The Spatial Arrangement of the Wild-Type and Mutant Tobacco Mosaic Virus Coat Proteins Assessed by Tritium Planigraphy

Mutant ts21-66 of the tobacco mosaic virus (TMV) differs from the wild-type TMV-U1 by two mutations (Ile21 Thr and Asp66 Gly) in the coat protein (CP) gene and in symptoms produced in infected N" plants. The CP structure in TMV-U1 and ts21-66 virions was probed by tritium planigraphy. Compared with the wild-type CP, labeling of the N-terminal region of mutant CP was half as high and suggested its greater shielding. The role of this CP region in virus interactions with the N" resistance system is discussed.     

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.     

Differences in the spatial structure of an envelope protein from tobacco mosaic virus and its mutant, detected by tritium planigraphy]

Mutant ts21-66 of the tobacco mosaic virus (TMV) differs from the wild-type TMV-U1 by two mutations (Ile-21-->Thr and Asp-66-->Gly) in the coat protein (CP) gene and in symptoms produced in infected N' plants. The CP structure in TMV-U1 and ts21-66 virions was probed by tritium planigraphy. Compared with the wild-type CP, labeling of the N-terminal region of mutant CP was half as high and suggested its greater shielding. A role of this CP region in virus interactions with the N' resistance system is discussed.     

Photosynthetic oxygen evolution: H/D isotope effects and the coupling between electron and proton transfer during the redox reactions at the oxidizing side of Photosystem II

The oxygen evolving complex (OEC) of Photosystem II (PS II) incorporates a Mn-cluster and probably a further redox cofactor, X. Four quanta of light drive the OEC through the increasingly oxidized states S0 S1 S2 S3 S4 to yield O2 during the transition S4 S0. It has been speculated that the oxidation of water might be kinetically facilitated by the abstraction of hydrogen. This implied that the respective electron acceptor is deprotonated upon oxidation. Whether YZ and X fulfill this expectation is under debate. We have previously inferred a 'chemical' deprotonation of X based on the kinetics of proton release (Haumann M, Drerenstedt W, Hundelt M and Junge W (1996) Biochim Biophys Acta 1273: 237–250. Here, we investigated the rates of electron transfer and proton release as function of the D2O/H2O ratio, the pH, and the temperature both in thylakoids and PS II core particles. The largest kinetic isotope effect on the rate of electron transfer (factor of 2.1–2.4) and the largest pH-dependence (factor of about 2 between pH 5 and 8) was found on S2 S3 where X is oxidized. During the other transitions both factors were much smaller ( 1.4). Electron transfer is probably kinetically steered by proton transfer only during S2 S3. These results corroborate the notion that X serves as a hydrogen acceptor for bound water during S4 S0. We propose a consistent scheme for the final reaction with water to yield dioxygen: two two-electron (hydrogen) transfers in series with a peroxide intermediate.     

Appearance of “β-Like” Circular Dichroism Spectra on Protein Aggregation That Is not Accompanied by Transition to β-Structure

CD spectra in the 200 to 250 nm spectral region for small ordered aggregates (trimers-pentamers) of tobacco mosaic virus (TMV) coat protein (CP) and for long virus-like helical aggregates of TMV CP were compared. It was found that small (4S) TMV CP aggregates have a CD spectrum typical of a protein with high -helix content, which agrees well with results of X-ray diffraction studies. But in the long helical aggregates (and in the TMV virions) TMV CP gives -like CD spectra similar to those of many other aggregated proteins. From X-ray diffraction data, it is well known that TMV CP subunits do not change their secondary or tertiary structure on assembly into virions or the helical repolymerized protein. Thus, the change in the shape of 200 to 250 nm CD spectra cannot be employed as the sole criterion of the conversion of a protein to -structure in the course of aggregation.     

外壳蛋白羧端序列缺失对烟草花叶病毒侵染性的影响

对野生型烟草花叶病毒(TMV-U1)的外壳蛋白羧端序列进行系列缺失突变,观察到TMV-U1株系的外壳蛋白羧端序列缺失6个氨基酸(保留152个氨基酸),仍能较强系统侵染烟草并高水平表达外壳蛋白,且能在新生叶里复制大量完整的病毒粒子。该研究结果表明：外壳蛋白羧端6个氨基酸序列并非烟草花叶病毒感染和复制所必需,并对利用外壳蛋白羧端缺失型病毒载体表达外源多肽具有一定的启示性。     

Adenylyl Cyclase Signaling Mechanism of Relaxin Action

In connection with our discovery of the adenylyl cyclase signaling mechanism (ACSM) of action of some peptides belonging to the insulin superfamily, a possibility of its involvement in action of another insulin superfamily peptide, relaxin, was studied. It was shown for the first time that human relaxin-2 (10^–12–10^–8 M) activated adenylyl cyclase (AC) in a dose-dependent manner. The maximal peptide effect was revealed at a concentration of 10^–8 M. Under condition of the hormonal action the basal enzyme activity increased by +310% in human myometrium, by +117%, in rat skeletal muscles, and by +49%, in foot smooth muscles of the bivalve mollusc Anodonta cygnea. Insulin and mammalian insulin-like growth factor-I (IGF-I) also produced the AC activating effect in these muscles. The order of efficiency of these peptides, based on their ability to induce the maximal AC stimulating effect, was as follows: relaxin > IGF-I > insulin (human myometrium); IGF-I > relaxin > insulin (rat skeletal muscle); insulin-like peptide of Anodonta (ILPA) > IGF-I > insulin > relaxin (molluscan muscle). The relaxin activating effect on AC was potentiated by a guanine nucleotide, the non-hydrolyzed analog of GTP, guanylylimidodiphosphate (Gpp[NH]p), which indicates participation of G_s-protein in realization of this effect. This effect was inhibited by a tyrosine kinase selective blocker, tyrphostin 47, and a phosphatidylinositol-3-kinase (PI-3-K) selective blocker, wortmannin. Thus, for the first time, participation of ACSM in the relaxin action has been established. This mechanism, as suggested at the present time state of its study, includes the following signal pathway: receptor-tyrosine kinase PI-3-K G_s-protein AC.     

Effect of triparanol (Mer 29) on rat liver lysosomes: cytochemical and biochemical study

Summary Rats treated with triparanol (MER-29) develop numerous membranous inclusions-myeloid bodies in the cytoplasm of liver cells. The myeloid bodies did not show cytochemically demonstrable acid phosphatase. Instead diffuse activity was observed throughout the cytoplasm. Biochemically, acid phosphatase was found in the liver lysosomal fraction obtained from triparanol treated rats. This fraction, however, did not show the structure-linked latency of acid phosphatase which is characteristic of normal lysosomes. It is suggested that myeloid bodies are lysosomes with altered membranes.     

Recruitment of the Host Plant Heat Shock Protein 70 by Tomato Yellow Leaf Curl Virus Coat Protein Is Required for Virus Infection

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.     

Low sodium dodecyl sulfate concentrations inhibit tobacco mosaic virus coat protein amorphous aggregation and change the protein stability

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 52 degrees C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 microM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four alpha-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.     

Assay for doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 1. In vitro anther culture

Sunflower anthers placed on solid medium developed calli and embryos after 12 days. Embryogenesis was improved by the addition of 0.1% polyvinylpyrrolidone (PVP) that alleviated anther and medium browning. As in other species, genotypic variability was an important parameter in the anther response and a medium genotype interaction was suggested with a different PVP effect depending on the genotype. Embryo germination was largely increased by the successive use of germination media with decreasing sucrose concentrations (10%6%3%). Histological examination of the anthers during the first ten days of culture showed that, under our conditions, the embryos were of somatic origin, arising directly from the anther wall on the outside or inside of the anther loculus, or indirectly from proliferating anther wall- or connective tissue-derived callus. Finally, the ploidy status of 78 embryo-derived plants was determined by Feulgen stain or flow cytometry: all plants were diploid (2n=34).Abbreviations PVP polyvinylpyrrolidone     

Proton release during the redox cycle of the water oxidase

Old and very recent experiments on the extent and the rate of proton release during the four reaction steps of photosynthetic water oxidation are reviewed. Proton release is discussed in terms of three main sources, namely the chemical production upon electron abstraction from water, protolytic reactions of Mn-ligands (e.g. oxo-bridges), and electrostatic response of neighboring amino acids. The extent of proton release differs between the four oxidation steps and greatly varies as a function of pH both, but differently, in thylakoids and PS II-membranes. Contrastingly, it is about constant in PS II-core particles. In any preparation, and on most if not all reaction steps, a large portion of proton transfer can occur very rapidly (<20 s) and before the oxidation of the Mn-cluster by Y_z ⁺ is completed. By these electrostatically driven reactions the catalytic center accumulates bases. An additional slow phase is observed during the oxygen evolving step, S₃S₄S₀. Depending on pH, this phase consists of a release or an uptake of protons which accounts for the balance between the number of preformed bases and the four chemically produced protons. These data are compatible with the hypothesis of concerted electron/proton-transfer to overcome the kinetic and energetic constraints of water oxidation.Abbreviations BBY-membranes Photosystem II-enriched membrane fragments prepared after Berthold, Babcock and Yocum (1981) - BSA bovine serum albumin - Chl chlorophyll - CAB-protein chlorophyll a/b-binding protein - core particles oxygen evolving reaction center core particles of Photosystem II - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - IML intermittent light - P-680 primary electron donor of Photosystem II - PS II Photosystem II - Y_z tyrosine residue on the D1 polypeptide, electron carrier between manganese and P-680 - photochemical reaction      

Distribution of catecholamines,indoleamines, and their precursors and metabolites in the scallop,Placopecten magellanicus (Bivalvia,Pectinidae)

Summary 1. Although monoamines are well-known to play important roles in molluscan physiology, we are far from fully understanding the synthetic and degradative pathways of these substances, particularly in commercially important bivalve species. In the present study endogenous catecholamines, indoleamines, and their possible precursors and metabolites were detected in the scallop,Placopecten magellanicus, by high-performance liquid chromatography coupled to electrochemical detection.2. Chromatographic analysis of CNS (cerebral, pedal, and parietovisceral combined), gill, gonad, kidney, mantle, liver, heart, fast adductor muscle, and foot disclosed the presence of the catecholamines 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, and epinephrine and their metabolites normetanephrine, metanephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid.3. Dopamine was detected most frequently and most consistently among all catecholamines. The concentrations of dopamine (1400 pg/mg wet weight) and its major metabolite 3,4-dihydroxyphenylacetic acid (300 pg/mg wet weight) were highest in the CNS. Following the CNS, dopamine was also abundant in other tissues such as heart, foot, and gill. The concentration of norepinephrine (1000 pg/mg wet weight) was highest in the CNS followed by the heart (700 pg/mg wet weight) and gill (600 pg/mg wet weight).4. The indoleamine, 5-hydroxytryptamine, was present in considerable amounts in all tissues, but its content was highest in the foot (2700 pg/mg wet weight) followed by the CNS (1150 pg/mg wet weight) and gonad (1000 pg/mg wet weight). The precursor 5-hydroxytryptophan was also abundant in the foot followed by the gonad, CNS, and heart.5. The oxidative metabolite 5-hydroxy-3-indole acetic acid was detected in the largest amount in CNS (200 pg/mg wet weight), whereasN-acetyl-5-hydroxytryptamine was detected in trace amounts in CNS, gonad and foot. This study also presents evidence for -glutamyl dopamine and -glutamyl 5-hydroxytryptamine as the possible alternate catabolic products of dopamine and 5-hydroxytryptamine, respectively, as previously described in gastropods.6. Thus, the detection of monoamines and their precursors and metabolites in scallop strongly suggests the presence of mammalian-type enzymic action of hydroxylation, oxidation, and methylation pathways leading to synthesis and degradation of detected compounds. Furthermore, this is the first study to disclose the evidence of nonconventional metabolic pathways for dopamine (-glutamyl dopamine dopamine dihydroxyphenylacetic acid homovanillic acid) and 5-hydroxytryptamine (-glutamyl 5-hydroxytryptamine 5-hydroxytryptamine 5-hydroxy-3-indoleacetic acid) inactivation in a bivalve species.     

Influence of protein structure on selection of nitrous acid induced mutants of TMV

Summary Calculated values for amino acid replacements in nitrous acid induced mutants of TMV are compared with experimentally found data. 60 of 68 analysed mutants (Wittmann, 1962, 1964; Tsugita and Fraenkel-Conrat, 1962) may be explained by transitions CU and A(H)G (Schuster, Schramm, 1958; Mundry, Gierer, 1958). Replacements of non-polar amino acids are less frequent than replacements of polar amino acids. Threonine is the most frequently replaced amino acid. Since substitutions are predominantly by leucine it is assumed that the non-polar character of threonine is predominant. 66% of all replacements are found in the tryptic peptides I, IV, and XI, indicating that these peptides or at least part of them are of less importance for the structure than the others. Based on the results on hemoglobins and myoglobin (Perutz, Kendrew, and H. C. Watson, 1965), and the amino acid replacements of TMV, one may predict helical parts of the TMV protein chain. A correlation of amino acid replacements and symptoms on host plants is evident. It is concluded that special types of replacements were selected preferentially because symptoms were used as the selection system.     

Kinetics of thermal aggregation of tobacco mosaic virus coat protein

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]₀. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]₀ (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]₀ and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A _lim; t} (t is time and A _lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.     

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.     

Fractal dimension of humic acids

Small-angle neutron scattering experiments have been made on solutions of humic acid aggregates with an acidity corresponding to pH 5.0 and at 0.1 M ionic strength. We observe power-law decay of the intensity over one decade of the scattering vector, Q, indicating that the aggregates are fractal. We explain the normalized intensity in the entire Q-range by assuming that the humic acid particles can be described by building units of a radial size, 25 Å, aggregated into clusters with an average radius of 400–500 Å. For humic acids obtained from two different sources, we determine the fractal dimension, D = 2.3 ± 0.1. For small values of Q, the measured data of one of the samples extend into the Guinier range giving an average radius of gyration of 320 ± 20 Å. Correspondence to: R. Österberg     

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.Members of the genus Crinivirus are emerging plant viruses in many parts of the world. An important factor contributing to the increase in the incidence of these viruses is their association with and transmission by whitefly vectors that have increased in distribution in the last several decades. Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus (family Closteroviridae), is specifically transmitted by the sweet potato whitefly, Bemisia tabaci biotype A, in a semipersistent, noncirculative manner (6). The virus is confined to phloem cells within infected plants and is not transmissible to plants by leaf rub inoculation. The bipartite single-stranded positive-sense LIYV genome components, consisting of RNA 1 (approximately 8.1 kb) and RNA 2 (approximately 7.2 kb), are separately encapsidated in flexuous filamentous particles that are characteristic of the family Closteroviridae (8, 11). These virions are comprised of four protein components: the major coat protein (CP), the minor coat protein (CPm), an Hsp70 homolog (Hsp70h), and a 59-kDa protein (P59). Like other viruses in the family Closteroviridae, LIYV has bipolar virions with a “body” composed mainly of the CP and a “head” that is formed by the assembly of CPm subunits (2, 4, 7, 22, 28). Hsp70h and P59 are detected in LIYV virions (22), but their locations have not been identified, as they are not readily detected by immunogold labeling and transmission electron microscopy (IGL-TEM). For two members of the family Closteroviridae, Citrus tristeza virus (CTV) and Beet yellows virus (BYV), the combination of Hsp70h, P61 (the homolog of LIYV P59 in CTV) or P64 (the homolog of LIYV P59 in BYV), and CPm encapsidates the 5′ end (∼630 to 650 nucleotides [nt]) of the RNA genome, demonstrating the complex interactions that exist among the capsid proteins and the genomic RNA (15, 21).In our previous studies, we demonstrated the transmission of LIYV using an in vitro acquisition and whitefly transmission system (13, 22). Results from previous work implicated a role for LIYV CPm in whitefly transmission. Antibodies to CPm blocked the in vitro acquisition/transmission of LIYV virion preparations by B. tabaci biotype A, while antibodies to CP, Hsp70h, and P59 did not (22). The in vitro whitefly membrane-feeding system had also been used to demonstrate B. tabaci biotype A transmission of virions that were derived from cloned infectious cDNAs of LIYV RNA 1 and RNA 2 of several genotypes, including pR6 (the first cloned wild-type [WT] infectious cDNA of LIYV RNA 2 [10]), establishing for the first time that these cloned constructs contained all of the information necessary for protoplast infection, virion formation, whitefly transmission, and infection in plants (12). In that study, the mutant p1-5b was among the cloned LIYV RNA 2 cDNAs derived from B. tabaci biotype A-transmitted virus maintained in Chenopodium murale plants.p1-5b contains a single-adenine-residue deletion in the CPm open reading frame (ORF) at nucleotide 592, a deletion that is predicted to result in a frameshift, 14 new amino acids, and premature termination of the protein (12). The predicted p1-5b CPm has 211 amino acids, compared to 453 amino acids in the wild-type (pR6 genotype) protein. The p1-5b genotype also contains three other nucleotide changes in the CPm ORF relative to the pR6 infectious clone sequence (27), all of which result in amino acid changes. In contrast, the p1-5b CP, Hsp70h, and P59 sequences are identical to that of pR6 (12). Possible polymorphisms throughout the rest of the p1-5b clone were not characterized. In a prior study, B. tabaci biotype A transmission of p1-5b virions was not observed, even though the mutation did not affect its infectivity in protoplasts (as determined by virion yields) and apparent particle morphology (12). However, those studies were disadvantaged by the necessity of propagation in protoplasts to obtain specific genotypes from infectious cloned cDNAs. Protoplasts yield low quantities of virion relative to plants, and virion concentration is a critical parameter in whitefly transmission (13). Although virion concentrations in those experiments were above typical thresholds for whitefly transmission (12, 13), low concentrations may still be limiting for transmission, making negative transmission results difficult to interpret. Obtaining adequate virion concentrations of specific genotypes for whitefly transmission to plants has therefore been a significant hurdle to LIYV transmission studies.The recently developed agroinoculation method for LIYV (24) permits the study of systemic plant infection by distinct LIYV genotypes, including those that are whitefly transmission deficient, and the recovery of higher virion yields than were possible using protoplasts. The objective of this study was to further examine the function of the LIVY CPm by extending our observations of p1-5b. We constructed mutants with the CPm frameshift restored to determine if engineered mutations that either restored or disrupted the formation of an intact CPm also affected systemic plant infection, virion formation, and B. tabaci biotype A transmission. Our study revealed that a mutant engineered with the restored CPm ORF produced a WT infection profile characterized by systemic virus movement within agroinoculated plants and the generation of CPm-containing virions that were whitefly transmissible. Intriguingly, systemic virus movement was also observed for a mutant engineered to express the 1-5b CPm, but the virions lacked an identifiable CPm and were defective in whitefly transmission. These results represent a significant advance in addressing challenging questions and hypotheses about Crinivirus whitefly transmission properties not testable using earlier systems.     

HSP70 and Its Cochaperone CPIP Promote Potyvirus Infection in Nicotiana benthamiana by Regulating Viral Coat Protein Functions

This study demonstrates that heat shock protein 70 (HSP70) together with its cochaperone CPIP regulates the function of a potyviral coat protein (CP), which in turn can interfere with viral gene expression. HSP70 was copurified as a component of a membrane-associated viral ribonucleoprotein complex from Potato virus A–infected plants. Downregulation of HSP70 caused a CP-mediated defect associated with replication. When PVA CP was expressed in trans, it interfered with viral gene expression and replication-associated translation (RAT). However, CP produced in cis interfered specifically with RAT. CPIP binds to potyviral CP, and overexpression of CPIP was sufficient to restore RAT inhibited by expression of CP in trans. Restoration of RAT was dependent on the ability of CPIP to interact with HSP70 since expression of a J-domain mutant, CPIP^Δ66, had only a minor effect on RAT. CPIP-mediated delivery of CP to HSP70 promoted CP degradation by increasing its ubiquitination when assayed in the absence of virus infection. In conclusion, CPIP and HSP70 are crucial components of a distinct translation activity that is associated with potyvirus replication.