Mycoviruses of an endophytic fungus can replicate in plant cells: evolutionary implications.

So far there is no record of a specific virus able to infect both fungal and plant hosts in nature. However, experimental evidence shows that some plant virus RdRPs are able to perform replication in trans of genomic or DI RNAs in the yeast Saccharomyces cerevisiae. Furthermore, tobacco mosaic virus was recently shown to replicate in a filamentous ascomycetous fungus. Thus, at least experimentally, some plant viruses can infect some fungi. Endophytic fungi have been reported from many plants and several of these fungi have been shown to contain viruses. Here we tested if mycoviruses derived from a marine plant endophyte can replicate in plant cells. For this purpose, we used partially purified viral particles from isolate MUT4330 of Penicillium aurantiogriseum var. viridicatum which harbors six virus species, some having dsRNA and some positive-strand ssRNA genomes. These were transfected into three distinct plant protoplast cell systems. Time-course analysis of absolute RNA accumulation provided for the first time evidence that viruses of two species belonging to the Partitiviridae and Totiviridae families, can replicate in plant cells without evidence of host adaptation, i.e, changes in their nucleotide sequence.

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs.

RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA.

Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA.

A new tobamovirus infecting tomato crops in Jordan.

In this study, we completed the whole genome sequence of a new tobamovirus isolated from tomato plants grown in greenhouses in Jordan during the spring of 2015. The 6393-nt single-stranded RNA (ssRNA) genome encodes four proteins, as do other tobamoviruses: two replication-related proteins of 126 kDa and 183 kDa, a 30-kDa movement protein (MP) and a 17.5-kDa coat protein (CP). Phylogenetic analysis showed that this virus does not group with either the tomato mosaic virus (ToMV) or the tobacco mosaic virus (TMV) clades. Instead, it stems from a branch leading to the TMV clade. Analysis of possible recombination events between this virus and representative isolates of closely related tomato-infecting tobamoviruses showed that at least one region originated by recombination. We provide evidence that we have identified a new tobamovirus, for which we propose the name "tomato brown rugose fruit virus".

Molecular characterization and detection of plum bark necrosis stem pitting-associated virus.

The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' and 3' ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.

The Lettuce infectious yellows virus (LIYV)-encoded P26 is associated with plasmalemma deposits within LIYV-infected cells.

Cytological, immunological, and mutagenesis approaches were used to identify the viral factors associated with the formation of plasmalemma deposits (PLDs) in whole plants and protoplasts infected by Lettuce infectious yellows virus (LIYV). Transmission electron microscopy and immunogold labeling using polyclonal antibodies to four of the five LIYV RNA 2-encoded large proteins, capsid protein (CP), minor capsid protein (CPm), HSP70 homolog (HSP70h), and P59, showed specific labeling of LIYV virions or virion aggregates around the vesiculated membranous inclusions, but not PLDs in LIYV-infected Nicotiana benthamiana, Nicotiana clevelandii, Lactuca sativa, and Chenopodium murale plants, and Nicotiana tabacum protoplasts. In contrast, antibodies to the RNA 2-encoded P26 showed specific labeling of PLDs but not virions in both LIYV-infected plants and protoplasts. Virion-like particles (VLPs) were seen in protoplasts infected by all LIYV RNA 2 mutants except for the CP (major capsid protein) mutant. PLDs were more difficult to find in protoplasts, but were seen in protoplasts infected by the CP and CPm mutants, but not in protoplasts infected by the P26, HSP70h, or P59 mutants. Interestingly, although the CPm mutant showed VLPs and PLDs, the PLDs did not show associated virions/virion-like particles as was always observed for PLDs seen in protoplasts infected by wild-type LIYV. Immunoblot analyses performed on purified LIYV virions showed that P26 was not detected with purified virions, but was detected in the cell wall, 1000 g and 30,000 g pellet fractions of LIYV-infected plants. These data suggest that P26 is associated with the LIYV-induced PLDs, and in contrast to the other RNA 2-encoded large proteins, P26 is not a virion protein.

Interaction between HSP70 homolog and filamentous virions of the Beet yellows virus.

An HSP70 homolog (HSP70h), encoded by the Closterovirus Beet yellows virus (BYV), functions in viral movement from cell to cell. A previous study revealed that in infected cells, HSP70h colocalizes with the masses of BYV filamentous virions. Here we demonstrate that HSP70h forms a physical complex with BYV virions. This conclusion is based on both the comigration of HSP70h with BYV virions in sucrose density gradients and the coimmunoprecipitation of the HSP70h and BYV capsid protein using anti-HSP70h serum. The HSP70h-virion complex is stable at high concentrations of sodium chloride; its dissociation using sodium dodecyl sulfate, lithium chloride, or alkaline pH was accompanied by virion disassembly. However, the complex formation does not involve covalent bonds between HSP70h and virion components. Each BYV virion contains approximately 10 molecules of HSP70h. The possible role of HSP70h interaction with the virions in cell-to-cell movement of BYV is discussed.

A heterogeneous population of defective RNAs is associated with lettuce infectious yellows virus.

Preparations of dsRNAs and virion RNAs extracted from Nicotiana clevelandii plants infected with the bipartite Lettuce infectious yellows virus (LIYV) were found to contain multiple LIYV RNA species. In addition to the two LIYV genomic RNAs, three types of RNAs were observed: (a) 3' coterminal subgenomic RNAs; (b) RNAs containing LIYV RNA 1 or RNA 2 5' terminus but lacking the 3' terminus; and (c) RNAs with both LIYV RNA 2 3' and 5' termini but each with a central extensive deletion, a structure typical of defective RNAs (D RNAs). No D RNA-like RNAs were detected for LIYV RNA 1. A reverse transcription followed by polymerase chain reaction (RT-PCR) strategy was used to clone from virion RNAs several LIYV RNA 2 D RNAs as cDNAs. Nucleotide sequence analysis of 43 cloned cDNAs showed in some D RNAs the presence of a stretch of 1-5 nt in the junction site that is repeated in the genomic RNA 2 in the two positions flanking the junction site or in close proximity. Some D RNAs contained in the junction site one or several extra nucleotides not present in the LIYV genomic RNA 2. Two of the cloned cDNAs were used to generate in vitro transcripts, and infectivity studies showed that both D RNAs were replication competent in protoplasts when coinoculated with LIYV RNAs 1 and 2 or with only LIYV RNA1. Neither D RNA showed obvious effects upon LIYV RNA 1 and RNA 2 accumulation in coinfected protoplasts. These data suggest that LIYV infections contain a heterogeneous population of LIYV RNA 2 D RNAs, and some are encapsidated into virions.

Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts.

Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.

The satellite RNA of barley yellow dwarf virus-RPV is supported by beet western yellows virus in dicotyledonous protoplasts and plants.

The subgroup II luteovirus barley yellow dwarf virus-RPV (BYDV-RPV) acts as a helper virus for a satellite RNA (satRPV RNA). The subgroup II luteovirus beet western yellows virus (BWYV) and the ST9-associated RNA (ST9a RNA), a BWYV-associated RNA that encodes a polymerase similar to those of subgroup I luteoviruses, were assayed for their ability to support replication of satRPV RNA. SatRPV RNA was replicated in tobacco protoplasts in the presence of BWYV RNA or a mixture of BWYV plus the ST9a RNA, but not in the presence of ST9a RNA alone. ST9a RNA stimulated BWYV RNA accumulation which, in turn, increased the accumulation of satRPV RNA. SatRPV RNA was encapsidated in BWYV capsids primarily as circular monomers, which differs from the linear monomers found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted to Capsella bursa-pastoris plants by aphids only in the presence of BWYV and ST9a RNA. SatRPV RNA reduced accumulation of both BWYV helper and ST9a nonhelper RNAs in plants but did not affect symptoms. The replication of satRPV RNA only in the presence of subgroup II luteoviral RNAs but not in the presence of RNAs with subgroup I-like polymerase genes, in both monocotyledonous and dicotyledonous hosts, suggests that the specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants.

In vitro transcripts from cloned cDNAs of the lettuce infectious yellows closterovirus bipartite genomic RNAs are competent for replication in Nicotiana benthamiana protoplasts.

Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived capped in vitro transcripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned cDNAs of a closterovirus genome.

Maize stripe tenuivirus RNA2 transcripts in plant and insect hosts and analysis of pvc2, a protein similar to the Phlebovirus virion membrane glycoproteins.

The complete sequence of the maize stripe tenuivirus (MStV) RNA2 was determined (3337 nucleotides). RNA2 contains two large open reading frames (ORFs) arranged in an ambisense orientation and specific RNAs of ca. 700 and 2600 nucleotides corresponding to the ORFs were detected in MStV-infected plants and planthoppers. The deduced amino acid sequence of the 23,500 MW protein (pv2) encoded by viral RNA2 (vRNA2) was similar to proteins encoded by the rice stripe (RStV) and rice hoja blanca tenuiviruses vRNA2. Sequence analysis suggested that pv2 is membrane associated. The 93,900 MW protein (pvc2) encoded by viral complementary MStV RNA2 (vcRNA2) was similar to the 94,000 MW protein of RStV RNA2 and to the virion membrane glycoproteins for Phlebovirus members of the Bunyaviridae. The phlebovirus glycoprotein cleavage site was similar to a region in the MStV and RStV proteins suggesting that the tenuivirus pvc2 may be processed analogous to the phlebovirus glycoproteins.

Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.

Symptom severity of beet western yellows virus strain ST9 is conferred by the ST9-associated RNA and is not associated with virus release from the phloem.

The ST9 strain of beet western yellows virus (BWYV ST9) is unique among BWYV strains because it encapsidates not only its 5.6-kb genomic RNA but also a 2.8-kb RNA of distinct nucleotide sequence, designated as the ST9-associated RNA. We obtained isolates of BWYV ST9 that are free of the associated RNA by transfecting Nicotiana tabacum protoplasts with transcripts of an ST9 genomic cDNA clone. Aphids were fed on extracts of infected protoplasts and were transferred to young Shepherd's Purse (Capsella bursa-pastoris) plants. When the protoplast inoculum was ST9 genomic transcript or virion RNA of the L-1 strain of BWYV (free of the associated RNA), symptoms were mild and characteristic of BWYV L-1. When ST9-associated RNA was included in the inoculum with genomic RNA of either source, subsequently infected Shepherd's Purse plants showed the severe symptoms that are characteristic of BWYV ST9. Inclusion of ST9-associated RNA in the inoculum with ST9 genomic RNA increased the accumulation of capsid antigen and ST9 genomic RNA, relative to infections initiated with ST9 genomic RNA alone. Using gold-labeled antibody and electron microscopy, we assessed the distribution of virions in Shepherd's Purse plants. Regardless of whether the associated RNA was present, sites showing immunoreactivity above background levels were restricted to the phloem, suggesting that the increased BWYV ST9 titer and symptom severity that are correlated with the presence of the ST9-associated RNA are not due to escape of the infection from phloem limitation.

Beet western yellows virus-associated RNA: an independently replicating RNA that stimulates virus accumulation.

Infections of plants by subviral RNA agents, alone or in association with virus genomic RNA molecules, are well known. The ST9 strain of beet western yellows virus encapsidates not only the 5.6-kilobase genomic RNA that is typical of luteoviruses, but also a 2.8-kilobase-associated RNA that has a distinct nucleotide sequence. The ST9-associated RNA has been postulated to be a satellite RNA, which by definition would be capable of replicating only in coinfections with beet western yellows virus or closely related viruses. To characterize the associated RNA, we inoculated protoplasts and leaves with in vitro transcripts of the virus genomic RNA and the ST9-associated RNA separately and in combination. Surprisingly, the ST9-associated RNA alone replicated efficiently in both protoplasts and leaves, and it stimulated accumulation of the virus genomic RNA in protoplasts. Thus, the ST9-associated RNA is a newly discovered type of plant infectious agent, which depends on its associated virus, beet western yellows virus, for encapsidation but not for replication.

Maize stripe virus RNA5 is of negative polarity and encodes a highly basic protein.

The entire 1317 nucleotide sequence of the maize stripe virus (MStV) RNA5 was determined. Only one open reading frame (ORF) was identified and was found in the viral complementary RNA (vcRNA). This ORF appears to encode a protein of M(r) 44237, hereafter referred to as NS5 delta. In vitro translation of transcripts representing nearly full-length RNA5 vcRNA yielded products of the predicted size, as well as some smaller, less prominent products. No products were identified from transcripts of viral polarity. RNA hybridization analyses of MStV-infected Zea mays revealed RNAs corresponding only to full-length RNA5, but both positive and negative polarity RNAs were abundant. Analysis of the NS5 amino acid sequence revealed that it is extremely basic, containing 21% arginine and lysine. Database comparisons showed that NS5 had no significant similarity to other protein sequences.

Nucleotide sequence and RNA hybridization analyses reveal an ambisense coding strategy for maize stripe virus RNA3.

The 2357-nt sequence of maize stripe virus (MStV) RNA3 was determined. Two nonoverlapping open reading frames (ORFs) of opposite polarities are contained in RNA3. A 591-nt ORF is located near the 5' end of MStV RNA3, while a second ORF of 948 nt is located near the 3' end in the viral complementary RNA (vcRNA). In vitro translation of transcripts derived from cDNA clones representing regions of each ORF was used to identify the respective proteins. A ca. 22,000 Mr protein was translated from the 591-nt ORF transcript. This protein comigrated in SDS-PAGE with a protein produced by in vitro translation of MStV RNA and is referred to as the NS3 protein. The protein from the 948-nt ORF transcript was specifically immunoprecipitated by antiserum to the MStV nucleocapsid protein (N protein). RNA hybridization analyses identified two subgenomic RNAs in total RNA extracts from MStV-infected Zea mays plants. These RNAs are ca. 650 and 1350 nt in size, are of opposite polarities, and correspond to regions of RNA3 containing the two ORFs. These data suggest that MStV RNA3 has an ambisense coding strategy similar to that found for the S RNA of the vertebrate-infecting phleboviruses, uukuviruses, and arenaviruses, as for tomato spotted wilt virus.

Identification and sequence analysis of the maize stripe virus major noncapsid protein gene.

The maize stripe virus (MStV) major noncapsid protein (NCP) gene was characterized, and the location of the NCP gene was identified among the 5-RNA, 18-kb genome. A 12-amino-acid sequence of the NCP was compared with nucleotide sequence data for MStV RNAs 3 and 4 and was found to align perfectly within a 528-nucleotide open reading frame (ORF) of RNA 4. The amino acid composition of purified NCP was almost identical to that deduced from the putative coding region. The deduced NCP molecular weight was 19,815, very similar to that determined by SDS-PAGE analysis of the purified NCP. In vitro transcription and translation analysis of the cDNA representing this region showed unequivocally that this region encoded the NCP. Primer extension analysis using a synthetic oligonucleotide complementary to a sequence near the 5' end of the coding region revealed that the NCP ORF is located 61 nucleotides from the 5' end of RNA 4.

The two capsid proteins of maize rayado fino virus contain common peptide sequences.

Virions of maize rayado fino virus (MRFV) were purified and two major capsid proteins (ca. Mr 29,000 and 22,000) were resolved by SDS-PAGE. When the two major capsid proteins were isolated from gels and compared by one-dimensional peptide mapping after digestion with Staphylococcus aureus V-8 protease, indistinguishable peptide maps were obtained, suggesting that these two proteins contain common peptide sequences. Some preparations also showed minor protein components that were intermediate between the Mr 22,000 and Mr 29,000 capsid proteins. One of the minor proteins, ca. Mr 27,000, gave a peptide map indistinguishable from the major capsid proteins. In vitro ageing of partially purified preparations or virion treatment with proteolytic enzymes failed to show conversion of the Mr 29,000 protein to a Mr 22,000. Protease inhibitors added to the buffers used for virion purification did not affect the apparent 1:3 ratio of 29,000 to 22,000 proteins in the purified preparations.