All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

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The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5′-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5′-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.nonsegmented negative-strand RNA virus | polyribonucleotidyltransferase | RNA-dependent RNA polymerase | L protein | mRNA modification A structural hallmark of eukaryotic mRNA is the presence of the 5′-terminal cap structure ½m 7 Gð5 0 Þpppð5 0 ÞN-, in which 7-methylguanosine (m 7

Increasing exposure of vesicular stomatitis virus particles to ultraviolet radiation caused differential inhibition of the synthesis in vitro of individual mRNA species which code for the viral structural proteins L, G, M, NS, and N. The synthesis of each mRNA species showed single hit kinetics. Target sizes for the expression of each gene were derived from kinetic data, and the molecular weight of the mIRNA coding for the N protein was found to be proportional to the target size of its corresponding gene. The remaining genes had target sizes larger than expected, ranging up to the molecular weight of the entire genome of the vesicular stomatitis virus. These results suggest that RNA transcription is initiated at a single site on the RNA genome and that individual mRNA species are synthesized sequentially. This analysis has allowed the mapping of the genes of this virus in the order 5'-L-G4(M-NS)N-3'.Vesicular stomatitis virus (VSV) contains a single-stranded RNA genome of negative polarity with a molecular weight of approximately 4 X 106 (1). A virion-associated RNA polymerase transcribes the genome RNA in vitro or in vivo into monocistronic mRNAs which can be translated into viral polypeptides in protein-synthesizing systems in vitro (2). mRNA species of molecular weights 2.1, 0.7, and 0.55 X 106 code for the large protein (L) (3), the surface glycoprotein (G), and the nucleocapsid protein (N) of the virion, respectively. Two mRNA species of similar molecular weights (0.38 X 106) code for the remaining membrane (M) and minor (NS) proteins (2). Together, these five mRNAs account for almost all of the coding potential of the VSV genome (4).The precise mechanism for the synthesis of VSV mRNAs is not known. The five monocistronic mRNAs may be synthesized by initiation at multiple sites on the genome RNA, or alternatively, the RNA polymerase may initiate transcription at a single site with subsequent cleavage of the mRNAs from a precursor RNA molecule. The latter model, involving RNA processing, gains indirect support from the following experimental observations. (a) The 5'-termini of the VSV mRNA species synthesized in vitro are blocked, having the structure GpppAp ... (5). Since only the a phosphate of ATP and both the a and ,3 phosphates of GTP are incorporated into the blocked structure, the biosynthesis of this structure may involve the cleavage of a larger RNA molecule generating a free 5'-phosphate which is then blocked with GDP. (b) The 5'-terminal sequence of all of the VSV mRNA species is the same: GpppApApCpApGp ...(6) and the 3'-terminal sequence of the VSV genome is ... PypGpUOH (7). Since these two sequences are not complementary, it follows that synthesis of the mRNAs must commence distal to the 3'-terminus of the genome RNA. Furthermore, to synthesize the complementary 42 S + strand (a required intermediate in the replication process), the RNA polymerase must initiate transcription at the 3'-terminus. These observations together suggest that both types of RNA may be initiated at the same sit...

Autophagy is a multistep process in which cytoplasmic components, including invading pathogens, are captured by autophagosomes that subsequently fuse with degradative lysosomes. Negative-strand RNA viruses, including paramyxoviruses, have been shown to alter autophagy, but the molecular mechanisms remain largely unknown. We demonstrate that human parainfluenza virus type 3 (HPIV3) induces incomplete autophagy by blocking autophagosome-lysosome fusion, resulting in increased virus production. The viral phosphoprotein (P) is necessary and sufficient to inhibition autophagosome degradation. P binds to SNAP29 and inhibits its interaction with syntaxin17, thereby preventing these two host SNARE proteins from mediating autophagosome-lysome fusion. Incomplete autophagy and resultant autophagosome accumulation increase extracellular viral production but do not affect viral protein synthesis. These findings highlight how viruses can block autophagosome degradation by disrupting the function of SNARE proteins.