Site Directed Mutagenesis of a Putative Protease Cleavage Site Within the 105 Kilodation Protein of Southern Bean Mosaic Virus

There are four open reading frames (ORF) in three positive-sense translational phases of Southern Bean Mosaic Virus (SBMV). ORF2 codes for a 105 KDa polyprotein which is proposed to be autocatalytically cleaved into smaller functional proteins. Amino acid sequence homology between the SBMV and poliovirus, foot and mouth disease virus, and cowpea mosaic virus reveal that SBMV has a similar genomic organization to picornaviruses with a conserved order: Vpg (Viral protein, genome-linked )-protease-replicase. Two Gln-Ser (QS) amino acid pairs within the 105 KDa polyprotein, C75= 930-936, C60= 1305-1310, are proposed to be the cleavage sites based on 1) similar structural arrangement around the two QS pairs to that of known cleavage sites of picornaviruses and potyviruses; 2) computer prediction of the cleavage products from the SBMV RNA sequence; 3) observed in vitro translation products. In order to investigate the role of the C60 QS pair in the proteolytic processing of the 105 KDa polyprotein, a mutagenic oligo was designed to create various amino acid substitutions in the QS pair. A coupled in vitro transcription/translation system was used to produce the protein products of both wildtype and mutants. During the procedure, tritiated leucine was incorporated into the protein products. The protein products were separated by SDS-PAGE and the cleavage patterns were detected after autoradiography. Two substitutions of the C60 QS pair-Pro-Val (PS) in mutant6, Gin-Pro (QP) in mutant19-reduced the yield of the 60 KDa protein by different levels. This result supports the hypothesis that the C60 QS is a cleavage site

Multiple Interactions among Proteins Encoded by the Mite-Transmitted Wheat Streak Mosaic Tritimovirus

AbstractThe genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins. To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system. Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast. Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins. The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI). Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another. The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected. In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3. The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells. The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes

Distribution and multiplication of iris severe mosaic potyvirus in bulbous Iris in relation to metabolic activity : implications for ISMV detection

During cultivation of iris, several viruses may cause severe damage like yield reduction and discoloration of the plant. In commercial stocks in the Netherlands virtually all plants are infected with iris mild mosaic virus (IMMV) while iris severe mosaic virus (ISMV) and narcissus latent virus (NLV) can also be present at high incidence. ISMV and IMMV both belong to the genus Potyvirus of the largest plant virus family, the Potyviridae.As the quality of virus-free iris is superior and methods are available now to produce and grow virus-free iris for commercial practice, it is of great importance to control the spread of iris viruses. Therefore rapid and reliable assays for the detection of these viruses are needed. In the Netherlands such tests are being developed mainly at the Bulb Research Centre in Lisse. At the start of the research described in this thesis, detection of ISMV in the iris bulb was problematic, in contrast to that of IMMV It has been difficult to detect ISMV reliably in iris bulbs from lifting in late August to planting in October by means of ELISA or electron microscopy.The aim of the research was to develop a reliable detection protocol suitable for monitoring ISMV infection, and, to understand the behaviour of this virus in the iris plant, especially the bulb, with respect to the multiplication and movement of the virus in relation to the metabolic activity of the plant. Preliminary results concerning the development of a test protocol are presented in Chapter 2, and further elaborated in Chapter 3, 7 and 8. In Chapters 4, 6 and 7 the behaviour of ISMV in relation to the metabolic activity of the plant is elaborated. In Chapters 5 and 8 a further characterisation of ISMV is presented.In freshly-lifted bulbs secondarily infected with ISMV, the virus was not always detected in the basal plate and rarely in bulb scale tissue (Chapter 4), but it gradually became better detectable in the bulb scale tissue when bulbs were incubated during several months at a temperature of circa 17°C (Chapter 2). When a wounding method was applied on the iris bulb by cutting a slice of bulb scale tissue from a side of the bulb, ISMV became readily detectable in all bulbs, though only in tissue adjacent to the cut surface, if the cut bulbs were incubated for three weeks at an optimal temperature of 17-20°C (tested within a range of 5 to 30°C; Chapters 2 and 3). It was concluded that stress followed by a recovery period is favourable for an enhanced detection of the virus. Indeed, high temperature treatment, applied as an alternative stress, also gave rise to improved detection of ISMV (Chapter 3).To investigate whether the virus became better detectable by multiplication rather than by modification of the antigenicity of the coat protein, the levels of the viral antigen as well as those of the viral RNA were followed after wounding. From this analysis it was concluded that the increase of the virus titre was due to multiplication (Chapter 4). For the detection of the viral RNA, a cDNA done corresponding to a part of the 3'-terminal region of the ISMV genome was used. The availability of this clone led to the determination of the nucleotide sequence of the ISMV coat protein (CP) gene, thus allowing a definitive classification of the virus. Phylogenetic comparisons of potyviral CP sequences revealed that ISMV is a taxonomically distinct potyvirus not closer related to other bulbous or monocotyledonous infecting potyviruses than to other potyviruses. The sequence data also allowed to conclude that the CP is probably cleaved off from the NIb protein at an unusual glutamine acid-glycine (E/G) dipeptide cleavage site. Furthermore the N-terminus of the CP appeared to be only 15 amino acids long, being the shortest found among potyvirus CPs studied so far.Further research on the localisation of the virus after high-temperature treatment showed that the virus was well detectable in the bulb base and usually also in the vascular bundles and surrounding tissue. This suggested that the virus did spread from the basal plate towards the bulb scales. However when the wounded (cut) apical parts of infected, but in ELISA negative reacting, bulbs were incubated at an optimal recovery temperature, the virus became detectable in these upper parts of the bulbs (Chapter 4). Thus, virus must have been originally present in the scales, albeit at a very low and at non-detectable concentration. This provides another indication that multiplication is likely to be the main factor involved in the improved sensitivity of viral detection. It is, therefore, hypothesised that the multiplication is enhanced by increased metabolic activity after stress. A possible correlation between metabolism and ISMV multiplication was further investigated in Chapter 6, with oxygen uptake as a measure for the metabolic activity after application of wounding, high-temperature stress or ethylene treatment.An increased level of total oxygen uptake was found after wounding as well as high- temperature treatment, thus positively correlating with the enhancement of ISMV detection. Application of ethylene, an important plant hormone in relation to stress, caused a limited increase in respiration and a slight improvement of ISMV detectability. After wounding, the mitochondrial respiration, the residual respiration and the capacity of the alternative pathway had increased, while after high-temperature treatment there was mainly an increase in residual respiration measured. These findings suggest that increased production of metabolic intermediates, possibly by the pentose phosphate pathway, rather then an increase in energy is important for the observed stress-induced multiplication of ISMV in iris bulbs.For the development of a satisfactory test method, it is imperative that virus is reliably detected not only in secondarily infected bulbs but also in primarilyinfected bulbs. To obtain primarily infected bulbs, virus-free plants were mechanically inoculated with ISMV at different times during the growing season. At lifting the level of ISMV in primarily infected bulbs appeared to be dependent on the date of inoculation. Surprisingly, it was found that early infections were scarcely detectable in the bulbs in contrast to late infections. The later in the season infection took place, the better ISMV was detectable in (untreated) bulbs (Chapter 7). Wounding of these primarily infected bulbs generally resulted in an increased detection in bulbs of the early infected plants, but the virus titre in bulbs of late infections decreased. However, these infections were still reliably detectable. Another potential problem for implementation of the developed test for routine use could be the existence of differently reacting isolates of ISMV. In spite of causing slightly different symptoms and serological reactions, all could be detected by the wounding method (Chapter 8).The reason why ISMV is so difficult to detect in secondarily infected and in early primarily infected bulbs was investigated further in Chapter 7. The virus titre was monitored in the whole plant, including the bulb, during the growing season for both secondarily and primarily infected plants. The distribution of ISMV in the above-ground parts of secondarily infected as well as primarily infected plants correlated with the nutrient flow via the vascular system. This implied that above-ground parts of secondarily infected plants were totally infected while in primary infections the presence of virus was dependent on the time of infection: during an early infection virus still spread to the upper leaves of the plant and only later to the new bulb, while in late infected plants the virus was found mainly in the new bulb. However, in secondarily infected plants hardly any virus could be detected in the new bulbs at any time during the whole growing season. Besides, the detectability of ISMV in bulbs of early infected plants decreased considerably towards the end of the growing season. This might be explained by assuming that the plant develops a barrier at some time after infection blocking virus avenue to the bulb, or that the virus in secondarily and early primarily infected plants is no longer available for transport anymore (Chapters 7 and 9). It must be concluded that detection of ISMV in these secondarily and early primarily infected bulbs immediately after lifting is unreliable due to impeded import of virus into the bulb

Engineering resistance against potato virus Y

Potato virus Y is the type species of the potyvirus genus, the largest genus of the plant virus family Potyviridae. The virus causes serious problems in the cultivation of several Solanaceous crops and although certain poly- and monogenic resistances are available, these can not always be employed, e.g. R y genes in potato cv. 'Bintje'. The aim of the research described in this thesis was to establish new forms of resistance against PVY by genetic modification of host plants. One such form of genetic engineered resistance is 'coat protein-mediated resistance', whereby expression of a viral coat protein (CP) in a transgenic plant may confer resistance against infection with the homologous virus, and some closely related viruses.At the start of this investigation no sequence data on the RNA genome of PVY were available, therefore cDNA synthesis and subsequent sequence determination was performed to obtain the necessary PVY CP gene sequence as well as additional sequences from the 3'-terminal region of the viral genome (Chapter 2 and Van der VIugt et al., 1989). This enabled the determination of the exact taxonomic position of the PVY N('tobacco veinal necrosis strain') isolate used in these experiments, among other PVY isolates from at least two different strains. Detailed comparisons of the PVY NCP and 3'-non translated (3'-NTR) sequences with those from a large number of geographically distinct PVY isolates that became available during the course of this investigation, showed that these sequences, in addition to distinguish between different potyvirus species (Ward and Shukla, 1991; Frenkel et al., 1989), can also be used for the distinction between strains of one potyvirus (Chapter 3, Van der VIugt et al., 1992a). Several strain specific amino acid sequences in the CPs and nucleotide sequences in the 3'-NTRs could be discerned, that are possibly involved in virulence and/or symptom expression. Further experiments are required to elucidate the precise biological significance of these sequence motifs. Interestingly the sequence comparisons as complied in Chapter 3 also confirmed the high levels of CP and 3'-NTR sequence identity between the PVY isolates at one hand and one putative isolate of pepper mottle virus (PepMoV, Dougherty et al., 1985) at the other, as described previously (Van der VIugt et al., 1989; Van der Vlugt, 1992). Initially described as an atypical strain of PVY (PVY-S, Zitter, 1972) PepMoV was later found to be serologically and biologically distinct from PVY (Purcifull et al., 1973, 1975; Zitter and Cook, 1973). Recent determination of the complete genomic RNA sequence of a Californian isolate of pepper mottle virus (PepMoV-C; Bowman-Vance et al, 1992a,b) and comparisons between a Florida isolate of PepMoV and PVY (Hiebert and Purcifull, 1992) however, suggest that PepMoV represents a distinct potyvirus though more closely related to PVY than to any other potyvirus. Additional sequence information of other, biologically well characterized, isolates of PepMoV, like a virus isolate apparently intermediate between PepMoV and PVY (Nelson and Wheeler, 1978), will hopefully aid in establishing the exact taxonomic position of this pepper infecting virus in the genus Potyvirus. Generally it is to be recommended that of all virus isolates whose (partial) sequences are under investigation, precise origin and other relevant biological characteristics are also accurately documented.In contrast to all other viruses for which 'CP-mediated resistance' has been described sofar, potyviruses do not express their CPs from a distinct, separate gene but through proteolytic cleavage of a polyprotein precursor. This necessitated theaddition of translational start signals, directly upstream of the CP encoding sequence, in order to enable expression of the PVY NCP in transgenic potato and tobacco plants. Potato tuber disc and tobacco leaf disc transformations with these constructs resulted in large numbers of transgenic plants (Chapters 4 and 5). Despite the fact that a large number of transgenic plants was tested for CP expression, using a highly sensitive enzyme-amplification based ELISA format, in none of the plants significant amounts of viral CP could be detected. Whether this is caused by the extra N-terminal methionine residue, or improper folding of the CP, resulting in decreased stability of the protein, or by inefficient protein extractions, possibly resulting from protein insolubility, is not known. It remains to be tested whether transformation of plants with a construct in which a functional protease domain is coupled to a potyviral CP with an intact protein processing sequence, will result in high levels of expression of the CP. For more practical purposes however, PVY CP expression levels appear not to be of significant importance since the protection against PVY, observed in the transgenic tobacco plants (Chapter 5 and 6), is apparently RNA-mediated, i.e. prima rily based on the presence of the CP encoding RNA rather than on the coat protein itself. Transgenic tobacco lines expressing PVY CP transcripts devoid of a translational start signal (CP -ATG), possess equal levels of protection against both mechanically inoculated virus and virus transmitted by the natural aphid vector Myzuspersicae (Chapter 5 and 6). It seems highly unlikely that the protection in these CP -ATGplants is based on minute amounts (i.e. less then 0.0001 % of the total soluble protein) of a truncated viral polypeptide since the presence of six translational stopcodons preceding the first in-frame AUG startcodon, 162 nucleotides down stream the 5'-end of the CP encoding sequence, will prevent expression of such a polypeptide.Analysis of the transgenic potato lines (Chapter 4) showed that most lines, as the transgenic tobacco lines, expressed CP specific RNA transcripts. Under the given greenhouse conditions, however, in none of the transgenic plants protection to PVY could be determined. In view of the results obtained with the transgenic tobacco lines, it may be anticipated that virus challenging of additional transgenic potato lines, under more optimal greenhouse conditions, will reveal similar levels of RNA-mediated virus resistance as observed in tobacco. For all practical purposes genetically engineered resistance based on the presence of RNA molecules is to be preferred over forms of resistance that are based on the expression of a (foreign) protein. Apart from being energetically more favourable for the plant, it is likely to aid in the acceptance of genetically modified crop plants by both politicians and the public, something which might, in the next few years, turn out to be the major obstacle in the successful application of plant transformation techniques.At this stage one can only speculate on the mechanism(s) on which this RNAmediated resistance is based. Transformation of plants with partial CP or other PVY Ngenomic sequences will help in identifying the protection mechanism(s) involved and show whether regions other than the CP-encoding domain can be equally effective in conferring virus resistance. If the resistance is based on a 'sense-RNA' effect, i.e. hybridization of the positive sense transgenic RNA to negative-sense viral RNA replication intermediates, thereby blocking further virus replication, the ribozyme technology might prove an efficient expansion of this genetically engineered type of resistance. Ribozymes, RNA sequences capable of specific and catalytic cleavage of other RNA-sequences, are able to cleave target RNAs efficiently and catalytically in vitro . The antiviral application of ribozymes in transgenic plants however has sofar demonstrated not to be very successful and reported protection levels are not yet exceeding those obtained with antisense RNAs (Edington and Nelson, 1992). Chapter 7 describes the design and synthesis of hammerhead ribozymes capable to cleave a highly conserved region from the PVY RNA dependent RNA-polymerase cistron. It was shown that the correct formation of the hammerhead cleavage complex, determined at least in part by the lengths of the antisense arms of the ribozyme, forms an important factor in the efficiency of cleavage. Cellular and full-length viral RNA molecules generally posses extended, unknown secondary structures which are likely to hamper precise formation of hammerhead structures, which requires bimolecular basepairing. Correct hammerhead formation and efficient cleavage of these RNAs will therefore require ribozymes with rather long basepairing arms. These long antisense arms however will make catalytic cleavage rather unlikely since complex dissociation will probably become the rate limiting factor. For this reason one can assume that ribozymes will only be successful when introduced into specific antisense RNA molecules, directed against the less abundant viral complementary strands, rather than as highly efficient RNA cleaving "enzymes"

Identification of two potyviruses of phaseolus vulgaris in South Africa

Summary in English.Bibliography: pages 105-125.A survey was conducted by researchers at ARC-PPRI on dry beans (Phaseolus vulgaris) during 1993. All the viruses known to occur on dry beans in South Africa were found, as well as a few unidentified viruses. Of these, samples 93/1 and 93/65 form the basis of this thesis. Electron microscopy (EM) indicated that these viruses could be potyviruses, as they were flexuous particles of approximately 700 to 800 nm. Observation of pinwheels in ultrathin sections of Nicotiana benthamiana infected with isolate 93/1 and Phaseolus vulgaris infected with isolate 93/65, confirmed that the viruses probably belonged to the Potyvirus genus, family Potyviridae. Further serological tests indicated that the viruses were related but not homologous to strains of clover yellow vein (CIYW) and blackeye cowpea mosaic (BICMV) viruses respectively. None of these viruses have previously been described as occurring in South Africa. As we were unable to positively identify the viruses with serological methods, we needed to characterise these viruses on a molecular level. Potyvirus specific oligonucleotide primers were used for PCR amplification of viral eDNA The primers amplify an approximately 700 bp fragment of the virus genome, spanning the 3' noncoding region as well as a part of the coat protein gene: one primer is complementary to the poly(A) tail, and the other to a sequence coding for a conserved block of amino acid sequences (also known as the WCIEN block) in the mid-region of the coat protein. The nucleic acid sequences of the PCR products were compared to that of other potyviruses to positively identify these isolates

The characterisation of Ornithogalum mosaic virus

Bibliography: pages 155-179.Ornithogalum mosaic virus (OMV) is the most serious pathogen of commercially grown Ornithogalum and Lachenalia species in South Africa. Although omithogalum mosaic disease was first reported as early as 1940, attempts to purify or characterise the virus(es) were not successful. The extremely mucilaginous nature of omithogalum and lachenalia plant extracts severely hampered virus purification from these hosts. No alternative propagation host for OMV is known: a virus purification protocol for systemically infected ornithogalum and lachenalia was therefore developed. This method eliminated the mucilage in leaf extracts by hemicellulase digestion. Physicochemical characterisation of purified particles suggested that a single virus was present: it had elongated, filamentous particles with a modal length in the range 720- 760 nm; a single major coat protein of Mᵣ30 000, and a single genomic ssRNA of Mᵣ2.90 x 10⁶ daltons. Oligo(dT)-cellulose chromatography confirmed that the genomic RNA was polyadenylated