Characterization of a new densovirus infecting the green peach aphid Myzus persicae.

A new icosahedral DNA virus was isolated from aphids (Myzus persicae) that showed abnormal growth and development. The purified virus particles have a diameter of 20 nm and contain a single-stranded DNA molecule of approximately 5.7 kb. The viral particles are composed of five structural proteins (92, 85, 68, 64, and 57 kDa). As the main biophysical properties of this virus are similar to those of the members of the genus Densovirus it was tentatively named Myzus persicae densovirus (MpDNV). A PCR-based detection method and a polyclonal antiserum raised against MpDNV allowed the detection of the virus in a single-infected aphid. MpDNV is immunologically related to Junonia coenia densovirus, but not to other members of the subfamily Densovirinae. Biological assays showed that MpDNV could be both transmitted transovarially and horizontally via honeydew and saliva. MpDNV was able to infect whiteflies but not other aphid species tested.

A new virus infecting Myzus persicae has a genome organization similar to the species of the genus Densovirus.

The genomic sequence of a new icosahedral DNA virus infecting Myzus persicae has been determined. Analysis of 5499 nt of the viral genome revealed five open reading frames (ORFs) evenly distributed in the 5' half of both DNA strands. Three ORFs (ORF1-3) share the same strand, while two other ORFs (ORF4 and ORF5) are detected in the complementary sequence. The overall genomic organization is similar to that of species from the genus DENSOVIRUS: ORFs 1-3 most likely encode the non-structural proteins, since their putative products contain conserved replication motifs, NTP-binding domains and helicase domains similar to those found in the NS-1 protein of parvoviruses. The deduced amino acid sequences from ORFs 4 and 5 show sequence similarities with the structural proteins of the members of the genus DENSOVIRUS: These data indicate that this virus is a new species of the genus Densovirus in the family PARVOVIRIDAE: The virus was tentatively named Myzus persicae densovirus.

Sequence analysis and genomic organization of Aphid lethal paralysis virus: a new member of the family Dicistroviridae.

The complete nucleotide sequence of the genomic RNA of an aphid-infecting virus, Aphid lethal paralysis virus (ALPV), has been determined. The genome is 9812 nt in length and contains two long open reading frames (ORFs), which are separated by an intergenic region of 163 nt. The first ORF (5' ORF) is preceded by an untranslated leader sequence of 506 nt, while an untranslated region of 571 nt follows the second ORF (3' ORF). The deduced amino acid sequences of the 5' ORF and 3' ORF products respectively showed similarity to the non-structural and structural proteins of members of the newly recognized genus Cripavirus (family Dicistroviridae). On the basis of the observed sequence similarities and identical genome organization, it is proposed that ALPV belongs to this genus. Phylogenetic analysis showed that ALPV is most closely related to Rhopalosiphum padi virus, and groups in a cluster with Drosophila C virus and Cricket paralysis virus, while the other members of this genus are more distantly related. Infectivity experiments showed that ALPV can not only infect aphid species but is also able to infect the whitefly Trialeurodes vaporariorum, extending its host range to another family of the order Hemiptera.

Nucleotide sequence and genomic organization of an ophiovirus associated with lettuce big-vein disease.

The complete nucleotide sequence of an ophiovirus associated with lettuce big-vein disease has been elucidated. The genome consisted of four RNA molecules of approximately 7.8, 1.7, 1.5 and 1.4 kb. Virus particles were shown to contain nearly equimolar amounts of RNA molecules of both polarities. The 5'- and 3'-terminal ends of the RNA molecules are largely, but not perfectly, complementary to each other. The virus genome contains seven open reading frames. Database searches with the putative viral products revealed homologies with the RNA-dependent RNA polymerases of rhabdoviruses and Ranunculus white mottle virus, and the capsid protein of Citrus psorosis virus. The gene encoding the viral polymerase appears to be located on the RNA segment 1, while the nucleocapsid protein is encoded by the RNA3. No significant sequence similarities were observed with other viral proteins. In spite of the morphological resemblance with species in the genus Tenuivirus, the ophioviruses appear not to be evolutionary closely related to this genus nor any other viral genus.

Mechanical transmission of poleroviruses.

Previously, transmission of poleroviruses has relied solely on the use of their aphid vectors. Biolistic inoculation allowed for the first time the mechanical transmission of Beet western yellows virus (BWYV) and Potato leafroll virus (PLRV) to several host plants. Inoculation with purified preparations and viral RNA extracts of PLRV resulted in 30-50% systemically infected Nicotiana occidentalis P1 plants and 15-30% infected Nicotiana clevelandii plants. Particle bombardment was also used successfully to infect N. clevelandii plants with in vitro RNA transcripts of full-length cDNA of BWYV.

Identifying the determinants in the equatorial domain of Buchnera GroEL implicated in binding Potato leafroll virus.

Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (Buchnera sp.). Mutational analysis of GroEL from the primary endosymbiont of Myzus persicae (MpB GroEL) revealed that the amino acids mediating binding of Potato leafroll virus (PLRV; Luteoviridae) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain.

Isolation and characterization of APSE-1, a bacteriophage infecting the secondary endosymbiont of Acyrthosiphon pisum.

A bacteriophage infecting the secondary endosymbiont of the pea aphid Acyrthosiphon pisum was isolated and characterized. The phage was tentatively named bacteriophage APSE-1, for bacteriophage 1 of the A. pisum secondary endosymbiont. The APSE-1 phage particles morphologically resembled those of species of the Podoviridae. The complete nucleotide sequence of the bacteriophage APSE-1 genome was elucidated, and its genomic organization was deduced. The genome consists of a circularly permuted and terminally redundant double-stranded DNA molecule of 36524 bp. Fifty-four open reading frames, putatively encoding proteins with molecular masses of more than 8 kDa, were distinguished. ORF24 was identified as the gene coding for the major head protein by N-terminal amino acid sequencing of the protein. Comparison of APSE-1 sequences with bacteriophage-derived sequences present in databases revealed the putative function of 24 products, including the lysis proteins, scaffolding protein, transfer proteins, and DNA polymerase. This is the first report of a phage infecting an endosymbiont of an arthropod.

Faba bean necrotic yellows virus (genus Nanovirus) requires a helper factor for its aphid transmission.

Purified faba bean necrotic yellows virus (FBNYV; genus Nanovirus) alone is not transmissible by its aphid vector, Acyrthosiphon pisum, regardless of whether it is acquired from artificial diets or directly microinjected into the aphid's hemocoel. The purified virus contains all of the genetic information required for its infection cycle as it readily replicated in cowpea protoplasts and systemically infected Vicia faba seedlings that were biolistically inoculated using gold particles coated with intact virions or viral DNA. The bombarded plants not only developed the typical disease syndrome, thus indicating that FBNYV is the sole causal agent of the disease, but also served as a source from which the virus was readily acquired and transmitted by A. pisum. The defect of the purified virus in aphid transmissibility suggests that FBNYV requires a helper factor (HF) for its vector transmission that is either nonfunctional or absent in purified virus suspensions. The requirement for an HF was confirmed in complementation experiments using two distinct isolates of the virus. These experiments revealed that aphids transmitted the purified virus isolate from artificial diets only when they had fed previously on plants infected with the other FBNYV isolate. Also, microinjected FBNYV, which persisted to the same extent in A. pisum as naturally acquired virus, was transmissible when aphids had acquired the HF from infected plants. This suggests that one of the functions of the HF in the transmission process is to facilitate virus transport across the hemocoel-salivary gland interface.

Recognition and receptors in virus transmission by arthropods.

Fundamental knowledge of the molecular mechanisms underlying virus transmission by arthropods is a prerequisite for the creation of new strategies to modulate vector competence. There have been several recent advances in identifying the viral and vector determinants involved in virus recognition, attachment and retention.

The genome-linked protein (VPg) of southern bean mosaic virus is encoded by the ORF2.

The sequence of the 20 N-terminal amino acids of the viral protein (VPg) which is covalently attached to the genomic RNA of the bean strain of Southern bean mosaic virus (SBMV-B) has been determined. The obtained VPg sequence mapped to position 327 to 346 of the SBMV-B ORF2 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Thus indicating that the sobemovirus genomic arrangement is similar to that of subgroup II luteoviruses. Comparison with other viral sequences revealed a high similarity with the sequence of the ORF2-product of the cowpea strain of SBMV (SBMV-C). No significant similarities were detected with amino acid sequences derived of other sobemoviruses or non-related viruses.

Potato leafroll virus binds to the equatorial domain of the aphid endosymbiotic GroEL homolog.

A GroEL homolog with a molecular mass of 60 kDa, produced by the primary endosymbiotic bacterium (a Buchnera sp.) of Myzus persicae and released into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV). Like other luteoviruses and pea enation mosaic virus, PLRV readily binds to extracellular Buchnera GroEL, and in vivo interference in this interaction coincides with reduced capsid integrity and loss of infectivity. To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL, the groE operon of the primary endosymbiont of M. persicae (MpB groE) and its flanking sequences were characterized and the PLRV-binding domain of Buchnera GroEL was identified by deletion mutant analysis. MpB GroEL has extensive sequence similarity (92%) with Escherichia coli GroEL and other members of the chaperonin-60 family. The genomic organization of the Buchnera groE operon is similar to that of the groE operon of E. coli except that a constitutive promoter sequence could not be identified; only the heat shock promoter was present. By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB GroEL (expressed in E. coli) as it did to wild-type MpB GroEL. Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain and not in the apical domain which is generally involved in polypeptide binding and folding. Buchnera GroEL mutants lacking the entire equatorial domain or parts of it lost the ability to bind PLRV. The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide. Both the N- and C-terminal regions of the equatorial domain were implicated in virus binding.

Nucleotide sequence and genomic organization of Acyrthosiphon pisum virus.

The nucleotide sequence of the genomic RNA of Acyrthosiphon pisum virus was determined. The APV genome is 10,016 nucleotides in length, excluding the 3'-end poly(A) track, and contains two large open reading frames (ORFs), encoding proteins of 296,340 and 63,279 Da. The ORF1 is preceded by an untranslated leader sequence of 267 nucleotides. The ORF1 product contains sequence motifs characteristic of RNA-dependent RNA polymerases, chymotrypsin-like proteases, and helicases. Interviral sequence comparison revealed significant similarities with viruses belonging to the so-called picornavirus superfamily. The ORF2 is most likely expressed by a -1 translational frameshift and is followed by an untranslated sequence of 222 nucleotides. Internal amino acid sequences of three capsid proteins (66K, 34K, 23/24K) were determined. Comparison of the obtained amino acid sequences with the APV sequence disclosed that the structural proteins are located in the 3'-terminal half of the genome. The 34K protein is encoded by the ORF1, while the 66K protein contains both ORF1-(34K) and ORF2-derived sequences and is probably expressed by a translational frameshift. The 23/24K proteins most likely arise by proteolytic breakdown of the 34K protein. Although the deduced APV genomic organization in some aspects resembles that of the picornaviruses, its overall genomic organization indicates that APV is a distinct species only distantly related to the Picornaviridae.

Characteristics of acyrthosiphon pisum virus, a newly identified virus infecting the pea aphid.

A new virus was isolated from the pea aphid, Acyrthosiphon pisum, and tentatively named Acyrthosiphon pisum virus (APV). The isometric virus particles were approximately 31 nm in diameter and contained a single-stranded RNA molecule of approximately 10 kb. Four structural proteins were observed with molecular masses of approximately 23.3, 24.2, 34.5, and 66.2 kDa. The 34.5-kDa capsid protein was the most abundant product in purified virions. Computer-assisted analysis revealed no significant homology between an internal sequence of 37 amino acids of the 34.5-kDa protein of APV and other polypeptides of viral origin. APV was not immunologically related to other ssRNA viruses from hemipteroid insects, such as aphid lethal paralysis virus, Rhopalosiphum padi virus, and Nezara viridula virus type 1. Immunolocalization on ultrathin sections of 3-day-old nymphs of A. pisum showed that APV antigen was predominantly present in the epithelial cells of the digestive tract. Virus particles were also observed associated with the microvilli of the intestine. Occasionally, muscle cells and mycetocyte cells were found infected. Purified APV, fed to 1-day-old A. pisum nymphs, significantly reduced the growth of the aphid and increased the time needed to reach maturity.

The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera GroEL and is essential for virus persistence in the aphid.

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.

The genome-linked protein of potato leafroll virus is located downstream of the putative protease domain of the ORF1 product.

The sequence of the 32 N-terminal amino acids of the protein (VPg) which is covalently linked to the RNA of potato leafroll virus has been determined. The obtained VPg sequence mapped to position 400 to 431 of the PLRV ORF1 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Comparison with other viral sequences revealed significant similarities with the ORF1 products of beet western yellows virus, cucurbit aphid-borne yellows virus, and beet mild yellowing virus.

Expression of the potato leafroll virus ORF0 induces viral-disease-like symptoms in transgenic potato plants.

The role of the open reading frame 0 (ORF0) of luteoviruses in the viral infection cycle has not been resolved, although the translation product (p28) of this ORF has been suggested to play a role in host recognition. To investigate the function of the potato leafroll luteovirus (PLRV) p28 protein, transgenic potato plants were produced containing the ORF0. In the lines in which the ORF0 transcripts could be detected by Northern (RNA) analysis, the plants displayed an altered phenotype resembling virus-infected plants. A positive correlation was observed between levels of accumulation of the transgenic transcripts and severity of the phenotypic aberrations observed. In contrast, potato plants transformed with a modified, untranslatable ORF0 sequence were phenotypically indistinguishable from wild-type control plants. These results suggest that the p28 protein is involved in viral symptom expression. Southern blot analysis showed that the transgenic plants that accumulated low levels of ORF0 transcripts detectable only by reverse transcription-polymerase chain reaction, contained methylated ORF0 DNA sequences, indicating down-regulation of the transgene provoked by the putatively unfavorable effects p28 causes in the plant cell.

Endosymbiotic bacteria associated with circulative transmission of potato leafroll virus by Myzus persicae.

In order to understand the molecular mechanisms underlying circulative transmission of potato leafroll virus (PLRV) by aphids, we screened Myzus persicae proteins as putative PLRV binding molecules using a virus overlay assay of protein blots. In this way, we found that purified PLRV particles exhibited affinity for five aphid proteins. The one most readily detected has an M(r) of 63K, and was identified as symbionin. This is the predominant protein synthesized by the bacterial endosymbiont of the aphid and is released into the haemolymph. Since further studies clearly showed that PLRV particles also bind to native symbionin, it was envisaged that virus particles when acquired into the haemocoel of an aphid interact with symbionin. Inhibition of prokaryotic protein synthesis by feeding M. persicae nymphs on an antibiotic-containing artificial diet prior to PLRV acquisition reduced virus transmission by more than 70%. The major coat protein of the virus was found to be degraded in the antibiotic-treated aphids; this would obviously have resulted in an increased exposure of viral RNA to enzymic breakdown and concomitant loss of infectivity. For these reasons we conclude that endosymbiotic bacteria play a crucial role in determining the persistent nature of PLRV in the aphid haemolymph and that symbionin is probably the key protein in this interaction.

Expression of the potato leafroll luteovirus coat protein gene in transgenic potato plants inhibits viral infection.

Transgenic potato plants, cultivar Désirée, were produced that contained the coat protein gene of potato leafroll luteovirus (PLRV). The transformed potato plants expressed the PLRV coat protein (CP) RNA sequences but accumulation of coat protein in transgenic tissues could not be detected. Upon inoculation with PLRV, the PLRV CP RNA expressing potato plants showed a reduced rate of virus multiplication.

Nucleotide sequence and organization of potato leafroll virus genomic RNA.

The nucleotide sequence of the genomic RNA of potato leafroll virus was determined and its genetic organization deduced. The RNA is 5882 nucleotides long and contains 6 open reading frames (ORFs) encoding proteins of 70, 70, 56, 28, 23 and 17 kDa. The putative genes for the coat protein (23 kDa) and the RNA-dependent RNA polymerase (70 kDa) were identified by interviral amino acid sequence homologies. For expression of the different ORFs, translational frameshift and readthrough events are proposed.