Phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro

We have investigated the functional significance of phosphoserine residues that lie in the L protein-binding domain between amino acids 213 and 247 of the phosphoprotein (NS) of vesicular stomatitis virus. A series of mutant NS proteins were made by cell-free translation of mRNAs transcribed from the cloned gene. Site-directed substitution of alanine for both serine 236 and serine 242 essentially abolished RNA synthesis catalyzed by the NS-L complex. Substitution of either of these serines reduced RNA synthesis by 75%. Serine 218 played no major role in RNA synthesis. Phosphorylation of NS by the L protein was abrogated by substitution of either serine 236 or serine 242. These results indicate that phosphorylation of serines 236 and 242 in the NS protein regulates its binding with the L protein and the N-RNA template and is essential for activation of viral RNA synthesis.     

Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase

The minimal RNA synthesis machinery of non-segmented negative-strand RNA viruses comprises a genomic RNA encased within a nucleocapsid protein (N-RNA), and associated with the RNA-dependent RNA polymerase (RdRP). The RdRP is contained within a viral large (L) protein, which associates with N-RNA through a phosphoprotein (P). Here, we define that vesicular stomatitis virus L initiates synthesis via a de-novo mechanism that does not require N or P, but depends on a high concentration of the first two nucleotides and specific template requirements. Purified L copies a template devoid of N, and P stimulates L initiation and processivity. Full processivity of the polymerase requires the template-associated N protein. This work provides new mechanistic insights into the workings of a minimal RNA synthesis machine shared by a broad group of important human, animal and plant pathogens, and defines a mechanism by which specific inhibitors of RNA synthesis function.     

Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.     

Sensitivity of the Polymerase of Vesicular Stomatitis Virus to 2′ Substitutions in the Template and Nucleotide Triphosphate during Initiation and Elongation

The RNA synthesis machinery of non-segmented negative-sense RNA viruses comprises a ribonucleoprotein complex of the genomic RNA coated by a nucleocapsid protein (N) and associated with polymerase. Work with vesicular stomatitis virus (VSV), a prototype, supports a model of RNA synthesis whereby N is displaced from the template to allow the catalytic subunit of the polymerase, the large protein (L) to gain access to the RNA. Consistent with that model, purified L can copy synthetic RNA that contains requisite promoter sequences. Full processivity of L requires its phosphoprotein cofactor and the template-associated N. Here we demonstrate the importance of the 2′ position of the RNA template and the substrate nucleotide triphosphates during initiation and elongation by L. The VSV polymerase can initiate on both DNA and RNA and can incorporate dNTPs. During elongation, the polymerase is sensitive to 2′ modifications, although dNTPs can be incorporated, and mixed DNA-RNA templates can function. Modifications to the 2′ position of the NTP, including 2′,3′-ddCTP, arabinose-CTP, and 2′-O-methyl-CTP, inhibit polymerase, whereas 2′-amino-CTP is incorporated. The inhibitory effects of the NTPs were more pronounced on authentic N-RNA with the exception of dGTP, which is incorporated. This work underscores the sensitivity of the VSV polymerase to nucleotide modifications during initiation and elongation and highlights the importance of the 2′-hydroxyl of both template and substrate NTP. Moreover, this study demonstrates a critical role of the template-associated N protein in the architecture of the RNA-dependent RNA polymerase domain of L.     

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B post-transfusion hepatitis. Its genome, a (+)-stranded RNA molecule of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)-strand RNA viruses, the replication of HCV is thought to proceed via the initial synthesis of a complementary (-) RNA strand, which serves, in turn, as a template for the production of progeny (+)-strand RNA molecules. An RNA-dependent RNA polymerase has been postulated to be involved in both of these steps. Using the heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA-dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non-structural protein 5B (NS5B). The characterization of the HCV RNA-dependent RNA polymerase product revealed that dimer-sized hairpin-like RNA molecules are generated in vitro, indicating that NS5B-mediated RNA polymerization proceeds by priming on the template via a 'copy-back' mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA- or DNA oligonucleotide primer-dependent RNA synthesis on templates with a blocked 3' end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.     

Translation of Vesicular Stomatitis Messenger RNA by Extracts from Mammalian and Plant Cells

RNA was isolated from polyribosomes of vesicular stomatitis virus (VSV)-infected cells and tested for its ability to direct protein synthesis in extracts of animal and plant cells. In cell-free, non-preincubated extracts of rabbit reticulocytes, the 28S VSV RNA stimulated synthesis of a protein the size of the vesicular stomatitis virus L protein whereas the 13 to 15S RNA directed synthesis of the VSV M, N, NS, and possibly G proteins. In wheat germ extracts, 13 to 15S RNA also directed synthesis of the N, NS, M, and possibly G proteins. Analysis of extracts labeled with formyl [(35)S]methionine showed that the 28S RNA directed the initiation of synthesis of one protein, whereas the 13 to 15S RNA directed initiation of at least four proteins. It is concluded that the 28S RNA encodes only the L protein, whereas the 13 to 15S RNA is a mixture of species, presumably monocistronic, which code for the four other known vesicular stomatitis virus proteins.