VPg Gene Research Articles

Bayesian phylogeographic inference of wheat yellow mosaic virus in China and Japan suggests that the virus migration history coincided with historical events

Wheat yellow mosaic virus (WYMV) is one of the most serious viral pathogens causing reductions in wheat yield in East Asia. We investigated the phylodynamics of WYMV by analysing the CP, VPg and P1 genes to understand the origin and dispersal of the virus. A Bayesian phylogenetic analysis revealed that the most recent common WYMV ancestor occurred in approximately 1742 (95% credibility interval, 1439–1916) CE (Common Era), and the evolutionary rates of the VPg, CP and P1 genes were 6.669 × 10−4 (95% credibility interval: 4.575 × 10−4–8.927 × 10−4), 2.468 × 10−4 (95% credibility interval: 1.667 × 10−4–3.338 × 10−4) and 5.765 × 10−5 (95% credibility interval: 3.285 × 10−6–1.252 × 10−4), respectively. Our phylogeographic analysis indicated that the WYMV population may have originated in Henan Province, China, first spreading to Japan in the mid-19th century and stopping after the Japanese surrender in World War II. The second wave spread to Japan from Shandong Province, China, in approximately 1977, a few years after the establishment of diplomatic relations between China and Japan. Before the founding of the People's Republic of China, Henan Province was the emigration centre of WYMV in East Asia, and after the late 20th century, Jiangsu and Shandong Provinces were also the virus emigration centres in East Asia. In addition, there were two migration pathways from Japan to Jiangsu and Shandong Provinces, China, in approximately 1918 and approximately 1999 respectively. Our results suggest that the wide spread of WYMV in East Asia is strongly related to human factors.

Broad-spectrum resistance against multiple PVY-strains by CRSIPR/Cas13 system in Solanum tuberosum crop

ABSTRACT Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.

Molecular Evolutionary Analysis of Potato Virus Y Infecting Potato Based on the VPg Gene.

Potato virus Y (PVY) is an important plant pathogen infecting solanaceous crops, causing significant losses to global potato and tobacco production. Some aspects of the plant pathology and molecular biology of PVY have been studied intensively, but the evolutionary dynamics of this virus are poorly understood. Here, we performed a comprehensive set of rigorous evolutionary analyses using 177 nucleotide sequences of the viral genome linked protein (VPg) gene, which interacts with the plant eukaryotic translation initiation factor 4E (eIF4E). Our Bayesian analysis reveals that the VPg gene of PVY has been evolving at a rate of 5.60 × 10–4 subs/site/year (95% credibility interval 3.35 × 10–4–8.17 × 10–4), which is equivalent to those of other plant-infecting RNA viruses. We identified different evolutionary constraints on the two clades of PVY, clade N and clade O, whose diverge time were estimated at the year 1861 CE (95% credibility interval 1750–1948 CE). We also found that genetic variations were correlated with geographic regions, suggesting that the evolution of this pathogen is strongly affected by geographical associated factors. Taken together, the results of our study have potential implications for the control strategies of PVY.

Molecular characterization of banana bract mosaic virus from India reveals recombination and positive selection in the VPg gene

Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is an important viral pathogen affecting banana and plantains in India. The present study was undertaken to determine the genetic variation and molecular evolution of BBrMV infecting different cultivars of banana and plantain in the Indian subcontinent, based on the viral genome-linked protein (VPg) gene. Sequence identity of this gene from 29 BBrMV isolates showed a range of nucleotide (nt) and amino acid (aa) identity of 75–100% and 95–100%, respectively. Phylogenetic analysis based on nt revealed that except for two isolates (TN1 and TN2), all the other Indian isolates clustered together. Different functional motifs of the VPg gene previously reported were found to be conserved. A single possible recombination event was detected using a recombination detection programme. A codon-based selection analysis revealed that most of the codons in the VPg gene were under purifying selection except for those at position 46, 47, 71, 107, 149, 153, 156, 175, 176, and 178, which were under positive selection. Gene flow between different Indian populations of BBrMV from banana was relatively low. This is the first report on genetic diversity and evolution of the VPg gene of BBrMV. Since VPg is identified as a virulence determinant in resistance mediated by the eukariotic intiation factor 4E (eIF4E) in several plant-potyvirus interactions, any mutation in the VPg interacting domain may abolish the interaction with eIF4E or its isoforms in vitro and prevent viral infection in planta. Therefore, understanding the population structure of BBrMV based on VPg would potentially provide insight into the diversification and infection cycle of this virus.

A multigene phylogentic analysis of Potato virus Y and its application in strain identification.

The objective of this study is to develop a rapid and accurate multigene phylogenetic analysis to identify Potato virus Y (PVY) strains. The phylogenetic relationships of strains within the PVY species were evaluated with isolate-strain association using five datasets of concatenated sequences from the P1, HC-pro, VPg and CP genes to determine the best dataset for PVY strain identification. Results from phylogenetic analyses and Bayesian tip-association significance (BaTS) tests indicated that the major PVY strains could be distinguished using the P1, VPg and CP concatenated sequences datasets but not the remaining concatenated sequence datasets. Phylogenetic trees reconstructed from the concatenated sequences of P1, VPg and CP genes revealed that the ML and NJ trees had broadly similar topologies and that both were better than the maximum clade credibility tree (MCC). Additionally, the full genome of HLJ26, one isolate randomly selected for the multigene phylogenetic analysis, was clustered with high confidence among members of the PVYNTN-NW (SYR-Ⅱ) strain, which includes isolates of SYR-Ⅱ-2-8, SYR-Ⅱ-Be1 and SYR-Ⅱ-DrH. This suggests that it was a PVYNTN-NW (SYR-Ⅱ) isolate. Recombination analysis of this isolate identified four putative recombination joints in the P1, HC-pro/P3, VPg and the 5'-terminus of CP. This pattern is similar to that observed in the genomic structure of PVYNTN-NW (SYR-I), supporting the classification of this isolate as the PVYNTN-NW strain (SYR-Ⅱ). Simultaneously, two expected fragments of approximately 1 000 and 400 bp in size were also amplified from the isolate by a multiplex RT-PCR, consistent with the expected band pattern of the PVYNTN-NW (SYR-Ⅱ) strain. This further supports the utility of the multigene phylogenetic method in identifying PVY strains. We propose that the major PVY strains could be distinguished accurately using multigene phylogenetic analysis based on the concatenated sequences from the P1, VPg and CP genes.

Prediction of Host-Derived miRNAs with the Potential to Target PVY in Potato Plants.

Potato virus Y has emerged as a threatening problem in all potato growing areas around the globe. PVY reduces the yield and quality of potato cultivars. During the last 30 years, significant genetic changes in PVY strains have been observed with an increased incidence associated with crop damage. In the current study, computational approaches were applied to predict Potato derived miRNA targets in the PVY genome. The PVY genome is approximately 9 thousand nucleotides, which transcribes the following 6 genes:CI, NIa, NIb-Pro, HC-Pro, CP, and VPg. A total of 343 mature miRNAs were retrieved from the miRBase database and were examined for their target sequences in PVY genes using the minimum free energy (mfe), minimum folding energy, sequence complementarity and mRNA-miRNA hybridization approaches. The identified potato miRNAs against viral mRNA targets have antiviral activities, leading to translational inhibition by mRNA cleavage and/or mRNA blockage. We found 86 miRNAs targeting the PVY genome at 151 different sites. Moreover, only 36 miRNAs potentially targeted the PVY genome at 101 loci. The CI gene of the PVY genome was targeted by 32 miRNAs followed by the complementarity of 26, 19, 18, 16, and 13 miRNAs. Most importantly, we found 5 miRNAs (miR160a-5p, miR7997b, miR166c-3p, miR399h, and miR5303d) that could target the CI, NIa, NIb-Pro, HC-Pro, CP, and VPg genes of PVY. The predicted miRNAs can be used for the development of PVY-resistant potato crops in the future.

Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein

Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV.

Association of VPg and eIF4E in the host tropism at the cellular level of Barley yellow mosaic virus and Wheat yellow mosaic virus in the genus Bymovirus

Barley yellow mosaic virus (BaYMV) and Wheat yellow mosaic virus (WYMV) are separate species in the genus Bymovirus with bipartite plus-sense RNA genomes. In fields, BaYMV infects only barley and WYMV infects only wheat. Here, we studied the replicative capability of the two viruses in barley and wheat mesophyll protoplasts. BaYMV replicated in both barley and wheat protoplasts, but WYMV replicated only in wheat protoplasts. The expression of wheat translation initiation factor 4E (eIF4E), a common host factor for potyviruses, from the WYMV genome enabled WYMV replication in barley protoplasts. Replacing the BaYMV VPg gene with that of WYMV abolished BaYMV replication in barley protoplasts, whereas the additional expression of wheat eIF4E from BaYMV genome restored the replication of the BaYMV mutant in barley protoplasts. These results indicate that both VPg and the host eIF4E are involved in the host tropism of BaYMV and WYMV at the replication level.

Short communication. Molecular analysis of the genomic RNAs 1 and 2 of the first Arabis mosaic virus isolate detected in Spanish grapevines

The Arabis mosaic virus (ArMV) is one of the causative agent of the grapevine fanleaf disease, one of the most widespread and damaging viral diseases of grapevine. Recently, the ArMV has been detected in Spanish vineyards, and its determination and molecular characterization was undertaken. To this aim, the nucleotide sequence of the genomic RNAs 1 and 2 of the first isolate of ArMV infecting grapevine detected in Spain (ArMV-DU13) has been determined. The ArMV-DU13 genomic sequences were compared to the corresponding sequences of other isolates of ArMV, or nepoviruses. The most divergent genes among ArMV isolates were the X1 and VPg genes on the RNA 1, and the 2A gene on the RNA 2, with identity levels at the amino acid level of 78% (X1 and VPg) or 69% (2A) between the most distant isolates. Interestingly, the VPg genes were identical between the two grapevine isolates ArMV-Du13 and –NW, suggesting a possible implication of the host. The phylogenetic analysis of the RNA 2 showed that the Spanish isolate was close to Grapevine fanleaf virus isolates. The analysis of the full length RNA 2 suggests a recombination event between ArMV-DU13 and GFLV-GHu isolates between nucleotides 54 and 586 in the ArMV-DU13 isolate. Altogether, these results confirm the high variability between isolates of ArMV, and will be helpful to design more appropriate and reliable molecular diagnostic techniques for the control of this emerging virus in Spain.

Intrahost mechanisms governing emergence of resistance-breaking variants of Potato virus Y

The emergence of resistance breaking (RB) variants starting from the avirulent Potato virus Y NN strain (PVYNN) was analyzed after imposing different selective host constraints. Tobacco resistance to PVYNN is conferred by va in both NC745 and VAM genotypes, but VAM carries an extra resistance gene, va2. RB-variants emerged only in NC745 and unexpectedly accumulated higher in the original host, tobacco B21, than the parental PVYNN. However, the recovery of RB-variants was interfered by PVYNN in mixed infections. Further analysis indicated that RB-variants also arose in tobacco VAM, but they were limited to subliminal local infections. Their inability to breakout was associated with absence of a mutational adaptation in the viral VPg gene, which implied a loss of fitness in tobacco B21. Altogether, the emergence of RB-variants was conditioned by inherited host constraints, interference by co-infecting avirulent virus genotypes, and fitness tradeoff of virus adaptations.

Analysis of potyvirus terminal protein VPg-transgenic Arabidopsis thaliana plants.

Virus-coded VPg protein of Potato virus Y (PVY) does not have homologs apart from other VPgs. Since VPg is indispensable for the potyvirus life cycle, it appeared a good candidate for eliciting pathogen-derived resistance to PVY. Following agroinfection used to obtain PVY VPg-transgenic Arabidopsis thaliana plants, only few transgenic seeds were recovered giving rise to six transgenic plants that contained the VPg gene with the correct sequence. They generated VPg mRNA, but VPg protein was not detected. Some plants were immune to PVY infection suggesting post-transcriptional gene silencing. However, the likely PVY VPg toxicity exerted at an early stage of transformed seeds development precludes its use for engineering pathogen-derived resistance.

Inhibiting Virus Infection by RNA Interference of the Eight Functional Genes of the Potato Virus Y Genome

AbstractThe genome of Potato virus Y is a single‐stranded, positive‐sense RNA molecule that encodes a single large polypeptide that is cleaved by self‐encoded proteases into 10 functional proteins. Using specific primers designed based on the cloned genome sequence of the necrotic strain of Potato virus Y (PVYN), we amplified 400 bp and 500 bp cDNA fragments from the 3′ ends of P1, HC‐Pro, P3, CI, Vpg, NIa, NIb and CP genes. These cDNA fragments were then inserted into the binary vector pROKII to construct the vectors pROK‐P1, pROK‐HC‐Pro, pROK‐P3, pROK‐CI, pROK‐NIa‐Vpg, pROK‐NIa‐Pro, pROK‐NIb and pROK‐CP, all of which contained hairpin RNAs (hpRNAs). These recombinant binary vectors were introduced into Agrobacterium tumefaciens strain LBA4404 and then into Nicotiana tabacum L. var. NC89 plants, respectively. Virus challenge inoculation indicated that the plants transformed with each construct were resistant to PVY infection, and the percentage of resistant plants obtained ranged from 33–64%. Northern blot showed an inverse correlation between transgenic transcript accumulation and virus resistance. siRNAs could be detected in all the resistant plants. We also studied the relationship between the secondary structure and the gene‐silencing efficiency and found a positive correlation between local free energies (ΔGloc) and the virus‐resistance ratio. For each construct, one line of progeny T1 was randomly selected to analyse the inheritance of the transgene and the resistance. All transgenic single locus lines were completely resistant, and this resistance could be stably inherited.

First Report of Banana bract mosaic virus in Flowering Ginger in Hawaii.

Flowering ginger, Alpinia purpurata (Vieill.) K. Schum., is a popular cut flower and tropical landscape plant in Hawaii. In Hawaii, ginger flowers, including red and pink cultivars, are grown as field crops with an estimated annual sales of more than $1.6 million (USD) in 2006 (2). In June 2009, a commercial ginger flower grower from Waimanalo, Oahu, Hawaii reported plants with symptoms that included severe mosaic and stripes on the leaves. Flowers showed significant cupping and browning and growers report a reduction in size and shelf life. Symptomatic ginger was also identified at the Lyon Arboretum in Honolulu. Double-stranded RNAs (dsRNAs) were isolated from pooled leaf samples collected from 42 symptomatic plants at two locations on the island of Oahu to further characterize the pathogen associated with the symptomatic ginger. dsRNAs of approximately 0.7, 1.1, 1.8, 2.2, and 12 kb were present in the extractions from symptomatic plants but not in extractions from asymptomatic plants. Partial cloning and sequence analysis of the dsRNA revealed 95 to 98% nucleotide identity to sequences of P1, HC-Pro, C1, 6K2, VpG, NIb, and CP genes and the 3' untranslated region (total approximately 6 kb) of Banana bract mosaic virus (BBrMV). Total RNAs were also isolated from the symptomatic and asymptomatic plants from the Waimanalo farm and Lyon Arboretum. These RNA isolations were used in reverse transcription (RT)-PCR with primers Bract N1: 5'-GGRACATCACCAAATTTRAATGG-3' and Bract NR: 5'-GTGTGCYTCTCTAGCCCTGTT-3' (1), to amplify a 279-bp conserved region of the coat protein of BBrMV. Amplicons of the appropriate size were obtained from 38 of the symptomatic plants, whereas none were obtained from asymptomatic controls. RT-PCR amplicons of arbitrarily selected samples were cloned into pGEM-T Easy, sequenced, and found to be 99% identical to corresponding sequences of BBrMV. Furthermore, using double-antibody sandwich-ELISA assay and antibodies (3), we developed a system that can specifically detect BBrMV in infected flowering ginger plants and not in healthy appearing ginger. To our knowledge, this is the first report of BBrMV in flowering ginger in Hawaii. Further research is needed to determine if BBrMV infecting ginger poses a threat to banana, edible ginger, and other closely related ornamentals in Hawaii.

First Report of Nasturtium as a Natural Host of Cherry leaf roll virus on Amsterdam Island.

Cherry leaf roll virus (CLRV) is a well-known virus belonging to the genus Nepovirus, but unlike most members of this genus, it is not known to be transmitted by nematodes but only through seeds and pollen. Since its first description in 1955 on Prunus avium L. in England (1), CLRV has been shown to have a worldwide distribution and a wide natural host range. During a survey of plant viruses in the French sub-Antarctic islands, samples from nasturtium plants (Tropaeolum majus), an introduced plant species, showing symptoms of leaf mosaic, deformation, and veinal necrosis were collected on Amsterdam Island. Upon mechanical transmission with sap extracts, necrotic ringspot and oak-leaf symptoms typical of Nepovirus infection were observed on the leaves of inoculated Nicotiana clevelandii and N. tabacum plants. Inoculation of healthy nasturtium plants resulted in mosaic and pin-point necrosis symptoms. Electron microscopy on negatively stained sap extracts revealed the presence of icosahedral virions, 28 to 30 nm in diameter, in the symptomatic Nicotiana leaves. Amplification by reverse transcription (RT)-PCR with a polyvalent test, which identifies viruses belonging to the family Comoviridae (2), yielded the expected 248-bp fragment. Sequencing of the cloned amplicon showed 80% nucleotide and 90% amino acid identity with a part of the RNA dependent RNA polymerase (RdRp) of CLRV (CAE83562). To confirm the presence of CLRV, an approximate 4.6-kbp cDNA fragment was PCR amplified from double-stranded RNAs purifed from infected Nicotiana plants using the sense primer 5'-GTGGGACTGCCATGCACCTACTC-3' and an oligo-T25 as antisense primer. This PCR product (GenBank Accession No. GU167974) spans the region between the VPg gene and the polyA tail at the 3' end of the genome and thus provides approximately 2.8 kb of new internal sequence information on RNA1 of CLRV. The presence of CLRV in the initial nasturtium samples was confirmed with a CLRV-specific RT-PCR assay that amplifies the 3' non-coding region of the CLRV genome (3). Sequence of the amplified fragment showed it to be identical to the corresponding part of the 3' non-coding region of 4.6-kbp clone obtained from the CLRV isolate mechanically transmitted to the N. tabacum and N. clevelandii plants. Experimental infection of nasturtium by CLRV has been reported (4), but to the best of our knowledge these results represent the first report of natural infection of T. majus by CLRV. Given its seed transmissible character in many hosts, CLRV likely was introduced in infected seeds of T. majus imported to the remote sub-Antarctic Amsterdam Island.

Mutations in Turnip mosaic virus genomes that have adapted to Raphanus sativus

The genetic basis for virulence in potyviruses is largely unknown. Earlier studies showed that there are two host types of Turnip mosaic virus (TuMV); the Brassica/Raphanus (BR)-host type infects both Brassica and Raphanus systemically, whereas the Brassica (B)-host type infects Brassica fully and systemically, but not Raphanus. The genetic basis of this difference has been explored by using the progeny of an infectious clone, p35Tunos; this clone is derived from the UK1 isolate, which is of the B-host type, but rarely infects Raphanus systemically and then only asymptomatically. Two inocula from one such infection were adapted to Raphanus by passaging, during which the infectivity and concentration of the virions of successive infections increased. The variant genomes in the samples, 16 in total, were sequenced fully. Four of the 39 nucleotide substitutions that were detected among the Raphanus sativus-adapted variant genomes were probably crucial for adaptation, as they were found in several variants with independent passage histories. These four were found in the protein 1 (P1), protein 3 (P3), cylindrical inclusion protein (CI) and genome-liked viral protein (VPg) genes. One of four 'parallel evolution' substitutions, 3430G-->A, resulted in a 1100Met-->Ile amino acid change in the C terminus of P3. It seems likely that this site is important in the initial stages of adaptation to R. sativus. Other independent substitutions were mostly found in the P3, CI and VPg genes.

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication.

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.

Genomic mapping of a calicivirus VPg.

We identified a primate calicivirus (Pan-1) VPg in Pan-1-infected cells. The Pan-1 VPg was associated with both genomic and subgenomic RNAs. RNase digestion of Pan-1 RNA yielded a residual protein of 16 kDa. The N-terminal sequence of Pan-1 VPg was determined by direct amino acid sequencing and mapped to a region of the genome equivalent to picornavirus VPgs. Alignment of this protein sequence with similar regions of other calicivirus genomes allowed identification of conserved amino acid motifs and potential boundaries of the calicivirus VPg genes. Proteinase K treatment abolished the infectivity of Pan-1 RNA, suggesting that Pan-1 VPg is required for RNA infectivity.

VPg gene amplification correlates with infective particle formation in foot-and-mouth disease virus.

In order to analyze the function of VPg amplification in aphthoviruses, we have undertaken the first mutational analysis of the repetitive VPg-coding region using an improved foot-and-mouth disease virus (FMDV) cDNA clone from which infective viral RNA was synthesized. A set of VPg mutants was constructed by site-directed mutagenesis which includes different VPg deletion mutations, a VPg insertion mutation, and amino acid residue replacement mutations that interfere with binding of the VPg protein to the viral RNA and with its proteolytic processing. Our results revealed that an amazing flexibility in the number of VPgs is tolerated in FMDV. Optimal viability is given when three VPgs are encoded. Deletion as well as insertion of one VPg gene still resulted in infective particle production. Infective particle formation was observed as long as one VPg remained intact. No obvious differences in the individual VPg molecules with regard to their promoting viral RNA synthesis were observed, indicating that all three VPgs can act equally in FMDV replication. Mutant polyprotein processing was comparable to that of the wild-type virus. However, VPg mutants showed reduced viral RNA synthesis levels after infection. The levels of viral RNA synthesis and infective particle formation were found to correlate with the number of functional VPgs left in the mutant virus. These findings suggest a direct VPg gene dosage effect on viral RNA synthesis, with a secondary effect on infective particle formation.