RNA Triphosphatase Component of the mRNA Capping Apparatus of Paramecium bursaria Chlorella Virus 1

The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by three enzymatic reactions: (i) the 5′ triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA triphosphatase (RTP), (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by S-adenosylmethionine:RNA (guanine-N7) methyltransferase (27). DNA viruses have evolved diverse capping strategies. The mRNAs of papovaviruses, parvoviruses, adenoviruses, and herpesviruses are transcribed in the nucleus by RNA polymerase II (Pol II), and their 5′ ends are modified by the host cell's capping and methylating enzymes. However, vaccinia virus and other poxviruses, which replicate in the cytoplasm, encode and encapsidate with the virus particle a multisubunit RNA polymerase and a complete mRNA capping apparatus (26). African swine fever virus (ASFV), which has a cytoplasmic replication phase, also encodes and encapsidates an RNA polymerase and mRNA capping enzymes (24). Baculoviruses, which replicate in the nucleus of insect cells, use Pol II to transcribe early genes, then switch at later times to a virus-encoded transcription system that includes an RNA polymerase and two cap-forming activities— RTP and RNA guanylyltransferase (4, 5, 14). Paramecium bursaria chlorella virus 1 (PBCV-1) encodes an RNA guanylyltransferase (7), but it is not clear whether it encodes an RNA polymerase and additional mRNA-processing activities. The triphosphatase, guanylyltransferase, and methyltransferase components of the capping apparatus are organized differently in these DNA virus systems. The triphosphatase, guanylyltransferase, and methyltransferase active sites of the vaccinia virus capping enzyme reside in a single 844-amino-acid (844-aa) polypeptide, and the order of the active sites in the primary structure (H2N-triphosphatase/guanylyltransferase/ methyltransferase-COOH) mimics the temporal order of the cap-forming reactions. The triphosphatase and guanylyltransferase active sites of the 464-aa baculovirus capping enzyme are also arrayed in cis in the order H2N-triphosphatase/guanylyltransferase-COOH. The baculovirus capping enzyme is structurally related to the 60-kDa triphosphatase-guanylyltransferase domain of the vaccinia virus capping enzyme. However, baculovirus encodes no discernible homologue of a vaccinia virus RNA (guanine-7)-methyltransferase, and it remains unclear whether a cellular or viral enzyme is responsible for baculovirus cap methylation. PBCV-1 is the prototype of a family of large icosahedral DNA viruses that replicate in unicellular Chlorella-like green algae (29). The 330-kbp linear PBCV-1 genome encodes 375 polypeptides, which makes PBCV-1 one of the most genetically complex viruses known. Its gene expression strategy is understood only crudely (30). It is believed that the infecting DNA is targeted to the nucleus, where the transcription of early genes ensues within 5 to 10 min postinfection. Transcription of the late class of viral genes commences after the onset of viral DNA replication at 60 to 90 min postinfection. The cis-acting DNA signals that control PBCV-1 transcription and the proteins that comprise the viral transcription machinery are unknown. PBCV-1 encodes polypeptides that resemble the cellular transcription factors TFIIB and TFIIS, but there are no discernible PBCV-1 homologues of cellular or viral RNA polymerase subunits. Thus, PBCV-1 either encodes a novel RNA polymerase or its genome is transcribed by one or more of the RNA polymerases of the algal host cell. Almost nothing is known about Chlorella virus mRNA processing events, except for the fact that PBCV-1 encodes its own mRNA guanylyltransferase, which has been purified and characterized biochemically (7). Chlorella virus guanylyltransferase is a 330-aa monomeric polypeptide that catalyzes the transfer of GMP from GTP to the 5′ diphosphate end of RNA to form the GpppN cap structure. Its structure and mechanism have been illuminated in atomic detail by X-ray crystallography (6). Chlorella virus guanylyltransferase is monofunctional and has no intrinsic triphosphatase or methyltransferase activities. It is most closely related to the monofunctional yeast RNA guanylyltransferases, more so than to the multifunctional vaccinia virus capping enzyme or the bifunctional triphosphatase-guanylyltransferases of ASFV or baculovirus. In order to cap its mRNAs, PBCV-1 must either encode its own RTP or rely on the resident enzyme(s) of the host cell to remove the γ-phosphate of the primary transcript so that the diphosphate end can be modified by the PBCV-1 guanylyltransferase. The similarities between Chlorella virus and yeast guanylyltransferases prompted us to search the PBCV-1 proteome for a polypeptide resembling the well-characterized Saccharomyces cerevisiae RTP Cet1p (18). This exercise pinpointed the 193-aa A449R gene product as a candidate RTP. We exploited a yeast-based genetic system for analysis of viral mRNA capping enzymes (12) to show that the Chlorella virus RTP (cvRTP) is functional in vivo in S. cerevisiae in lieu of Cet1p. Purified recombinant cvRTP has intrinsic metal-dependent RTP and nucleoside triphosphatase activities. Mechanistic conservation between cvRTP and yeast RTPs is suggested by mutational analysis of the putative metal-binding site. Our results underscore a close evolutionary connection between the capping apparatus of fungi and Chlorella virus, and they suggest that the capping enzymes of metazoan DNA viruses arose by gene fusion events involving yeast/PBCV-1-like domains.