First report of saffron latent virus in Crocus sativus from Hungary.

In early April of 2018 we sampled asymptomatic autumn flowering Crocus plants (Fig. S1.) in a private collection in Hajdú-Bihar county, Hungary. From each species (Cr. kotschyanus subsp. kotschyanus, Cr. sativus, Cr. speciosus) 200 mg leaf sample was collected from 5 neighboring shoot, which were treated as one sample. ELISA tests were carried out in duplicates using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) on the samples (Agdia, Elkhart, IN, USA). A sample was considered positive if the absorbance was at least three times greater than that of the negative control. Only one sample tested positive; the absorbance values of Cr. sativus leaves were 0.013 and 0.014, while the negative controls were 0.002 and 0.003, respectively. The samples were further tested by RT-PCR for potyviruses (Salamon and Palkovics 2005), tomato spotted wilt virus (TSWV) (Nemes and Salánki 2020) and nepovirus subgroup A (Digiaro et al. 2007). Total nucleic acid was extracted with the phenol-chloroform method of White and Kaper (1989), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) using random hexamer primer. The samples were negative for TSWV and nepovirus subgroup A, but a single PCR product of ~ 1700 nucleotide (nt) was amplified with potyvirus specific primers and cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). The 1726 nt long insert sequence, including the complete coat protein region was determined and deposited in the NCBI GenBank database (Accession No: OR425160). Digestion of the original PCR products with restriction enzyme SacI yielded only the predicted restriction fragments (364 / 1362 bp), indicating the presence of only a single potyvirus in the infected sample. BLASTn analysis of the CP cistron revealed the highest nt identities to saffron latent virus (SaLV) Iranian isolates (GenBank AccNo.: MN990394 - 85.44%, MN990415 - 85.39% and RefSeq: NC_036802 - 84.05%). For phylogenetic analyses MEGA11 (Tamura et al. 2021) was used. The resulting Maximum Likelihood tree (Fig. S2) showed that all Iranian SaLV isolates grouped together, while the Hungarian isolate is on an adjacent branch, separate from other virus species, and supported with 100% bootstrap values. From these results, it appears that the Hungarian isolate has been separated from the Iranian clade, and has evolved separately as a distinct lineage. We were unable to fulfill Koch's postulates as all available Crocus sativus plants were infected with SaLV. Latent potyvirus infection of Crocus species, by bean yellow mosaic virus (BYMV), iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV) and turnip mosaic virus (TuMV) has been reported by Grilli Caiola and Faoro (2011). SaLV was first reported from Iran (Parizad et al. 2017), but to our knowledge has never been reported from Europe or from any current EPPO member state. Since Crocus species can be asymptomatic virus reservoirs, it is important that any certification scheme for production should require laboratory tests to prove the health of the plants; or advise growers to keep possible high value susceptible crops such as breeding material and nuclear stocks at a distance from crocuses to mitigate virus transmission between stocks. It is also advisable to grow infected lots far from healthy stocks and protected wild hosts. To our knowledge, this is the first report of SaLV from Hungary and from Europe.

First report of meadow saffron breaking virus on wild Colchicum autumnale from a stricly protected Natura2000 site at a Hungarian National Park.

In mid-April of 2018 light green to greenish yellow linear stripes (Fig. S1.) were observed on the foliage of meadow saffron (Colchicum autumnale) plants - which are native to Hungary - at a strictly protected Natura2000 site maintained by the Duna-Ipoly National Park (DNPI). By autumn, during the flowering season, flower breaking symptoms (Fig. S2.) were noticed, which indicated possible viral infection. With the permit of the Government Office of Pest County and the DNPI, 200 mg leaf sample was collected from one symptomatic plant in spring 2021 and stored at -70 °C until further processing. At the time of the sampling about 2.5 % of the ~ 5000 meadow saffron were symptomatic. Multiplex RT-PCR testing of the sample and an asymptomatic C. autumnale plant for cucumber mosaic virus, tomato spotted wilt virus (Nemes and Salánki 2020) and Nepovirus subgroup-A (Digiaro et al. 2007) gave negative results. The asymptomatic plant also tested negative for potyviruses (Salamon and Palkovics 2005). The asymptomatic (healthy) C. autumnale plant was inoculated with leaf sap of the sample (0.02M Sörensen's phosphate buffer pH 7.2 + 2 % PVP-40 (m/v)) resulting in symptoms of flower breaking in autumn of 2021, and linear stripes on the foliage in spring 2022, identical to symptoms on the originally infected plant. ELISA tests were carried out on the source plants in duplicate using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) (Agdia, Elkhart, IN, USA). Absorbance values were 1.519 and 1.530, while the negative controls were 0.003 and 0.007, respectively indicating potyvirus infection of the sample. Molecular tests were carried out on the source and inoculated plant samples in 2022. Total nucleic acid was extracted with the modified CTAB protocol of Xu et al. (2004), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) with poly T2 (5'-CGGGGATCCTCGAGAAGCTTTTTTTTTTTTTTTTT-3') primer (Salamon and Palkovics 2005). PCR amplification was carried out with poty7941 (5'-GGAATTCCCGCGGNAAYAAYAGYGGNCARCC-3') and poly T2 primers as described earlier (Salamon and Palkovics 2005). A PCR product of ~ 1.6 kb was obtained in each case (Fig. S3.), cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α strain. The obtained 1642 nucleotide (nt) sequence encompassing the complete coat protein (CP) was determined (Accession No: OP057214). The virus sequence present in the source and inoculated plants shared 100% nt identity. EcoRV digestion of the PCR products yielded two restriction fragments (369/1273 bp), indicating the presence of a single potyvirus in the infected plant tissue (Fig. S3.). BLASTN analysis of the CP cistron revealed highest nt identity (93.91 %) to meadow saffron breaking virus (MSBV) isolate FR GenBank Acc. No.: AY388995. MSBV was first reported in the Alsace region of France at an INRA research station in cultivated meadow saffron plants showing similar symptoms and the disease reached 100% incidence within a year (Poutaraud et al. 2004). Potyviruses are transmitted mechanically and by aphids (Inoue-Nagata et al. 2022). The spread of MSBV could lead to the infection and decline of the population of Colchicum in protected ecosystems. To our knowledge, this is the first report of MSBV on wild meadow saffron plant from a strictly protected Natura2000 site at a Hungarian National Park.

Peanut stunt virus movement protein is the limiting factor in Capsicum annuum infection.

Cucumber mosaic virus (CMV) is one of the most devastating plant viruses, with more than 1,200 species of host plants. The host range and economic importance of peanut stunt virus (PSV) are mostly limited to legumes, despite the similar taxonomy and genome structure with CMV. Since no data are available on the background of the limited host range of PSV, RNA 3 recombinant and reassortant viruses were generated (C12P3, P12C3, C12CP3, C12PC3, C12PΔC3) to study their infection phenotype on a common host (Nicotiana benthamiana) and on a selective host (Capsicum annuum cv. Brody). The PSV movement protein (MP) was not able to function with the coat protein (CP) of CMV unless the C-terminal 42 amino acids were deleted from the PSV MP. As a result of the inoculation experiments, MP was considered the protein influencing symptom phenotypes on N. benthamiana and responsible for the host range difference on the pepper. Since plasmodesmata (PD) localization of viral MPs is essential for cell-to-cell movement, subcellular localization of GFP-tagged MPs (CMV-MP-eGFP, PSV-MP-eGFP) was observed. In the case of CMV-MP-eGFP, clear colocalization with PD was detected in both hosts, but PSV-MP-eGFP was not tightly connected to the PD in N. benthamiana and barely localized to the PD in C. annuum epidermal cells. Measuring Pearson correlation coefficients (PCCs) also supported the visual observation.

The 28 Ser Amino Acid of Cucumber Mosaic Virus Movement Protein Has a Role in Symptom Formation and Plasmodesmata Localization.

Cucumber mosaic virus (CMV, Cucumovirus, Bromoviridae) is an economically significant virus infecting important horticultural and field crops. Current knowledge regarding the specific functions of its movement protein (MP) is still incomplete. In the present study, potential post-translational modification sites of its MP were assayed with mutant viruses: MP/S28A, MP/S28D, MP/S120A and MP/S120D. Ser28 was identified as an important factor in viral pathogenicity on Nicotiana tabacum cv. Xanthi, Cucumis sativus and Chenopodium murale. The subcellular localization of GFP-tagged movement proteins was determined with confocal laser-scanning microscopy. The wild type movement protein fused to green fluorescent protein (GFP) (MP-eGFP) greatly colocalized with callose at plasmodesmata, while MP/S28A-eGFP and MP/S28D-eGFP were detected as punctate spots along the cell membrane without callose colocalization. These results underline the importance of phosphorylatable amino acids in symptom formation and provide data regarding the essential factors for plasmodesmata localization of CMV MP.

Genetic Diversity of Potyviruses Associated with Tulip Breaking Syndrome.

Tulip breaking is economically the most important viral disease of modern-day tulip growing. It is characterized by irregular flame and feather-like patterns in the flowers and mosaic on the foliage. Thirty-two leaf samples were collected from cultivated tulip plants showing tulip breaking syndrome from Hungary in 2017 and 2018. Virus identification was performed by serological (ELISA) and molecular (RT-PCR) methods. All samples proved to be infected with a potyvirus and evidence was provided that three potyvirus species could be identified in the samples: Lily mottle virus (LMoV), Tulip breaking virus (TBV) and Rembrandt tulip-breaking virus (ReTBV). Recombination prediction accomplished with Recombination Detection Program (RDP) v4.98 revealed potential intraspecies recombination in the case of TBV and LMoV. Phylogenetic analyses of the coat protein (CP) regions proved the monophyletic origin of these viruses and verified them as three different species according to current International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria. Based on these results, we analyzed taxonomic relations concerning potyviruses associated with tulip breaking syndrome. We propose the elevation of ReTBV to species level, and emergence of two new subgroups in ReTBV.

A multiplex RT-PCR assay for the simultaneous detection of prevalent viruses infecting pepper (Capsicum annuum L.).

The aim of this work was to create an easy, fast and sensitive method for the simultaneous detection of the most frequent viruses known to infect pepper (Capsicum annuum L.) crops. A multiplex RT-PCR assay was developed that successfully achieved this aim. Using specifically designed primer pairs, the assay could simultaneously amplify the genomes of members of the two subgroups (I and II) of cucumber mosaic virus (CMV), two tobamoviruses, tobacco mosaic virus (TMV) and pepper mild mottle virus (PMMoV), potato virus Y (PVY), and tomato spotted wilt virus (TSWV) in a single assay. The multiplex RT-PCR assay was found to be a sensitive diagnostic tool for the detection of the viruses from the leaves and fruits of naturally infected pepper plants. This assay would provide prompt disease status information for pepper breeders.

Symptom recovery is affected by Cucumber mosaic virus coat protein phosphorylation.

Cucumber mosaic virus induces specific recovery phenotype, namely cyclic mosaic symptoms on tobacco plants. We provide further evidence that besides the 2b suppressor protein, the coat protein (CP) also has a role in symptom recovery and it is connected to its phosphorylation. We analyzed the impact of the phosphorylated (S148D) and the non-phosphorylated (S148A) state of CP148 Ser on symptom formation, virion stability and the effect of CP and its mutants on 2b-mediated local GFP-silencing. We demonstrated that a single aa change could be responsible for preventing the recovery phenomenon as replacing the phosphorylatable Ser with Ala in the 148aa position abolishing the cyclic phenomenon. CP/S148A mutation equilibrates the accumulation of the virus during the infection both at RNA and protein level in N. tabacum L. cv Xanthi plants. In summary, we determined a regulatory effect of the CMV CP on the self-attenuation mechanism and downregulation of the suppressor effect of the 2b protein.

Molecular Modeling for Better Understanding of Cucumovirus Pathology.

Cucumber mosaic virus (CMV) is a small RNA virus capable of infecting a wide variety of plant species. The high economic losses due to the CMV infection made this virus a relevant subject of scientific studies, which were further facilitated by the small size of the viral genome. Hence, CMV also became a model organism to investigate the molecular mechanism of pathogenesis. All viral functions are dependent on intra- and intermolecular interactions between nucleic acids and proteins of the virus and the host. This review summarizes the recent data on molecular determinants of such interactions. A particular emphasis is given to the results obtained by utilizing molecular-based planning and modeling techniques.

A single point mutation on the cucumber mosaic virus surface induces an unexpected and strong interaction with the F1 complex of the ATP synthase in Nicotiana clevelandii plants.

A previous study showed that a single amino acid difference in the cucumber mosaic virus (CMV) capsid protein (CP) elicits unusual symptoms. The wild-type strain (CMV-R) induces green mosaic symptoms and malformation while the mutant strain (CMV-R3E79R) causes chlorotic lesions on inoculated leaves and strong stunting with necrosis on systemic leaves. Virion preparations of CMV-R and CMV-R3E79R were partially purified from Nicotiana clevelandii A. Gray and analysed by two-dimensional gel electrophoresis. Their separated protein patterns showed remarkable differences at the 50-75 kDa range, both in numbers and intensity of spots, with more protein spots for the mutant CMV. Mass spectrometry analysis demonstrated that the virion preparations contained host proteins identified as ATP synthase alpha and beta subunits as well as small and large Rubisco subunits, respectively. Virus overlay protein binding assay (VOPBA), immunogold electron microscopy and modified ELISA experiments were used to prove the direct interaction between the virus particle and the N. clevelandii ATP synthase F1 motor complex. Protein-protein docking study revealed that the electrostatic change in the mutant CMV can introduce stronger interactions with ATP synthase F1 complex. Based on our findings we suggest that the mutation present in the CP can have a direct effect on the long-distance movement and systemic symptoms. In molecular view the mutant CMV virion can lethally block the rotation of the ATP synthase F1 motor complex which may lead to cell apoptosis, and finally to plant death.

Phosphorylation regulates the subcellular localization of Cucumber Mosaic Virus 2b protein.

The 2b protein of Cucumber mosaic virus has a role in nearly all steps of the viral cycle including cell-to-cell movement, symptom induction and suppression of antiviral RNA silencing. Previous studies demonstrated the presence of 2b protein in the nucleus and in cytoplasm as well. Phosphorylation site of 2b protein is conserved in all CMV isolates, including proposed constitute motifs for casein kinase II and cyclin-dependent kinase 2. To discern the impact of 2b protein phosphorylation, we created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. We compared these mutants to the wild-type (Rs-CMV) virus in terms of symptom induction, gene silencing suppressor activity as well as in cellular localization. Here, in this study we confirmed the phosphorylation of 2b protein in vivo, both in infected N. benthamiana and in infiltrated patches. Mutants containing aspartic acid in the phosphorylation site accumulated only in the cytoplasm indicating that phosphorylated 2b protein could not enter the nucleus. We identified a conserved dual phosphorylation switch in CMV 2b protein, which equilibrates the shuttling of the 2b protein between the nucleus and the cytoplasm, and regulates the suppressor activity of the 2b protein.

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper.

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.

Phylogenetic analysis of Tomato spotted wilt virus (TSWV) NSs protein demonstrates the isolated emergence of resistance-breaking strains in pepper.

Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistance-breaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010-2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.

Alanine scanning of cucumber mosaic virus (CMV) 2b protein identifies different positions for cell-to-cell movement and gene silencing suppressor activity.

The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa) to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA) overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA), and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA), which are not essential for suppressor activity.

Oral immunogenicity of a plant virus vector based porcine circovirus antigen - short communication.

A recombinant cucumber mosaic virus based expression system has been developed for the production of an immunogenic porcine circovirus epitope. The resulting nanoparticle was shown to elicit specific immune response in mice and pigs, when administered parenterally. To evaluate the oral applicability of this vaccine candidate, two experiments were performed. In the first one, the resistance of the vector itself to mucosal environment was tested in mice. Cucumber mosaic virus particles fed to mice were able to elicit specific mucosal and serum antibody production. In the second experiment, recombinant cucumber mosaic virus fed to piglets resulted in the appearance of porcine circovirus specific serum antibodies. The vector proved to be able to survive in the gastrointestinal tract, so that an epitope expressed on its surface could induce specific immune response. These results indicate that the developed plant virus based expression system offers an effective method for mucosal vaccine production.

The C-terminal domain of the 2b protein of Cucumber mosaic virus is stabilized by divalent metal ion coordination.

The main function of the 2b protein of Cucumber mosaic virus (CMV) is binding permanently the double stranded siRNA molecules in the suppression process of post-transcriptional gene silencing (PTGS). The crystal structure of the homologue Tomato aspermy virus (TAV) 2b protein is known, but without the C-terminal domain. The biologically active form is a tetramer: four 2b protein molecules and two siRNA duplexes. Regarding the complete 2b protein structure, we performed a molecular dynamics (MD) simulation of the whole siRNA-2b ribonucleoprotein complex. Unfortunately, the C-terminal domain is proved to be partially unstructured. Multiple sequence alignment showed a well conserved motif between residues 94 and 105. The negatively charged residues of the C-terminal domain are supposed to take part in coordination of a divalent metal ion and stabilize the three-dimensional structure of the C-terminal domain. MD simulations were performed on the detached C-terminal domains (aa 65-110). 0.15 M MgC2, CaCl2, FeCl2 and ZnCl2 salt concentrations were used in the screening simulations. Among the tested divalent metal ions Mg²âº proved to be very successful because Asp95, Asp96 and Asp98 forms a quasi-permanent Mg²âº binding site. However the control computations have resulted in any (at least) divalent metal ion remains in the binding site after replacement of the bound Mg²âº ion. A quadruple mutation (Rs2DDTD/95-98/AAAA) was introduced into the position of the putative divalent metal ion binding site to analyze the biological relevance of molecular modeling derived hypothesis. The plant inoculation experiments proved that the movement of the mutant virus is slower and the symptoms are milder comparing to the wild type virus. These results demonstrate that the quadruple mutation weakens the stability of the 2b protein tetramer-siRNA ribonucleoprotein complex.

A cucumber mosaic virus based expression system for the production of porcine circovirus specific vaccines.

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.

Identification a coat protein region of cucumber mosaic virus (CMV) essential for long-distance movement in cucumber.

To characterise the long-distance movement determinant of cucumoviral coat proteins (CPs), five mutants were engineered into the CMV CP bearing the corresponding tomato aspermy virus (TAV) loops exposed on the surface of the virion. Both viruses can move long-distance in Nicotiana clevelandii, but only CMV can move long-distance in cucumber. Investigation of the CMV chimeras identified three amino acids of the ßB-ßC loop that were essential for the CMV long-distance movement in cucumber. Introducing these mutations into the TAV CP was not sufficient for long-distance movement, indicating that this is not the sole region causing long-distance movement deficiency.

Comparison of the nucleotide sequences of wheat dwarf virus (WDV) isolates from Hungary and Ukraine.

Wheat dwarf virus (WDV) is the most ubiquitous virus in cereals causing huge losses in both Hungary and Ukraine. The presence of barley-and wheat-adapted strains has been confirmed, suggesting that the barley strain is restricted to barley, while the wheat strain is present in both wheat and barley plants. Five WDV isolates from wheat plants sampled in Hungary and Ukraine were sequenced and compared with known WDV isolates from GenBank. Four WDV isolates belonged to the wheat strain. Our results indicate that WDV-Odessa is an isolate of special interest since it has originated from wheat, but belongs to the barley-adapted strain, providing novel data on WDV biology and raising issues of pathogen epidemiology.

Nucleotide sequence analysis of peanut stunt virus Rp strain suggests the role of homologous recombination in cucumovirus evolution.

The complete nucleotide (nt) sequence of peanut stunt virus Robinia strain (PSV-Rp) was determined and compared to other PSV strains and to representatives of the genus Cucumovirus. Nt sequence comparison showed 74.1-84.6% identity with the known PSV strains. Phylogenetic analysis revealed the different origin of the two genes encoded by RNA3. While the 3a gene clustered with PSV-W, the coat protein gene clustered with PSV-Mi. Recombination breakpoint analysis revealed two recombination points on RNA3. Based on these results, the establishment of a fourth PSV subgroup is proposed. This work revealed that homologous recombination occurred during the evolution of PSV.

Modeling-based characterization of the elicitor function of amino acid 461 of Cucumber mosaic virus 1a protein in the hypersensitive response.

The Ns strain of Cucumber mosaic virus (CMV) induces hypersensitive response (HR) on Nicotiana tabacum cv. Xanthi-nc and on Nicotiana glutinosa. The genetic determinant of the HR induction was localized earlier to amino acid 461 of the 1a protein. The 3D structure of the 1a protein is still unknown and building a homology model is impossible. Nevertheless, on the basis of secondary structure predictions we have created partial protein models for the region surrounding residue 461 which can account structurally for the effect of aa 461 on elicitor function. Seven different amino acid mutations were designed and introduced to the position 461 of the 1a protein in RNA 1. Three of the mutations (proline, glutamic acid, asparagine) inhibited virus replication. Two of the mutants caused systemic symptom development (lysine and arginine). Two mutants (alanine and serine) resulted in localization of the virus, but strong necrosis similar to the original Ns-CMV strain was not observed. Inoculation of purified Ns-CMV virions at extremely high concentration provoked systemic symptoms.