Temperature requirements for initiation of RNA-dependent RNA polymerization

To continue the molecular characterization of RNA-dependent RNA polymerases of dsRNA bacteriophages (Cystoviridae), we purified and biochemically characterized the wild-type (wt) and a temperature-sensitive (ts) point mutant of the polymerase subunit (Pol) from bacteriophage phi12. Interestingly, initiation by both wt and the ts phi12 Pol was notably more sensitive to increased temperatures than the elongation step, the absolute value of the nonpermissive temperature being lower for the ts enzyme. Experiments with the Pol subunit of related cystovirus phi6 revealed a similar differential sensitivity of the initiation and elongation steps. This is consistent with the previous result showing that de novo initiation by RdRp from dengue virus is inhibited at elevated temperatures, whereas the elongation phase is relatively thermostable. Overall, these data suggest that de novo RNA-dependent RNA synthesis in many viral systems includes a specialized thermolabile state of the RdRp initiation complex.     

Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) ordinarily exists in electrophoretically distinct hypophosphorylated and hyperphosphorylated forms. Human cytomegalovirus infection induced forms of this subunit whose electrophoretic mobilities were intermediate without decreases in abundance of the original forms. Phosphatase treatment nearly eliminated the intermediate migrating forms. In vitro, the viral protein kinase, UL97, phosphorylated this subunit, a recombinant protein containing the CTD, and peptides containing the CTD consensus sequence, YSPTSPS. Phosphorylation occurred predominantly on serine 5 and was substantially reduced when either serine 2 or 5 was already phosphorylated. The abundance of the intermediate and hypophosphorylated forms was reduced at most twofold during infections in which UL97 was genetically or pharmacologically inhibited. These results identify a new pattern of RNA polymerase II modification induced by virus infection and a viral enzyme that phosphorylates the CTD in vitro, but only possibly in vivo.     

The mouse Pol I terminator is more efficient than the hepatitis delta virus ribozyme in generating influenza-virus-like RNAs with precise 3′ ends in a plasmid-only-based virus rescue system

Reverse genetics systems for generating recombinant influenza viruses are based on two different mechanisms for obtaining the 3′ end of the viral RNA: one uses the self-cleaving hepatitis delta virus ribozyme (HDVR), and the other uses the murine RNA polymerase I (Pol I) terminator. In this study, we employed EGFP and Renilla luciferase reporter constructs to compare the efficiency of both methods. Our results indicate that the murine Pol I terminator was more efficient than the HDVR, which will be helpful in choosing an influenza virus rescue system, as well as in establishing other RNA virus rescue systems.     

Structural comparison of the Autographa californica nuclear polyhedrosis virus-induced RNA polymerase and the three nuclear RNA polymerases from the host, Spodoptera frugiperda.

A partial subunit structure has been determined for the novel RNA polymerase that is induced in fall armyworm (Spodoptera frugiperda) cells upon infection with the Autographa californica nuclear polyhedrosis virus (AcNPV). The putative structure includes nine polypeptides; the complexity of this structure is in accord with the high sedimentation coefficient (15S) estimated for this enzyme. A comparison of the putative stucture of the virus-induced polymerase with those of the three host nuclear RNA polymerases shows that the structure of the viral polymerase is apparently unlike any of the host nuclear polymerases. This conclusion is reinforced by immunoblot experiments that show no cross-reactivity between the virus-induced polymerase and an antiserum directed against Drosophila RNA polymerase II. The virus-induced RNA polymerase appears at the onset of the late phase of infection and still appears when viral DNA synthesis is blocked by aphidicolin. Thus, the virus-induced polymerase seems to be composed of early viral products.