containing the partially complementary sequence found at the ends of viral RNA, endonuclease activity is stimulated in a concentration-dependent and sequence-specific manner. Once stimulated, the polymerase is able to elongate from the added viral template. Thus, addition of viral template is required for polymerase activity, while the presence of nucleoprotein is not required for limited transcription. Also, full activation of this recombinant viral polymerase is dependent on the presence of both the 3' and 5' ends of the viral genome, as model RNA containing either end alone could not effectively trigger the endonuclease. The RNA-dependent RNA polymerase of influenza virus is made up of a complex of three viral proteins (PB1, PB2, and PA), associated with the eight nucleoprotein (NP) encapsidated viral gene segments. The initial function of the polymerase complex is the transcription of virus-specific mRNAs. This is accomplished via three distinct activities. First, the PB2 subunit has been shown to bind specifically to the cap structure of host-encoded cellular mRNAs (6, 7, 46, 47). These mRNAs are then cleaved 9 to 15 bases downstream of the cap structure
Modulation of the biosynthesis of the vasoconstrictor peptide endothelin was studied in cultured endothelial cells. Immunoreactive endothelin (irET) levels were significantly elevated in conditioned medium from bovine pulmonary artery endothelial (BPAE) or human umbilical vein endothelial cells when coincubated with washed human platelets. Platelets (approximately 200,000 cells/microliters) enhanced irET levels approximately 250% over basal levels. Stimulation of irET levels in BPAE cell-conditioned medium by platelets was time and platelet number dependent. Platelets, as well as thrombin and transforming growth factor-beta 1, stimulated the expression of preproendothelin-1 mRNA in a time-dependent manner. Coincubation of low doses of thrombin (0.1 unit/ml) and subthreshold concentrations of platelets with BPAE cells resulted in a further enhancement of irET levels in conditioned medium. Platelet-mediated stimulation of irET production was not significantly affected by indomethacin (1 microM) or the platelet-activating factor receptor antagonist WEB 2086 (1 microM); however, coincubation of endotoxin (100 ng/ml) with platelets and BPAE cells resulted in significantly higher levels of irET. Whether direct contact or adhesion between platelets and endothelial cells is necessary for stimulating irET release was studied by separating platelets from BPAE cells with a 0.4 microns permeable membrane. Under these conditions, platelets still produced significant elevations (approximately 190% over basal levels) in irET levels in BPAE cell-conditioned medium. In addition, platelet-free buffer from agonist-induced platelet aggregation also significantly enhanced irET production (200% over basal values). These data indicate that a platelet-derived regulatory factor can induce the biosynthesis of endothelin from cultured endothelial cells and also suggest that platelets might play a role in vasomotor regulation via a novel intercellular interaction with the endothelium.
An in vitro cleavage system was established to measure HCV NS3 protease trans-processing activity. This system utilizes purified NS3-4A protein from baculovirus, purified substrates expressed by in vitro transcription and translation and defined buffer components. The 41-residue substrates, 5A/5B and 4A/4B, were processed efficiently in trans by wild-type NS3 but not by a catalytically inactive mutant protease; radiolabel sequencing confirmed that NS3-mediated cleavage occurred at the correct cysteine/serine sites, thereby authenticating this system. Two striking features of this in vitro assay are: (1) analogous 4B/5A and 3/4A substrates cannot be processed in trans under the same conditions, and (2) in vitro cleavage of the 5A/5B and 4A/4B sites is highly dependent on the presence of NS4A, which we show is not the case in vivo.
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