The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2

Abstract

The genome of pea enation mosaic virus (PEMV) consists of two plus-sense RNAs, both of which are required for mechanical transmission. RNA 1 (5706 nucleotides) has strong sequence similarity with members of the luteovirus group, a similarity that is also manifested in the symptomatology, cytopathology and vector transmission of this virus. RNA 2 (4253 nucleotides) is hypothesized to facilitate systemic invasion and mechanical transmission, attributes that distinguish PEMV from the phloem-limited luteoviruses. Sequence analysis of RNA 2 has demonstrated that PEMV is unique among multicomponent viruses in that it lacks Tand 5'-terminal homology between its genomic RNAs. Sequence analysis of RNA 2 has identified an open reading frame encoding a putative product of 65K that contains a series of polymerase-like motifs typical of viral RNA-dependent RNA polymerases. This protein sequence lacks homology with the polymerase encoded on RNA 1 of PEMV, instead being more closely affiliated with the polymerases of viruses related to the carmoand tombusvirus groups. Inoculation of pea protoplasts with RNA transcripts derived from a full-length cDNA clone ofRNA 2 has demonstrated that RNA 2 replicates autonomously in the absence of RNA 1, although comparable inoculation of whole plants failed to establish a systemic infection. There is no evidence that RNA 2 encodes structural proteins, suggesting that encapsidation functions are supplied in trans by RNA 1, comparable to the helper-dependent complexes occurring within the luteovirus group. These data suggest that the PEMV genome can be characterized as a symbiotic association of two taxonomically distinct viral RNAs cooperatively interacting in the establishment of a systemic virus infection.

Mutational evidence that the VPg is involved in the replication and not the movement of Pea enation mosaic virus-1

“…Pea protoplasts were isolated using a modification of the methods of Loesch-Fries & Hall (1980), Demler & de Zoeten (1989) and Demler et al (1993). Briefly, 5 g portions of fully expanded leaves were collected from dark-conditioned, 2-4-week-old pea seedlings.…”

“…8221). Pea seedlings (n 40 plants per mutant) were inoculated with 5-10 µg of combinations of synthetic RNA transcripts of WT-PEMV-2 and WT-PEMV-1 or transcripts of PEMV-1 with mutated VPgs according to Demler et al (1993). Total RNA was extracted from 100 mg newly formed leaves of both symptomatic and asymptomatic plants using the protocol described by Demler et al (1994), or the SV Total RNA Isolation System (Promega).…”

“…Briefly, 5 g portions of fully expanded leaves were collected from dark-conditioned, 2-4-week-old pea seedlings. The leaves were surface sterilized as described by Demler et al (1993), and sliced with a sterile razor blade into 1-3 mm strips. The strips were preplasmolysed by gentle shaking in 100 ml 10 % mannitol for 1 h at room temperature.…”

Molecular biology of umbraviruses: phantom warriors

“…One or both of the carrot-infecting umbraviruses probably occurs worldwide wherever carrots are grown, but they are uncommon in commercial crops because the insecticides used to control carrot fly also control the aphid vectors of CMoV and CMoMV. However, because of their peculiar properties and the attendant difficulties in working with them, the molecular virology of umbraviruses has been developed only in the last decade, when nucleotide sequencing has revealed unique 'umbravirus' features (Demler et al, 1993;Taliansky et al, 1996;Gibbs et al, 1996).Umbraviruses, and in particular their taxonomy, classification and evolution, and their biological properties, such as host range, pathology, geographical distribution, mechanisms of aphid transmission, interaction with assistor luteoviruses and satellite RNAs, have been reviewed in detail (Murant, 1993;Demler et al, 1996b;Naidu et al, 1998;Robinson & Murant, 1999;Taliansky et al, 2000). In this article we will focus on new molecular findings that highlight the distinctive features of umbraviruses that follow from the absence of a capsid protein and virions.…”

Complete Nucleotide Sequence and Organization of the RNA Genome of Groundnut Rosette Umbravirus

“…No motif typical of a helicase domain could be identified in the putative products of GRV ORFs I or 2 (or of ORFs 3 or 4). The overlapping arrangement of ORFs 1 and 2, together with the lack of an AUG initiation codon near the 5' end of ORF2, suggest that ORF2 may be expressed by a -1 ffameshift from ORF1, as has been proposed for the analogous ORFs of PEMV (Demler et al, 1993) and of CMoMV (Gibbs, 1995). This idea is supported by the presence, immediately upstream of the UAG stop codon of GRV ORF 1, of the' shifty' heptanucleotide AAAUUUU.…”