Size Restriction Is Required for Proper Functioning of a Bipartite Begomovirus AC4 Protein.

Many geminiviruses, including members of the genus Begomovirus, produce a protein known as C4 or AC4. Whereas C4/AC4 typically consists of more than 80 amino acid residues, a few are much shorter. The significance of these shorter C4/AC4 proteins in viral infection and why the virus maintains their abbreviated length is not yet understood. The AC4 of the begomovirus Tomato leaf curl Hsinchu virus contains only 65 amino acids, but it extends to 96 amino acids when the natural termination codon is replaced with a normal codon. We discovered that both interrupting and extending AC4 were harmful to tomato leaf curl Hsinchu virus (ToLCHsV). The extended AC4 (EAC4) also showed a reduced ability to promote the infection of the heterologous virus Potato virus X than the wild-type AC4. When the wild-type AC4 was fused with yellow fluorescent protein (AC4-YFP), it was predominantly found in chloroplasts, whereas EAC4-YFP was mainly localized to the cell periphery. These results suggest that ToLCHsV's AC4 protein is important for viral infection, and the virus may benefit from the abbreviated length, because it may lead to chloroplast localization. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rice Stripe Tenuivirus Has a Greater Tendency To Use the Prime-and-Realign Mechanism in Transcription of Genomic than in Transcription of Antigenomic Template RNAs.

Most segmented negative-sense RNA viruses employ a process termed cap snatching, during which they snatch capped RNA leaders from host cellular mRNAs and use the snatched leaders as primers for transcription, leading to the synthesis of viral mRNAs with 5' heterogeneous sequences (HSs). With traditional methods, only a few HSs can be determined, and identification of their donors is difficult. Here, the mRNA 5' ends of Rice stripe tenuivirus (RSV) and Rice grassy stunt tenuivirus (RGSV) and those of their host rice were determined by high-throughput sequencing. Millions of tenuiviral HSs were obtained, and a large number of them mapped to the 5' ends of corresponding host cellular mRNAs. Repeats of the dinucleotide AC, which are complementary to the U1G2 of the tenuiviral template 3'-U1G2U3G4UUUCG, were found to be prevalent at the 3' termini of tenuiviral HSs. Most of these ACs did not match host cellular mRNAs, supporting the idea that tenuiviruses use the prime-and-realign mechanism during cap snatching. We previously reported a greater tendency of RSV than RGSV to use the prime-and-realign mechanism in transcription with leaders cap snatched from a coinfecting reovirus. Besides confirming this observation in natural tenuiviral infections, the data here additionally reveal that RSV has a greater tendency to use this mechanism in transcribing genomic than in transcribing antigenomic templates. The data also suggest that tenuiviruses cap snatch host cellular mRNAs from translation- and photosynthesis-related genes, and capped RNA leaders snatched by tenuiviruses base pair with U1/U3 or G2/G4 of viral templates. These results provide unprecedented insights into the cap-snatching process of tenuiviruses.IMPORTANCE Many segmented negative-sense RNA viruses (segmented NSVs) are medically or agriculturally important pathogens. The cap-snatching process is a promising target for the development of antiviral strategies against this group of viruses. However, many details of this process remain poorly characterized. Tenuiviruses constitute a genus of agriculturally important segmented NSVs, several members of which are major viral pathogens of rice. Here, we for the first time adopted a high-throughput sequencing strategy to determine the 5' heterogeneous sequences (HSs) of tenuiviruses and mapped them to host cellular mRNAs. Besides providing deep insights into the cap snatching of tenuiviruses, the data obtained provide clear evidence to support several previously proposed models regarding cap snatching. Curiously and importantly, the data here reveal that not only different tenuiviruses but also the same tenuivirus synthesizing different mRNAs use the prime-and-realign mechanism with different tendencies during their cap snatching.

The complete genome sequence of a novel cytorhabdovirus identified in strawberry (Fragaria ananassa Duch.).

A cytorhabdovirus, tentatively named "strawberry-associated virus 1" (SaV1), was identified in strawberry (Fragaria ananassa Duch.), and its complete genome sequence was determined. Its negative-sense single-stranded RNA genome is composed of 14,159 nucleotides and contains eight open reading frames (ORFs) in the canonical order 3'-N-P-P3-M-G-P6-P7-L-5. The ORFs are separated by conserved intergenic sequences, and the genome coding region is flanked by 3' and 5' untranslated regions of 179 and 856 nt, respectively. SaV1 N and L genes shares 32-57% and 38-64% amino acid sequence identity with those of nine reported cytorhabdoviruses, respectively. Phylogenetic analysis showed that SaV1 clustered with high confidence with representative cytorhabdoviruses and is most closely related to tomato yellow mottle-associated virus. There are two additional small genes of unknown function between the G and L genes. We propose that SaV1 should be considered a member of a novel species in the genus Cytorhabdovirus, family Rhabdoviridae.

Genetic diversity of begomoviruses in Pakistan captured through a vector based survey.

Begomoviruses (Geminiviridea), transmitted by whiteflies, constitute one of the most dangerous groups of plant viruses posing a severe threat to economically important crops in tropical and sub-tropical areas. In this study, whiteflies were collected from various locations all over Pakistan. The begomoviruses carried by these whiteflies were detected by PCR with the degenerative primers pair AV94/Dep3. Analysis of the 177 sequences obtained in our study, revealed 14 distinct begomovirus species, including five which were not previously reported in this country. Putative novel strains of Corchorus yellow vein virus (CoYVV) and Chilli leaf curl virus (ChiLCV) showing less than 90% identity with the previously available taxa were also identified. The greatest number of begomoviruses per single site was detected in Sindh province, where up to five different begomovirus species were identified from the same cropping field. Moreover, Cotton leaf curl Multan virus - Rajasthan (CLCuMuV-Ra) was found prevalent in all the cotton growing areas. The data reported here may be useful in the development of control measures against begomoviruses.

Complete genome sequences of three novel cycloviruses identified in a dragonfly (Odonata: Anisoptera) from China.

Three cycloviruses (genus Cyclovirus, family Circoviridae) were recovered from a dragonfly (Odonata: Anisoptera) captured in Fuzhou, China. The three cycloviruses, named dragonfly associated cyclovirus 9, 10 and 11 (DfCyV-9, -10, -11), respectively, show 56.1-79.6% genome-wide identity to known cycloviruses and 61.6-65.1% among themselves. Thus, according to the current species demarcation criteria, they represent three novel cycloviruses. Notably, DfCyV-10 has a predicted replication-associated protein (Rep) that is most similar to that of bat associated cyclovirus 2 (BatACyV-2), a cyclovirus discovered in China, with 79.4% amino acid sequence identity, but a putative capsid protein (Cp) most similar to that of BatACyV-10, a cyclovirus discovered in Brazil, with 71.7% amino acid sequence identity. These data are useful for understanding the diversity and evolution of cycloviruses, especially those found in insects.

The genome sequence of Brassica campestris chrysovirus 1, a novel putative plant-infecting tripartite chrysovirus.

A putative chrysovirus recovered from Brassica campestris var. purpurea and provisionally named "Brassica campestris chrysovirus 1" (BrcCV1) was sequenced. The genome of the putative BrcCV1 consists of three double-stranded RNAs (dsRNAs) comprising 3,639 (dsRNA 1), 3,567 (dsRNA 2) and 3,337 (dsRNA 3) base pairs, respectively, each containing a single open reading frame (ORF 1-3). The putative proteins encoded by ORF 1-3 show homologies to RdRp, CP and chryso-P3 of approved or tentative chrysoviruses. In addition, the three dsRNAs of BrcCV1 contain highly conserved 5' and 3' untranslated regions (UTRs) in a way similar to known chrysoviruses. In a phylogenetic tree based on the conserved amino acid sequences of the RdRps of chrysoviruses, totiviruses and partitiviruses, the putative BrcCV1 formed a separate clade with Raphanus sativus chrysovirus 1 (RasCV1), a putative trisegmented, plant-infecting chrysovirus, in the family Chrysoviridae.

A new putative alphapartitivirus recovered from the powdery mildew fungus Erysiphe palczewskii.

Two double-stranded RNAs (dsRNA) likely representing the genome of a novel alphapartitivirus which we provisionally named Erysiphe palczewskii alphapartitivirus 1 (EpV1) were recovered from the powdery mildew fungus E. palczewskii infecting Sophora japonica in Jingzhou, Hubei province of China. The two dsRNAs, 1955 (dsRNA1) and 1917 (dsRNA2) bp in size, respectively, each contains a single open reading frame (ORF) encoding a 585- and 528-aa protein, respectively. The 585-aa protein contains a conserved RNA-dependent RNA polymerase (RdRp) domain and shows significant homology to RdRps of approved or putative partitiviruses, particularly those belonging to the genus Alphapartitivirus. However, it shares an aa sequence identity lower than 80% with its closest relative, the RdRp of the putative alphapartitivirus Grapevine partitivirus, and lower than 60% with the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRp aa sequences of selected partitiviruses, the putative virus EpV1 clustered with Grapevine partitivirus and formed a well-supported monophyletic clade with known or putative alphapartitiviruses.

Rice grassy stunt virus p5 interacts with two protein components of the plant-specific CBL-CIPK Ca+2 signaling network of rice.

Rice grassy stunt virus (RGSV) is a tenuivirus posing a threat to rice production in many South, Southeast, and East Asian countries. To date, no host factor interacting with RGSV has been reported. In this study, we screened a rice cDNA library with the GAL4-based yeast two-hybrid system using RGSV p5 as the bait. One of the candidate host factors interacting with RGSV p5 was found to be CBL-interacting protein kinase 25 (OsCIPK25), a member of the plant-specific CBL-CIPK Ca2+ signaling network. The interaction between RGSV p5 and OsCIPK25, as well as OsCIPK5, which is closely related to OsCIPK25, was confirmed by their cellular co-localization and by a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Given the importance of CIPKs in the regulation of ion homeostasis and the resemblance of RGSV symptoms to potassium deficiency in rice, we evaluated potassium content of RGSV-infected rice and found it to be much lower than that in the healthy rice.

The speed of tubule formation of two fijiviruses corresponds with their dissemination efficiency in their insect vectors.

BACKGROUND: Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) are two closely related fijiviruses transmitted by the small brown planthopper (SBPH) and white-backed planthopper (WBPH), respectively. SRBSDV has a latent period 4 days shorter than that of RBSDV, implying a more efficient spread in insect vector. Currently, the mechanisms underlying this higher efficiency are poorly understood. However, our recent studies have implicated a role of virus induced tubular structures in the dissemination of fijiiruses within their insect vectors. METHODS: Immunofluorescence labeling was performed to visualize and compare the dynamics of P7-1 tubule formation of the RBSDV and SRBSDV in their own vector insects and nonhost Spodoptera frugiperda (Sf9) cells. RESULTS: Tubule formation of SRBSDV P7-1 was faster than that of RBSDV P7-1. For RBSDV, P7-1 formed tubules were observed at 3-days post-first access to diseased plants (padp) in SBPH. For SRBSDV, these structures were detected as early as 1 day padp in WBPH. Importantly, similar phenomena were observed when P7-1 proteins from the two viruses were expressed alone in Sf9 cells. CONCLUSIONS: Our research revealed a relationship between the speed of P7-1 tubule formation and virus dissemination efficiency and also supports a role of such tubular structures in the spread of reoviruses within insect vectors.

Complete sequence of a double-stranded RNA from the phytopathogenic fungus Erysiphe cichoracearum that might represent a novel endornavirus.

A double-stranded RNA (dsRNA) HBJZ1506 recovered from the phytopathogenic fungus Erysiphe cichoracearum infecting Calendula officinalis in Jingzhou, Hubei Province, China, was sequenced. HBJZ1506 comprises 11,908 nucleotides (nt) and contains a 11,859-nt-long open reading frame (ORF) coding for a polypeptide that is 61 % identical to that of a putative endornavirus named grapevine endophyte endornavirus (GeEV). The putative polyprotein has an RNA-dependent RNA polymerase (RdRp) domain and an RNA helicase domain, which show homology to and have an arrangement that is similar to that of their counterparts in approved or putative endornaviruses. In a phylogenetic tree constructed using amino acid sequences of the RdRp region of HBJZ1506 and selected endornaviruses, HBJZ1506 clustered with endornaviruses and formed a well-supported monophyletic branch with GeEV. These results suggest that HBJZ1506 might represent a novel endornavirus, for which the name Erysiphe cichoracearum endornavirus (EcEV) is proposed.

High genetic homogeneity points to a single introduction event responsible for invasion of Cotton leaf curl Multan virus and its associated betasatellite into China.

BACKGROUND: Cotton leaf curl Multan virus (CLCuMuV) is a Whitefly Transmitted Geminivirus (WTG) endemic to the India subcontinent and is notorious as a causal agent of cotton leaf curl disease (CLCuD), a major constraint to cotton production in south Asia. We found CLCuMuV infecting Hibiscus rosa-sinensis in Guangzhou, China in 2006. The spread and evolution of the invading CLCuMuV were monitored in the following nine years. FINDINGS: CLCuMuV spread rapidly in the last nine years and became established in Southern China. It infects at least five malvaceous plant species, H. rosa-sinensis, H. esculentus, Malvaiscus arboreus, Gossypium hirsutum and H. cannabinus. Complete nucleotide sequences of 34 geographically and/or temporally distinct CLCuMuV isolates were determined and analyzed together with six other publicly available genomes of CLCuMuV occurring in China. The 40 CLCuMuV isolates were found to share > 99 % nucleotide sequence identity with each other. In all cases tested, the CLCuMuVs were associated with a CLCuMuB. The 36 CLCuMuBs (30 sequenced by us) shared > 98 % nucleotide sequence identity. CONCLUSION: The high genetic homogeneity of CLCuMuV and CLCuMuB in China suggests the establishment of them from a single founder event.

Transcriptome profiling confirmed correlations between symptoms and transcriptional changes in RDV infected rice and revealed nucleolus as a possible target of RDV manipulation.

BACKGROUND: Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which limits rice production in many areas of south East Asia. Transcriptional changes of rice in response to RDV infection have been characterized by Shimizu et al. and Satoh et al.. Both studies found induction of defense related genes and correlations between transcriptional changes and symptom development in RDV-infected rice. However, the same rice cultivar, namely Nipponbare belonging to the Japonic subspecies of rice was used in both studies. METHODS: Gene expression changes of the indica subspecies of rice, namely Oryza sativa L. ssp. indica cv Yixiang2292 that show moderate resistance to RDV, in response to RDV infection were characterized using an Affymetrix Rice Genome Array. Differentially expressed genes (DEGs) were classified according to their Gene Ontology (GO) annotation. The effects of transient expression of Pns11 in Nicotiana benthaminana on the expression of nucleolar genes were studied using real-time PCR (RT-PCR). RESULTS: 856 genes involved in defense or other physiological processes were identified to be DEGs, most of which showed up-regulation. Ribosome- and nucleolus related genes were significantly enriched in the DEGs. Representative genes related to nucleolar function exhibited altered expression in N. benthaminana plants transiently expressing Pns11 of RDV. CONCLUSIONS: Induction of defense related genes is common for rice infected with RDV. There is a co-relation between symptom severity and transcriptional alteration in RDV infected rice. Besides ribosome, RDV may also target nucleolus to manipulate the translation machinery of rice. Given the tight links between nucleolus and ribosome, it is intriguing to speculate that RDV may enhance expression of ribosomal genes by targeting nucleolus through Pns11.

Identification and molecular characterization of a single-stranded circular DNA virus with similarities to Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1.

A putative circular single-stranded DNA (ssDNA) virus was recovered from Hypericum japonicum collected in Vietnam. The viral isolate was tentatively named Hypericum japonicum-associated circular DNA virus (HJasCV). HJasCV shares 58.7-65.4% nucleotide sequence identity with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and SsHADV-1-like viruses. Like this group of viruses, the genome of HJasCV (2 200 nt) has two large ORFs, one in the virion-sense and the other in the complementary-sense DNA. The proteins encoded in the virion-sense and complementary-sense ORFs share 39-46 % and 45-67 % amino acid sequence identity with the putative capsid and replication-associated proteins (Reps), respectively, of SsHADV-1 and SsHADV-1-like viruses. The putative Rep of HJasCV contains all of the motifs related to rolling-circle replication. Its 111-bp intergenic region (IR) contains a hairpin structure with a geminivirus-like nonanucleotide sequence, TAATGTTAT, at the apex of the loop. Phylogenetic analysis revealed that HJasCV forms a monophyletic clade with SsHADV-1 and SsHADV-1-like viruses.

Isolation and molecular characterization of a distinct begomovirus and its associated betasatellite infecting Hedyotis uncinella (Hook. et Arn.) in Vietnam.

A begomovirus isolate VN1 associated with symptomatic Hedyotis uncinella Hook. et Arn. from Vietnam was characterized. The virus, which we provisionally name H. uncinella yellow mosaic virus (HUYMV), has a monopartite genome of 2,749 nucleotides (nts). Pairwise comparisons of DNA-A sequences showed that HUYMV had a maximum nt sequence identity with Soybean crinkle leaf virus (SbLCV) and Premna leaf curl virus (PLCuV) at 82.1 and 81.9 %, respectively, which are less than the 89 % identity in the complete genome, which has been used as the threshold value for demarcation of species in the genus Begomovirus, the family Geminiviridae. One recombination event was detected for HUYMV, which involves an unknown begomovirus as the major parent and Tomato leaf curl Philippines virus (ToLCPV) as the minor parent, with nt 2163 and nt 2452 as the beginning and ending breakpoints, respectively. A betasatellite was found to be associated with HUYMV. The betasatellite showed the highest nt sequence identity (70 %) with Tomato leaf curl Philippine betasatellite--[Philippines:Laguna2:2006]. The name H. uncinella yellow mosaic betasatellite [Vietnam: Binh Dinh: 2013] was proposed for the betasatellite.

Transcription start site mapping of geminiviruses using the in vitro cap-snatching of a tenuivirus.

Geminiviruses are a family of single-stranded DNA viruses that cause significant yield losses in crop production worldwide. Transcription start site (TSS) mapping is crucial in understanding the gene expression mechanisms of geminiviruses. However, this often requires costly and laborious experiments. Rice stripe virus (RSV) has a mechanism called cap-snatching, whereby it cleaves cellular mRNAs and uses the 5' cleavage product, a capped-RNA leader (CRL), as primers for transcription. Our previous work demonstrated that RSV snatches CRLs from geminiviral mRNAs in co-infected plants, providing a convenient and powerful approach to map the TSSs of geminiviruses. However, co-infections are not always feasible for all geminiviruses. In this study, we evaluated the use of in vitro cap-snatching of RSV for the same purpose, using tomato yellow leaf curl virus (TYLCV) as an example. We incubated RNA extracted from TYLCV-infected plants with purified RSV ribonucleoproteins in a reaction mixture that supports in vitro cap-snatching of RSV. The RSV mRNAs produced in the reaction were deep sequenced. The CRLs snatched by RSV allowed us to locate 28 TSSs in TYLCV. These results provide support for using RSV's in vitro cap-snatching to map geminiviral TSSs.

The cap-snatching frequency of a plant bunyavirus from nonsense mRNAs is low but is increased by silencing of UPF1 or SMG7.

Bunyaviruses cleave host cellular mRNAs to acquire cap structures for their own mRNAs in a process called cap-snatching. How bunyaviruses interact with cellular mRNA surveillance pathways such as nonsense-mediated decay (NMD) during cap-snatching remains poorly understood, especially in plants. Rice stripe virus (RSV) is a plant bunyavirus threatening rice production in East Asia. Here, with a newly developed system allowing us to present defined mRNAs to RSV in Nicotiana benthamiana, we found that the frequency of RSV to target nonsense mRNAs (nsRNAs) during cap-snatching was much lower than its frequency to target normal mRNAs. The frequency of RSV to target nsRNAs was increased by virus-induced gene silencing of UPF1 or SMG7, each encoding a protein component involved in early steps of NMD (in an rdr6 RNAi background). Coincidently, RSV accumulation was increased in the UPF1- or SMG7-silenced plants. These data indicated that the frequency of RSV to target nsRNAs during cap-snatching is restricted by NMD. By restricting the frequency of RSV to target nsRNAs, NMD may impose a constraint to the overall cap-snatching efficiency of RSV. Besides a deeper understanding for the cap-snatching of RSV, these findings point to a novel role of NMD in plant-bunyavirus interactions.

The Spatial Diffusion of Cherry Leaf Roll Virus Revealed by a Bayesian Phylodynamic Analysis.

Cherry leaf roll virus (CLRV) is an important plant pathogen that causes severe and detrimental effects on cherry and other fruit plants. Despite recent progress in plant pathology, molecular biology, and population genetics of CLRV, the spatiotemporal spread of this virus remains poorly studied. In this study, we employed a Bayesian phylodynamics framework to investigate the spatial diffusion patterns of CLRV by analyzing the coat protein gene sequences of 81 viral isolates collected from five different countries. Consistent with the trade of cherry, our Bayesian phylodynamic analyses pointed to viral origins in New Zealand and identified multiple migration pathways between Germany and other countries, suggesting that Germany has played an important role in CLRV transmission. The results of our study will be useful in developing sustainable management strategies to control this pathogen.

Bayesian Phylogeographic Inference Suggests Japan as the Center for the Origin and Dissemination of Rice Stripe Virus.

Rice stripe virus (RSV) is one of the most important viral pathogens of rice in East Asia. The origin and dispersal of RSV remain poorly understood, but an emerging hypothesis suggests that: (i) RSV originates from Yunnan, a southwest province of China; and (ii) some places of eastern China have acted as a center for the international dissemination of RSV. This hypothesis, however, has never been tested rigorously. Using a data set comprising more than 200 time-stamped coat protein gene sequences of RSV from Japan, China and South Korea, we reconstructed the phylogeographic history of RSV with Bayesian phylogeographic inference. Unexpectedly, the results did not support the abovementioned hypothesis. Instead, they suggested that RSV originates from Japan and Japan has been the major center for the dissemination of RSV in the past decades. Based on these data and the temporal dynamics of RSV reported recently by another group, we proposed a new hypothesis to explain the origin and dispersal of RSV. This new hypothesis may be valuable for further studies aiming to clarify the epidemiology of RSV. It may also be useful in designing management strategies against this devastating virus.

The phylogeographic history of tomato mosaic virus in Eurasia.

Tomato mosaic virus (ToMV) is a tobamovirus affecting solanaceous crops worldwide. The process of its emergence, however, is poorly understood. Here, Bayesian phylogenetic framework was employed to reconstruct the phylogeography of ToMV in Eurasia. The results showed that the ToMV in Europe, Middle East and East Asia has been evolving at a rate of 4.05 × 10-4 substitutions/site/year (95% credibility interval 2.43 × 10-4 - 5.62 × 10-4). Their most recent common ancestor (MRCA), most probably first appeared in Europe, was dated to around 1757 Common Era. The first introduction of ToMV into Middle East occurred in 1920s, with Europe as the source, while the first introduction of ToMV into East Asia occurred shortly afterwards, with Middle East as the source. From about 1950 onwards, inter-regional migrations of ToMV between Europe, Middle East and East Asia have been common. Overall, these data provide a glimpse into the phylogeographic history of ToMV in Eurasia.

Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor.

RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus.