Accumulation Of Genomic RNA Research Articles

Beet Soil-Borne Virus Is a Helper Virus for the Novel Beta vulgaris Satellite Virus 1A.

Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.

The HC-Pro cistron of Triticum mosaic virus is dispensable for systemic infection in wheat but is required for symptom phenotype and efficient genome amplification

Triticum mosaic virus (TriMV), the type species of the genus Poacevirus in the family Potyviridae, is an economically important wheat curl mite-transmitted wheat-infecting virus in the Great Plains region of the USA. In this study, the functional genomics of helper component-proteinase (HC-Pro) encoded by TriMV was examined using a reverse genetics approach. TriMV with complete deletion of HC-Pro cistron elicited systemic infection in wheat, indicating that HC-Pro cistron is dispensable for TriMV systemic infection. However, TriMV lacking HC-Pro caused delayed systemic infection with mild symptoms that resulted in little or no stunting of plants with a significant reduction in the accumulation of genomic RNA copies and coat protein (CP). Sequential deletion mutagenesis from the 5′ end of HC-Pro cistron in the TriMV genome revealed that deletions within amino acids 3 to 25, except for amino acids 3 and 4, elicited mild symptoms with reduced accumulation of genomic RNA and CP. Surprisingly, TriMV with deletion of amino acids 3 to 50 or 3 to 125 in HC-Pro elicited severe symptoms with a substantial increase in genomic RNA copies but a drastic reduction in CP accumulation. Additionally, TriMV with heterologous HC-Pro from other potyvirids produced symptom phenotype and genomic RNA accumulation similar to that of TriMV without HC-Pro, suggesting that HC-Pros of other potyvirids were not effective in complementing TriMV in wheat. Our data indicate that HC-Pro is expendable for replication of TriMV but is required for efficient viral genomic RNA amplification and symptom development. The availability of TriMV with various deletions in the HC-Pro cistron will facilitate the examination of the requirement of HC-Pro for wheat curl mite transmission.

6K1, NIa-VPg, NIa-Pro, and CP of Wheat Streak Mosaic Virus are Collective Determinants of Wheat Streak Mosaic Disease in Wheat.

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is the causal agent of the most economically important wheat streak mosaic disease of wheat (Triticum aestivum L.) in the Great Plains region of the USA. WSMV determinants responsible for wheat streak mosaic disease in wheat are unknown. Triticum mosaic virus (TriMV), a wheat-infecting virus, was used as an expression vector for the transient expression of each of the WSMV-encoded cistrons in wheat. WSMV-encoded 6K1, NIa-VPg, NIa-Pro, and CP cistrons in TriMV elicited symptoms specific to different stages of wheat streak mosaic disease without significantly affecting the genomic RNA accumulation. WSMV 6K1 produced early wheat streak mosaic disease-like symptoms of severe chlorotic streaks and patches. NIa-VPg and CP caused severe chlorotic streaks, followed by moderate stunting (only with NIa-VPg) of wheat, mimicking early- and mid-stage symptoms of wheat streak mosaic disease. WSMV NIa-Pro caused mild chlorotic streaks, followed by dark green leaves with severe stunting, representing the late symptoms of wheat streak mosaic disease. Collectively, these data suggest that cumulative effects of WSMV-encoded 6K1, NIa-VPg, NIa-Pro, and CP are responsible for different stages of wheat streak mosaic disease symptoms in wheat. Furthermore, deletion analysis of wheat streak mosaic disease determinants revealed that complete 6K1 and NIa-Pro, amino acids 3-60 and 121-197 of NIa-VPg, and amino acids 101-294 of CP are responsible for wheat streak mosaic disease-like symptoms in wheat. This study suggests that management strategies for wheat streak mosaic disease in wheat should target WSMV determinants of the disease phenotype.

Nitrate-inducible MdBT2 acts as a restriction factor to limit apple necrotic mosaic virus genome replication in Malus domestica.

Apple necrotic mosaic virus (ApNMV) is highly associated with the occurrence of apple mosaic disease in China. However, ApNMV–host interactions and defence mechanisms of host plants against this virus are poorly studied. Here, we report that nitrate treatment restrains ApNMV genomic RNA accumulation by destabilizing viral replication protein 1a through the MdBT2‐mediated ubiquitin‐proteasome pathway. MdBT2, a nitrate‐responsive BTB/TAZ domain‐containing protein, was identified in a yeast two‐hybrid screen of an apple cDNA library using viral protein 1a as bait, and 1a was further confirmed to interact with MdBT2 both in vivo and in vitro. It was further verified that MdBT2 promoted the ubiquitination and degradation of viral protein 1a through the ubiquitin‐proteasome pathway in an MdCUL3A‐independent manner. Viral genomic RNA accumulation was reduced in MdBT2‐overexpressing transgenic apple leaves but enhanced in MdBT2‐antisense leaves compared to the wild type. Moreover, MdBT2 was found to interfere with the interaction between viral replication proteins 1a and 2apol by competitively interacting with 1a. Taken together, our results demonstrate that nitrate‐inducible MdBT2 functions as a limiting factor in ApNMV viral RNA accumulation by promoting the ubiquitination and degradation of viral protein 1a and interfering with interactions between viral replication proteins.

N6 -Methyladenosine Negatively Regulates Human Respiratory Syncytial Virus Replication.

N6-methyladenosine (m6A) is the most abundant internal modification described in eukaryotic mRNA and several viral RNA including human respiratory syncytial virus (HRSV). Here, we evaluated the impact of m6A writers, erasers and readers on HRSV genomic RNA accumulation and inclusion bodies assembly during viral replication. We observed that the METTL3/METTL14 m6A writer complex plays a negative role in HRSV protein synthesis and viral titers, while m6A erasers FTO and ALKBH5 had the opposite effect. We also observed that m6A readers YTHDF1-3 bind to the viral genomic RNA inducing a decrease in its intracellular levels and thus, inhibiting viral replication. Finally, we observed that overexpression of YTHDFs proteins caused a decrease in the size of inclusion bodies (IBs), accompanied by an increase in their number. METTL3 knockdown cells showed an opposite effect indicating that the dynamics of IBs assembly and coalescence are strongly affected by m6A readers in a mechanism dependent on m6A writers. Taken together, our results demonstrated that the m6A modification negatively affects HRSV replication, possibly through a mechanism involving the assembly of inclusion bodies, the main factories of viral genomic RNA synthesis.

Plant Immune System Activation Upon Citrus Leprosis Virus C Infection Is Mimicked by the Ectopic Expression of the P61 Viral Protein.

Citrus leprosis virus C (CiLV-C, genus Cilevirus, family Kitaviridae) is an atypical virus that does not spread systemically in its plant hosts. Upon its inoculation by Brevipalpus mites, only localized lesions occur, and the infection remains limited to cells around mite feeding sites. Here, we aimed to gain insights into the putative causes of viral unfitness in plants by expanding the limited knowledge of the molecular mechanisms underlying plant/kitavirid interactions. Firstly, we quantified the CiLV-C viral RNAs during the infection in Arabidopsis thaliana plants using RT-qPCR and systematized it by defining three stages of distinguishing subgenomic and genomic RNA accumulation: i) 0–24 h after infestation, ii) 2–4 days after infestation (dai), and iii) 6–10 dai. Accordingly, the global plant response to CiLV-C infection was assessed by RNA-Seq at each period. Results indicated a progressive reprogramming of the plant transcriptome in parallel to the increasing viral loads. Gene ontology enrichment analysis revealed the induction of cell growth-related processes at the early stages of the infection and the triggering of the SA-mediated pathway, ROS burst and hypersensitive response (HR) at the presymptomatic stage. Conversely, infected plants downregulated JA/ET-mediated pathways and processes involved in the primary metabolism including photosynthesis. Marker genes of unfolded protein response were also induced, suggesting a contribution of the endoplasmic reticulum stress to the cell death caused by the viral infection. Finally, we transiently expressed CiLV-C proteins in Nicotiana benthamiana plants to undertake their roles in the elicited plant responses. Expression of the CiLV-C P61 protein consistently triggered ROS burst, upregulated SA- and HR-related genes, increased SA levels, reduced JA levels, and caused cell death. Mimicry of responses typically observed during CiLV-C–plant interaction indicates P61 as a putative viral effector causing the HR-like symptoms associated with the infection. Our data strengthen the hypothesis that symptoms of CiLV-C infection might be the outcome of a hypersensitive-like response during an incompatible interaction. Consequently, the locally restricted infection of CiLV-C, commonly observed across infections by kitavirids, supports the thesis that these viruses, likely arising from an ancestral arthropod-infecting virus, are unable to fully circumvent plant defenses.

Genomic RNA accumulation of gentian ovary ring-spot virus and raspberry bushy dwarf virus in pollen tubes

The time course of genomic RNA (gRNA) accumulation of gentian ovary ring-spot virus (GORV) and raspberry bushy dwarf virus (RBDV) among total RNAs from germinating pollen grains and elongating pollen grains incubated in germination buffer was assessed by quantitative reverse transcription-polymerase chain reactions. GORV and RBDV gRNAs reached a maximum accumulation among the total RNAs after 3 and 1 h of incubation, respectively. Because the viral gRNAs could have been present in the germination buffer rather than in the pollen grains, GORV and RBDV might replicate and be discharged from the growing pollen tubes.

Wheat streak mosaic virus Coat Protein Deletion Mutants Elicit More Severe Symptoms Than Wild-Type Virus in Multiple Cereal Hosts.

Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV) (genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84 but not of 36 to 57 induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared with mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from the WSMV genome accelerated cell-to-cell movement, with increased accumulation of genomic RNAs and CP, compared with the wild-type virus. Microscopic examination of wheat tissues infected by green fluorescent protein-tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis, compared with mutants eliciting wild-type symptoms. All deletion mutants accumulated CP-specific major protein similarly to that in wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared with wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from the WSMV genome enhanced accumulation of CP and genomic RNA, altered CP-specific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts.

The C-terminus of Wheat streak mosaic virus Coat Protein Is Involved in Differential Infection of Wheat and Maize through Host-Specific Long-Distance Transport

Viral determinants and mechanisms involved in extension of host range of monocot-infecting viruses are poorly understood. Viral coat proteins (CP) serve many functions in almost every aspect of the virus life cycle. The role of the C-terminal region of Wheat streak mosaic virus (WSMV) CP in virus biology was examined by mutating six negatively charged aspartic acid residues at positions 216, 289, 290, 326, 333, and 334. All of these amino acid residues are dispensable for virion assembly, and aspartic acid residues at positions 216, 333, and 334 are expendable for normal infection of wheat and maize. However, mutants D289N, D289A, D290A, DD289/290NA, and D326A exhibited slow cell-to-cell movement in wheat, which resulted in delayed onset of systemic infection, followed by a rapid recovery of genomic RNA accumulation and symptom development. Mutants D289N, D289A, and D326A inefficiently infected maize, eliciting milder symptoms, while D290A and DD289/290NA failed to infect systemically, suggesting that the C-terminus of CP is involved in differential infection of wheat and maize. Mutation of aspartic acid residues at amino acid positions 289, 290, and 326 severely debilitated virus ingress into the vascular system of maize but not wheat, suggesting that these amino acids facilitate expansion of WSMV host range through host-specific long-distance transport.

ESCRT‐II's involvement in HIV‐1 genomic RNA trafficking and assembly

Several host proteins play crucial roles in the HIV-1 replication cycle. The endosomal sorting complex required for transport (ESCRT) exemplifies a large, multi-component host machinery that is required by HIV-1 for viral budding. ESCRT promotes the inward budding of vesicles from the membranes of late endosomes to generate multi-vesicular bodies. However, HIV-1 co-opts the ESCRT to enable outwards budding of virus particles from the plasma membrane, a phenomenon that is topologically similar to multi-vesicular body biogenesis. A role for ESCRTII in mRNA trafficking has been established in Drosophila in which the ESCRT-II components, Vps22 and Vps36, promote the localisation of the bicoid mRNA in the fertilised egg. This is achieved via specific interactions with the Staufen protein. In this work, we investigated a possible implication of ESCRT-II in the HIV-1 replication cycle. Co-immunoprecipitation analyses and live cell tri-molecular fluorescence complementation assays revealed that interactions between EAP30 and Gag and another between EAP30 and Staufen1 occur in mammalian cells. We then depleted EAP30 (the orthologue for Vps22) by siRNA to target ESCRT-II in HIV-1 expressing cells. This treatment disrupted ESCRT-II function and leads to the degradation of the two other ESCRT-II complex proteins, EAP45 and EAP20, as well as the associated Rab7-interacting lysosomal protein. The depletion of EAP30 led to dramatically reduced viral structural protein Gag and virus production levels, without any effect on viral RNA levels. On the contrary, the overexpression of EAP30 led to a several-fold increase in virus production. Unexpec-tedly, siRNA-mediated depletion of EAP30 led to a block to HIV-1 genomic RNA trafficking and resulted in the accumulation of genomic RNA in the nucleus and juxtanuclear domains. Our data provide the first evidence that the Staufen1-ESCRT-II interaction is evolutionarily conserved from lower to higher eukaryotes and reveal a novel role for EAP30 in the control of HIV-1 RNA trafficking and gene expression.

Tomato bushy stunt virus Co-Opts the RNA-Binding Function of a Host Metabolic Enzyme for Viral Genomic RNA Synthesis

Tomato bushy stunt virus (TBSV), a plus-stranded [(+)] RNA plant virus, incorporates the host metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the viral replicase complex. Here, we show that, during TBSV replication in yeast, the yeast GAPDH Tdh2p moves from the cytosol to the peroxisomal membrane surface, the site of viral RNA synthesis. In yeast cells lacking Tdh2p, decreasing the levels of its functionally redundant homolog Tdh3p inhibited TBSV replication and resulted in equivalent levels of (+) and minus-stranded [(-)] viral RNA, in contrast to the hallmark excess of (+)RNA. Tdh2p specifically bound an AU pentamer sequence in the (-)RNA, suggesting that GAPDH promotes asymmetric RNA synthesis by selectively retaining the (-)RNA template in the replicase complex. Downregulation of GAPDH in a natural plant host decreased TBSV genomic RNA accumulation. Thus, TBSV co-opts the RNA-binding function of a metabolic protein, helping convert the host cell into a viral factory.

Synergism between a mycoreovirus and a hypovirus mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713

Infection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys-->Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Deltap29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys(162). These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

The AAUAAA Motif of Bamboo Mosaic Virus RNA Is Involved in Minus-Strand RNA Synthesis and Plus-Strand RNA Polyadenylation

Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome with a 5'-cap structure and a 3' poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3' untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of Nicotiana benthamiana protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect N. benthamiana plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3' UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.

Involvement of a bovine viral diarrhea virus NS5B locus in virion assembly.

A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-to-proline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans-packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly.

Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane.

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.

Hypovirus papain-like protease p29 functions in trans to enhance viral double-stranded RNA accumulation and vertical transmission.

The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to approximately 50% for Deltap29, and 10 to 20% for Deltap69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for Deltap29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a Deltap69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys(70) or Cys(72), strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.

Tomato bushy stunt virus genomic RNA accumulation is regulated by interdependent cis-acting elements within the movement protein open reading frames.

This study on Tomato bushy stunt virus identified and defined three previously unknown regulatory sequences involved in RNA accumulation that are located within the 3'-proximal nested movement protein genes p22 and p19. The first is a 16-nucleotide (nt) element termed III-A that is positioned at the very 3' end of p22 and is essential for RNA accumulation. Approximately 300 nt upstream of III-A resides an approximately 80-nt inhibitory element (IE) that is obstructive to replication only in the absence of a third regulatory element of approximately 30 nt (SUR-III) that is positioned immediately upstream of III-A. Inspection of the nucleotide sequences predicted that III-A and SUR-III can form looped hairpins. A comparison of different tombusviruses showed, in each case, conservation for potential base pairing between the two predicted hairpin-loops. Insertion of a spacer adjacent to the predicted hairpins had no or a minimal effect on RNA accumulation, whereas an insertion in the putative III-A loop abolished genomic RNA multiplication. We conclude that the sequences composing the predicted III-A and SUR-III hairpin-loops are crucial for optimal RNA accumulation and that the inhibitory effect of IE surfaces when the alleged interaction between SUR-III and III-A is disturbed.

Effects of inactivation of the coat protein and movement genes of Tomato bushy stunt virus on early accumulation of genomic and subgenomic RNAs

Accumulation of RNA of Tomato bushy stunt virus (TBSV) was examined within the first few hours after infection of Nicotiana benthamiana protoplasts to determine the influence of the coat protein (CP), the movement-associated proteins P22 and P19 and RNA sequences at very early stages of replication. The results showed that P19 had no effect on early RNA replication, whereas the absence of CP and/or P22 expression delayed RNA accumulation only marginally. Removal of CP-coding sequences had no added negative effects, but when the deletion extended into the downstream p22 gene, it not only eliminated synthesis of subgenomic RNA2 but also delayed accumulation of genomic RNA by 10 h. At times beyond 20 h post-transfection, RNA accumulated to normal high levels for all mutants. This illustrates that TBSV RNA sequences that have negligible impact on overall RNA levels observed late in infection can actually have pronounced effects at very early stages.

Down-regulation of Groundnut Rosette Virus Replication by a Variant Satellite RNA

Symptom production in groundnut plants infected with groundnut rosette virus (GRV) depends on the presence of satellite RNA (sat-RNA) in the GRV culture, and sat-RNA variants that induce only mild symptoms are known. One such variant drastically diminished the replication of GRV genomic RNA in infectedNicotiana benthamianaplants. This down-regulating ability did not involve either of the two open reading frames in the sat-RNA but was controlled by a region near its 5′ end, which is required for sat-RNA replication. WhenN. benthamianaplants were inoculated with GRV and the mild satellite and challenged by inoculation with a GRV isolate (YB) containing a sat-RNA that induces yellow blotch symptoms, no symptoms appeared and little GRV genomic RNA or sat-RNA was detected in the plants, provided the two inoculations were no more than 2 days apart. A GRV isolate containing a sat-RNA that neither induces symptoms inN. benthamiananor affects genomic RNA accumulation also provided protection against yellow blotch symptom production if inoculated before or up to 2 days after isolate YB. However, in this case protection was incomplete and both GRV RNA and sat-RNA accumulated to normal levels. It is suggested that sequences from the mild sat-RNA may provide a novel source of resistance against rosette disease.