A Renaissance in Nepovirus Research Provides New Insights Into Their Molecular Interface With Hosts and Vectors.

Nepoviruses supplied seminal landmarks to the historical trail of plant virology. Among the first agriculturally relevant viruses recognized in the late 1920s and among the first plant viruses officially classified in the early 1970s, nepoviruses also comprise the first species for which a soil-borne ectoparasitic nematode vector was identified. Early research on nepoviruses shed light on the genome structure and expression, biological properties of the two genomic RNAs, and mode of transmission. In recent years, research on nepoviruses enjoyed an extraordinary renaissance. This resurgence provided new insights into the molecular interface between viruses and their plant hosts, and between viruses and dagger nematode vectors to advance our understanding of some of the major steps of the infectious cycle. Here we examine these recent findings, highlight ongoing work, and offer some perspectives for future research.

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015).

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

Cheravirus and Sadwavirus: two unassigned genera of plant positive-sense single-stranded RNA viruses formerly considered atypical members of the genus Nepovirus (family Comoviridae).

The genus Nepovirus (family Comoviridae) was known both for a good level of homogeneity and for the presence of atypical members. In particular, the atypical members of the genus differed by the number of capsid protein (CP) subunits. While typical nepoviruses have a single CP subunit with three structural domains, atypical nepoviruses have either three small CP subunits, probably corresponding to the three individual domains, or a large and a small subunit, probably containing two and one structural domains, respectively. These differences are corroborated by hierarchical clustering based on sequences derived from both genomic RNAs. Therefore, these atypical viruses are now classified in two distinct genera, Cheravirus (three CP subunits; type species Cherry rasp leaf virus) and Sadwavirus (two CP subunits; type species Satsuma dwarf virus).

Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor.

The 3C-like proteinase (Pro) from Tomato ringspot virus (genus Nepovirus) is responsible for the processing of the RNA1-encoded (P1) and RNA2-encoded (P2) polyproteins. Cleavage between the VPg and Pro domains is inefficient in vitro and in E. coli, resulting in the accumulation of the VPg-Pro. In this study, we have compared the trans-activity of the Pro and VPg-Pro on various P1- and P2-derived precursors. Recombinant Pro and VPg-Pro were partially purified using an E. coli expression system. A mutation of the VPg-Pro cleavage site was introduced into the VPg-Pro to prevent slow release of the Pro. The Pro was five to ten times more active than the VPg-Pro on two P2 cleavage sites (at the N- and C-termini of the movement protein domain) and was approximately two times more active than the VPg-Pro on the third P2 cleavage site (between the X3 and X4 domains). Neither the Pro nor the VPg-Pro could cleave in trans P1-derived substrates containing the three cleavage sites delineating the X1, X2, putative NTP-binding protein and VPg domains. These results are discussed in light of the possible regulation of the proteinase activity during virus replication.

Mutagenesis of amino acids at two tomato ringspot nepovirus cleavage sites: effect on proteolytic processing in cis and in trans by the 3C-like protease.

Tomato ringspot nepovirus (ToRSV) encodes two polyproteins that are processed by a 3C-like protease at specific cleavage sites. Analysis of ToRSV cleavage sites identified previously and in this study revealed that cleavage occurs at conserved Q/(G or S) dipeptides. In addition, a Cys or Val is found in the -2 position. Amino acid substitutions were introduced in the -6 to +1 positions of two ToRSV cleavage sites: the cleavage site between the protease and putative RNA-dependent RNA polymerase, which is processed in cis, and the cleavage site at the N-terminus of the movement protein, which is cleaved in trans. The effect of the mutations on proteolytic processing at these sites was tested using in vitro translation systems. Substitution of conserved amino acids at the -2, -1, and +1 positions resulted in a significant reduction in proteolytic processing at both cleavage sites. The effects of individual substitutions were stronger on the cleavage site processed in trans than on the one processed in cis. The cleavage site specificity of the ToRSV protease is discussed in comparison to that of related proteases.

Expression of the tomato ringsport nepovirus movement and coat proteins in protoplasts.

Tomato ringspot nepovirus (TomRSV) produces a 45 kDa movement protein and a 58 kDa coat protein in infected plants. Accumulation of the movement protein in relation to that of the coat protein was studied in infected protoplasts using a monoclonal antibody against the movement protein and polyclonal antibodies against the coat protein. Unlike most other viral movement proteins, the TomRSV movement protein was present at late stages of infection. Pulse-chase labelling experiments revealed that the release of the movement protein from the precursor polyprotein was coordinated with that of the coat protein. However, the movement protein was less stable than the coat protein in the extractable fraction of the protoplasts. The expression pattern of the TomRSV movement protein is discussed in the light of the proposed mechanism of cell-to-cell movement of virus-like particles through tubular structures composed of the movement protein.

Tomato ringspot nepovirus protease: characterization and cleavage site specificity.

We have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have demonstrated that proteins produced from these clones possess a proteolytic activity in trans on the cleavage site between the TomRSV movement and coat proteins. By amino acid homology of the TomRSV 3C-related protease with other nepo- and comovirus proteases, His1283, Glu1331 (or Asp1354) and Cys1433 have been predicted to constitute the catalytic triad. Site-directed mutagenesis of His1283 to Asp abolished the TomRSV protease activity, in vitro and in vivo. The cleavage site between the TomRSV movement and coat proteins has been determined to be Q/G, by direct protein sequencing. Previously, His1451 located in the substrate binding pocket of the TomRSV 3C-related protease has been suggested to be involved in the cleavage site specificity. We show that an inactive TomRSV 3C-related protease is obtained after substitution of His1451 with Leu. These results are discussed in light of the possible relation of the TomRSV 3C-related protease to 3C-related proteases of nepo-, como- and potyviruses.

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons.

A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll(®) gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the ß-glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.

Analysis of figwort mosaic virus (plant pararetrovirus) polyadenylation signal.

Analysis of the cauliflower mosaic virus (CaMV) polyadenylation (poly(A)) signal has revealed several striking differences to poly(A) signals from animal genes such as the absence of activating sequences downstream from the cleavage site. Instead, upstream sequences were shown to induce recognition of an AAUAAA sequence. To test whether these features are representative of other plant pararetrovirus poly(A) signals, a characterization of the figwort mosaic virus (FMV) poly(A) signal is presented here. The FMV RNAs were isolated from infected plants and mapped, and the different elements composing the FMV poly(A) signal were identified. Multiple upstream sequences were found to be essential for efficient processing at the FMV poly(A) site and could be replaced by the CaMV upstream elements. The FMV upstream sequences showed homologies to other characterized upstream sequences from CaMV, from animal viruses, and from plant poly(A) signals. Surprisingly, neither the FMV nor the CaMV upstream elements could induce recognition of an AAUAAA sequence present in the FMV poly(A) signal, instead a UAUAAA sequence 55 nucleotides further downstream was utilized. It is proposed that additional features may be required for appropriate cleavage such as the context of the AAUAAA-like sequence or perhaps the cleavage site itself.

Characterization and subcellular localization of tomato ringspot nepovirus putative movement protein.

Sequence comparison of the tomato ringspot nepovirus (TomRSV) genome with related viruses suggested that the region of the RNA-2-encoded polyprotein immediately upstream of the coat protein may be involved in the cell-to-cell movement of the virus (Rott et al., 1991, J. Gen. Virol. 72, 1505-1514). To further study the role of this portion of the genome, monoclonal antibodies against the putative movement protein were raised. Western blots of plant extracts allowed the detection of a viral nonstructural protein of M(r) 45K present only in TomRSV-infected tissues. Immunogold-labeling studies revealed that in Nicotiana clevelandii the putative movement protein was found only in infected cells immediately adjacent to the necrotic tissue, and that it was associated with tubular structures containing virus-like particles present in or near the cell wall. This provides further evidence that this protein is involved in the cell-to-cell movement of the virus and that this movement might take place via the formation of tubular structures.

Analysis of cauliflower mosaic virus RNAs in Brassica species showing a range of susceptibility to infection.

Cauliflower mosaic virus (CaMV) is a plant pararetrovirus i.e., a DNA virus that replicates through reverse transcription of its terminally redundant genomic RNA (the 35 S RNA). In this study, the absolute levels and relative ratios of CaMV-encoded RNA species were analyzed in Brassica host plants with different susceptibilities to infection. As reported previously, only very low levels of CaMV RNAs were detected in plants of low susceptibility such as cauliflower. Early in infection, a large proportion of these RNAs were the "short-stop" RNA: a 180-nucleotide RNA generated by mRNA 3' end processing at the first encounter of the polyadenylation [poly(A)] signal rather than at the second encounter by which the 35 S RNA is generated. In contrast, in highly susceptible plants such as turnip, high levels of CaMV RNAs were detected, and the short-stop RNA represented only a small fraction of the RNA. In leaf protoplasts, bypass of the poly(A) signal was similar in all Brassica species. Finally, the ratio of the 19 S RNA, a subgenomic RNA encoding a post-transcriptional trans-activator, to the 35 S RNA was lower in cauliflower than in turnip. These results are discussed in light of the CaMV life cycle.

Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.

A dissection of the cauliflower mosaic virus polyadenylation signal.

Mutagenesis analysis of the polyadenylation [poly(A)] signal from the cauliflower mosaic virus (CaMV), a plant pararetrovirus, revealed striking differences to known vertebrate poly(A) signals. Our results show that (1) the AATAAA sequence is necessary for efficient cleavage at the poly(A) site, although the requirement for an authentic AATAAA might be less stringent in plant than in vertebrate cells; (2) surprisingly and in contrast to the majority of vertebrate poly(A) signals, the sequences downstream of the CaMV poly(A) site do not influence processing efficiency drastically although they affect the precision of cleavage; and (3) deletion of sequences upstream of the CaMV AATAAA sequence decreased processing at the CaMV site dramatically, suggesting the presence of one or several positively acting upstream elements. An oligonucleotide consisting of CaMV upstream sequences could induce the recognition of a normally silent exogenous poly(A) signal when inserted upstream of its AATAAA motif.

Closely spaced and divergent promoters for an aminoacyl-tRNA synthetase gene and a tRNA operon in Escherichia coli. Transcriptional and post-transcriptional regulation of gltX, valU and alaW.

The transcription of the gltX gene encoding the glutamyl-tRNA synthetase and of the adjacent valU and alaW tRNA operons of Escherichia coli K-12 has been studied. The alaW operon containing two tRNA(GGCAla) genes, is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand. The valU operon, containing three tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN) genes, is adjacent to gltX and is transcribed from the opposite strand. Its only promoter is upstream from the gltX promoters. The gltX gene transcript is monocistronic and its transcription initiates at three promoters, P1, P2 and P3. The transcripts from one or more of these promoters are processed by RNase E to generate two major species of gltX mRNA, which are stable and whose relative abundance varies with growth conditions. The stability of gltX mRNA decreases in an RNase E- strain and its level increases with growth rate about three times more than that of the glutamyl-tRNA synthetase. The 5' region of these mRNAs can adopt a stable secondary structure (close to the ribosome binding site) that is similar to the anticodon and part of the dihydroU stems and loops of tRNA(Glu), and which might be involved in translational regulation of GluRS synthesis. The gltX and valU promoters share the same AT-rich and bent upstream region, whose position coincides with the position of the upstream activating sequences of tRNA and rRNA promoters to which they are similar. This suggests that gltX and valU share transcriptional regulatory mechanisms.

Proximity to the promoter inhibits recognition of cauliflower mosaic virus polyadenylation signal.

The replication of retroviruses and pararetroviruses (such as caulimo- and hepadnaviruses) involves the production of terminally redundant genomic-length RNA. The sequence repeated at both ends of the RNA (the R region) contains a polyadenylation signal, and for production of full-length RNA the version of this at the 5' end of the template must be bypassed by RNA polymerase, but the version at the 3' end must be recognized. This implies that the position of the polyadenylation signal determines its efficiency, and we report here experiments aimed at investigating the basis of this phenomenon. Our results with cauliflower mosaic virus suggest that proximity to the transcription initiation site inhibits messenger RNA 3'-end processing directed by polyadenylation signals.

Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA.

We have studied the influence of the 600 nt long leader sequence of cauliflower mosaic virus 35S RNA on downstream translation. Plant protoplasts were transfected with plasmids expressing a CAT reporter gene from a mRNA, containing wild-type or mutant forms of the 35S RNA leader. Deletion analysis revealed the presence of three separate stimulatory sequence regions, S1, S2 and S3. The latter two interact with each other to enhance downstream translation 5- to 10-fold. This enhancement was not observed in protoplasts from a non-host plant. In the absence of either S2 or S3, the region I2, located in between, exerts an inhibitory effect on downstream translation, probably due to the presence of short open reading frames. Expression of a reporter gene inserted into I2 increases 2-fold upon deletion of either S2 or S3. We propose that mRNA regions S2 and S3 form a complex with cellular factors that allows scanning ribosomes to bypass region I2.

Posttranscriptional trans-activation in cauliflower mosaic virus.

The ability of plant cells to translate dicistronic mRNAs that mimic a segment of the polycistronic 35S RNA from cauliflower mosaic virus has been tested. The chloramphenicol acetyltransferase and beta-glucuronidase open reading frames (ORFs) were fused in-frame to the second viral cistron (ORF I). Efficient reporter expression from the corresponding plasmids in plant protoplasts was observed only upon cotransfection with viral DNA. The trans-activating gene maps at ORF VI, which is expressed from a separate, monocistronic messenger (19S RNA). Deletion analysis shows that trans-activation selectively enhances downstream gene expression; the high expression of the upstream ORF is not further increased. The major reporter transcript remained bicistronic upon trans-activation, and its abundance varied only to a limited extent. Results indicate that trans-activation enhances the translation of downstream ORFs on polycistronic mRNAs derived from cauliflower mosaic virus.

Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader.

The effect of the 600 nucleotide-long CaMV 35S RNA 5' leader sequence on the expression of downstream genes was analyzed both in plant protoplasts and in vitro. For transient expression studies in protoplasts derived from host and nonhost plants, the bacterial chloramphenicol acetyl transferase (CAT) gene was fused to the initiation codon of ORF VII. The leader sequence reduced CAT expression two- to four-fold in protoplasts derived from three host species, but 10- to 50-fold in protoplasts derived from three different nonhost species. For in-vitro studies the 35S promoter was replaced by the SP6 promoter. The leader reduced in-vitro translation of SP6 transcripts approximately six-fold, indicating that at least part of the inhibition observed in protoplasts is directly due to the interference of the leader sequence with translation. Other steps in gene expression that may also be affected are discussed.

The leading sequence of caulimovirus large RNA can be folded into a large stem-loop structure.

The 600 nt long sequences preceeding the first large ORFs (ORF VII) of three caulimoviruses, although varying in primary sequence, can be folded into a large stem/loop structure centered around a conserved stretch of 36 nucleotides. Deletions of the conserved sequence delay symptom appearance considerably, but do not affect expression of a reporter gene in plant protoplasts. Another striking similarity between the leaders concerns the number and distribution of small open reading frames (sORF) they carry. Expression of two of these sORFs was tested by fusion of a reporter gene: both were expressed in plant protoplasts.

Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases.

The gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli and adjacent regulatory regions was isolated and sequenced. The structural gene encodes a protein of 471 amino acids whose molecular weight is 53,810. The codon usage is that of genes highly expressed in E. coli. The amino acid sequence deduced from the nucleotide sequence of the gltX gene was confirmed by mass spectrometry of large peptides derived from the glutamyl-tRNA synthetase. The observed peptides confirm 73% of the predicted sequence, including the NH2-terminal and the COOH-terminal segments. Sequence homology between the glutamyl-tRNA synthetase and other aminoacyl-tRNA synthetases of E. coli was found in four segments. Three of them are aligned in the same order in all the synthetases where they are present, but the intersegment spacings are not constant; these ordered segments may come from a progenitor to which other domains were added. Starting from the NH2-end, the first two segments are part of a longer region of homology with the glutaminyl-tRNA synthetase, without need for gaps; its size, about 100 amino acids, is typical of a single folding domain. In the first segment, containing sequences homologous to the HIGH consensus, the homology is consistent with the following evolutionary linkage: gltX----glnS----metS----ileS and tyrS.