Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA

Abstract

Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.

Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

“…1), and all of these are found in association with virus particles (42). A ribonucleoprotein core that can be released from virions (17), and from the nuclei of infected plants (44), consists of the nucleocapsid (N) protein, the phosphoprotein (M2), and the polymerase (L) protein complexed with SYNV RNA. The glycoprotein (G) is thought to traverse the viral lipid envelope and associate with the matrix (M1) protein, which probably also has a role in coiling of the ribonucleoprotein core (16).…”

“…Two putative regulatory regions, located at the 3Ј and 5Ј ends of the SYNV genome, flank the six minus-sense genes. The 144-nt leader sequence is located at the 3Ј end of the genome (44,51), and the 160-nt untranslated trailer sequence is present at the 5Ј end of the genomic RNA. The transcribed plus-sense leader RNA is encapsidated by the N protein, and the extent of encapsidation is thought to regulate transcription versus replication (49).…”

“…Incubation with tunicamycin, a glycosylation inhibitor, interrupts morphogenesis and leads to accumulation of a striking array of nucleocapsid cores at the periphery of the nuclei and at the outer edges of greatly enlarged viroplasms (43). An active polymerase complex contained within the nucleoprotein core has also been isolated from purified tobacco nuclei (20,44,45). This complex is involved in transcription because it is capable of synthesizing the polyadenylated leader RNA and each of the six mRNAs in a polar fashion to yield decreasing amounts as follows: (44,45).…”

“…An active polymerase complex contained within the nucleoprotein core has also been isolated from purified tobacco nuclei (20,44,45). This complex is involved in transcription because it is capable of synthesizing the polyadenylated leader RNA and each of the six mRNAs in a polar fashion to yield decreasing amounts as follows: (44,45). A temporal switch from transcription to replication is thought to occur at the intermediate stages of the infection process because SYNV genomic RNAs accumulate later than the mRNAs in infected protoplasts (22).…”

Strawberry crinkle virus, a Cytorhabdovirus needing more attention from virologists

“…The 3 ′ leader sequence is transcribed into positive sense RNA, however, positive strand trailer RNA has not been detected. In SYNV and RTYV, the leader sequence is polyadenylated (Wagner et al , 1996;Wang et al , 1999) but there is no evidence for polyadenylation of the LNYV leader (Wetzel et al , 1994). In animal rhabodoviruses, e.g.…”