Non-propagative translocation of velvet tobacco mottle virus in the mirid, Cyrtopeltis nicotianae

Nymphs of the mirid, Cyrtopeltis nicotianae became infective when injected with velvet tobacco mottle virus (VTMoV). Injections of amounts between 1 and 154 ng into the haemocoele induced 2/60 to infect test plants and these two nymphs contained 50 and 63 ng of virus respectively. Injection of amounts between 15 and 2400 ng rendered 11/47 nymphs infective. This observation is characteristic of a circulative association. However, there is no evidence that the salivary glands are involved in transmission and the virus is therefore defined as translocating, rather than circulating, in the mirid vector. Mirids which acquired infectivity by feeding lost it between 5 and 9 days after completion of acquisition, and the most rapid loss of infectivity occurred within 2 days. Nine days after acquisition none contained antigen detectable by ELISA, but detectable antigen decreased less rapidly than infectivity, and at all times more mirids contained antigen than were able to transmit. Mirids containing antigen carried between 150 and 3340 ng each. Thus, although VTMoV can be transmitted by its mirid vector following introduction of virus into the body cavity by injection, VTMoV is not propagative. Nor does the presence of virus within the mirid guarantee an ability to transmit.     

Studies on the transmission of velvet tobacco mottle virus by the mirid, Cyrtopeltis nicotianae

The minimum acquisition period of velvet tobacco mottle virus (VTMoV) by its mirid vector Cyrtopeltis nicotianae was about 1 min, with an increase in the rate of transmission (i.e. proportion of test plants infected) for acquisition periods up to 1000 min. Pre-acquisition starvation periods up to 18 h did not affect the rate of transmission. After an acquisition access period of 2 days, the minimum inoculation period was between 1 and 2 h and the rate of transmission increased with increasing inoculation time; when the acquisition access period was 1 h, or if vectors were fasted for 16 h after the 2 day acquisition, the rate of transmission was significantly lower. When mirids were transferred sequentially each day to a healthy plant after a 24 h acquisition feed, they transmitted intermittently for up to 10 days. Up to 50% of mirids transmitted after a moult and this was not due to the mirids probing the shed cuticles or exudates of infective insects. Mirids transmitted after a moult, following acquisition periods of 10, 100 or 1000 min. C. nicotianae transmitted solanum nodiflorum mottle virus (SNMV), sowbane mosaic virus (SoMV) and southern bean mosaic virus (SBMV), but not subterranean clover mottle virus (SCMoV), lucerne transient streak virus (LTSV), tobacco ringspot virus (TRSV), galinsoga mosaic virus (GMV), nor nicotiana velutina mosaic virus (NVMV). Tomato bushy stunt virus (TBSV) was transmitted to 1/58 test plants.     

Translational strategy of Solanum nodiflorum mottle virus RNA: synthesis of a coat protein precursor in vitro and in vivo.

Solanum nodiflorum mottle virus RNA (Mr = 1.5 X 10(6)) was translated in vitro in a wheat embryo extract. Four major products were synthesized: 2 related proteins of molecular weight 100K (P100) and 67K (P67), a protein of molecular weight 38K (P38), and a methionine-lacking protein of molecular weight 28K (P28). P38 was synthesized by a minor RNA component (Mr approximately 0.4 X 10(6)) and comigrated with the only viral product detected in SNMV-infected N. clevelandii protoplasts. Antiserum raised against purified SNMV virions precipitated both in vitro- and in vivo-synthesized P38, suggesting that it is either a precursor to or an intact form of SNMV coat protein whose apparent molecular weight in purified virus preparations is 30K.     

Structural transitions in Cowpea chlorotic mottle virus (CCMV)

Viral capsids act as molecular containers for the encapsulation of genomic nucleic acid. These protein cages can also be used as constrained reaction vessels for packaging and entrapment of synthetic cargos. The icosahedral Cowpea chlorotic mottle virus (CCMV) is an excellent model for understanding the encapsulation and packaging of both genomic and synthetic materials. High-resolution structural information of the CCMV capsid has been invaluable for evaluating structure-function relationships in the assembled capsid but does not allow insight into the capsid dynamics. The dynamic nature of the CCMV capsid might play an important role in the biological function of the virus. The CCMV capsid undergoes a pH and metal ion dependent reversible structural transition where 60 separate pores in the capsid open or close, exposing the interior of the protein cage to the bulk medium. In addition, the highly basic N-terminal domain of the capsid, which is disordered in the crystal structure, plays a significant role in packaging the viral cargo. Interestingly, in limited proteolysis and mass spectrometry experiments the N-terminal domain is the first part of the subunit to be cleaved, confirming its dynamic nature. Based on our fundamental understanding of the capsid dynamics in CCMV, we have utilized these aspects to direct packaging of a range of synthetic materials including drugs and inorganic nanoparticles.     

Distribution of velvet tobacco mottle virus in its mirid vector and its relationship to transmissibility

Following acquisition by feeding, velvet tobacco mottle virus (VTMoV) was detected in the gut, haemolymph and faeces of the mirid vector, Cyrtopeltis nicotianae, but not in the salivary glands. Virus antigen was detected in the gut and haemolymph for up to nine days following acquisition. Infective virus was detected in the secretions and excretions of the mirids immediately after acquisition and was also detected in the faeces of nymphs after six days. Insoluble nigrosin dye was eliminated intermittently from the gut up to six days after ingestion, in a manner similar to the loss of virus infectivity. Non-infective mirids were able to inoculate plants from infectious sap deposits on the upper epidermis. An ingestion-defecation model of insect transmission in which the salivary glands are not implicated is proposed as one explanation for the persistence of transmission in this mirid-virus association.     

Propagation of black raspberry necrosis virus (BRNV) in mixed culture with solanum nodiflorum mottle virus, and the production and use of BRNV antiserum

The concentration of particles of black raspberry necrosis virus (BRNV), which is normally extremely low in herbaceous plants, increased about 1000-fold when Nicotiana clevelandii plants were inoculated with a mixture of BRNV and an unrelated virus, solanum nodiflorum mottle (SNMV). In sap from N. clevelandii infected with the mixed culture, BRNV infectivity survived dilution to 10?⁴ but not 10?⁵, and storage for 6 but not 8 days at 20 ^oC, for 6 but usually not 10 days at 4 ^oC and for more than 13 days at – 15 ^oC. When plants were inoculated with the mixed culture, BRNV induced typical symptoms in several Chenopodium species and infected several previously unreported hosts. Purified preparations of particles of the mixed culture contained only a small proportion of BRNV particles, which sedimented in sucrose density gradients as two components, one, probably non-infective, of c. 505, and the other, infective, of 120-130S. An antiserum prepared to purified particles of the mixed culture was cross-absorbed with SNMV particles and used in indirect ELISA to detect BRNV in herbaceous plants infected with the mixed culture, and also in a wide range of Rubus species, cultivars and hybrids infected naturally, by grafting or by inoculation with the aphid Amphorophora idaei. The reliability of ELISA for detecting BRNV in raspberry leaves depended on the cultivar and time of year. Some cultivars, such as Glen Clova, had low concentrations of BRNV, which was detected reliably only in late spring/early summer, whereas other cultivars, such as Lloyd George and Mailing Enterprise, had greater BRNV concentrations. In small-scale surveys in eastern Scotland, BRNV was detected by ELISA in many raspberry cvs, including some that contain major gene resistance to the vector, A. idaei; in five of nine raspberry stocks entered for the Standard grade certificate but in none of five stocks entered for the Stock Cane certificate; and in 40% of wild raspberry and 14% of wild bramble plants growing near commercial raspberry crops. The significance of these findings for the control of BRNV is discussed.     

Detection and characterization of defective interfering RNAs associated with the cocksfoot mottle sobemovirus

A new RNA of about 900 nt was found in the virions of cocksfoot mottle virus (CfMV) and in infected plants by RNA hybridization and RT-PCR. Structural features suggested that this RNA is a defective interfering RNA (diRNA). The CfMV diRNA was shown to consist of a 35-nt 5′-terminal genomic region, which formed a hairpin, and a 3′-terminal genomic region, which included the coat protein (CP) gene lacking the first 120 nt.In vitro translation of the diRNA started at the third Met codon to produce truncated CP. The CfMV diRNA was assumed totrans-activate synthesis of the CP subgenomic RNA (sgRNA).     

Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.     

Pepper veinal mottle virus associated with a streak diseaseof tomato in Nigeria

A destructive streak disease of tomato (Lycopersicon esculentum) was observed on the University of Ife farm. The disease killed many plants and greatly diminished the quantity and quality of fruit produced by the other plants. A virus that is readily transmitted by mechanical inoculation, by the green peach aphid (Myzus persicae) and by grafting was isolated consistently from diseased plants. The virus was propagated in Nicotiana occidentalis and assayed in Physalis angulata. It was infective after dilution to io^-3 but not io^-4; after iomin at 55 but not 60^oC; or after 5 days but not 7 days at 20–26^oC. Electron microscope examination of sap from infected N. occidentalis leaves revealed flexuous rods with a modal length of about 780 nm. Based on the host range and symptomatology, particle morphology and size, properties in vitro and serology, the virus is shown to be related to, and possibly indistinguishable from, pepper veinal mottle virus.     

Phytophagous insects associated with cadang-cadang infected and healthy coconut palms in South-eastern Luzon, Philippines

Abstract. 1. The most common phytophagous insects associated (as adults) with the leaves of coconut palms in the Southeastern part of Luzon have been identified as part of a study on the natural method of spread of cadangcadang disease. Sixty-three species, of which at least twenty individuals were caught alive or with sticky boards, and five colony-forming Homoptera are listed. The sixty-three species belonged to the orders of Orthoptera (two species), Coleoptera (eight species), Hemiptera (one species) and Homoptera (fifty-two species, mainly Cicadellidae and Derbidae).
2. Three beetles ( Oryctes rhinoceros, Plesispa reichei and Hemipeplus sp.) and a lace bug ( Stephanitis typicus ) were found to be significantly more abundant in areas with a high incidence of cadangcadang disease. O.rhinoceros and P.reichei were also significantly more common on old palms (which are predisposed to the disease), and more abundant on cadang-cadang infected than on neighbouring healthy palms. Hemipeplus sp. apparently feeds by scraping the cuticle layer of the youngest fronds.     

The interaction of sodium dodecyl sulfate with cucumber green mottle mosaic virus protein and tobacco mosaic virus protein

The binding of sodium dodecyl sulfate to coat protein subunits of cucumber green mottle mosaic virus and tobacco mosaic virus was studied by equilibrium dialysis. The amount of dodecyl sulfate bound to the cucumber virus protein in 0.1 m phosphate buffer (pH 7.2) was found to be 1.55 g/g, which was the same value as that obtained with the tobacco virus protein. The presence of 8 m urea markedly decreased the degree of binding of dodecyl sulfate to the proteins. The amount of binding to the cucumber virus protein was reduced to 0.56 g/g, and that to the tobacco virus protein decreased to 0.8 g/g. The net charges of both proteins were negative at neutral pH and the amount of negative charge of the cucumber virus protein, obtained from the potentiometric titration curves, was larger than that of the tobacco virus protein, either in the native state or in the denatured state. In dodecyl sulfate/polyacrylamide gel electrophoresis the cucumber virus protein migrated faster than the tobacco virus protein. On the other hand, in the presence of 8 m urea, the electrophoretic migration rate of the cucumber virus protein was equal to that of the tobacco virus protein. Sedimentation equilibrium experiments in 6 m guanidinium chloride gave molecular weights of 17,700 and 17,200 for the tobacco mosaic virus and the cucumber virus proteins, respectively. These results suggest that the effective negative charge density of the cucumber virus protein-dodecyl sulfate complex is higher than that of the tobacco virus proteindodecyl sulfate complex in 0.1% dodecyl sulfate solution. The conformation of both proteins was investigated by circular dichroism measurements. Both proteins have a slightly higher degree of α-helix content in dodecyl sulfate solution than in the native state. The addition of 8 m urea to both proteins while in this solution induced a change in conformation to one having a much smaller degree of ordered structure, although the change in the cucumber virus protein was more intense than that in the tobacco virus protein.     

Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.

Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry. This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L. We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB. We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes. PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L. PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6. These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements. Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation.     

Direct visualization of the RNA location in cucumber green mottle mosaic virus and tobacco mosaic virus protein disk

The location of RNA in cucumber green mottle mosaic virus and tobacco mosaic virus protein disks was visualized by a negative staining method as a narrow ring localized at a radius of 4 nm, which corresponds to the location of RNA obtained by X-ray diffraction studies of tobacco mosaic virus. The same ring-shaped stains were observed in the end views of helical rods prepared in acidic solutions from viral protein without RNA. Since such a ring-shaped image could not be observed in end views of natural particles and reconstituted particles composed of protein and RNA, the narrow ring was concluded to indicate the RNA location on the basis of X-ray analysis.     

Structural transitions associated with changes in the thickness and density of phospholipid bilayers

Relationship between a change of bilayer density and thickness and dissociation degree of the polar groups of phospholipid molecules was studied. It has been stated that with a decrease of ionization level a transition of bilayer from liquid to ordered state should occur. The latter is accompanied by a decrease of thickness and increase of density of the bilayer.     

Aphid-injection experiments with carrot mottle virus and its helper virus, carrot red leaf

Carrot mottle virus (CMotV) and its helper virus, carrot red leaf (CRLV), were not transmitted by aphids (Cavariella aegopodii) that had fed through membranes on, or had been injected with, sap from mixedly infected chervil plants or partially purified preparations of CMotV. However, the viruses were transmitted by recipient aphids injected with haemolymph from donor aphids that had fed on mixedly infected plants but not by a second series of recipients injected with haemolymph from the first series. Some of the first series of recipients transmitted both viruses for up to 11 days but others transmitted erratically and many lost ability to transmit after a few days. The results confirm that both viruses are circulative but provide no evidence for multiplication in the vector. Non-viruliferous aphids, or aphids that had acquired CRLV by feeding, did not transmit CMotV when they were injected with haemolymph from aphids that had fed on a source of CMotV alone, confirming that they can only transmit CMotV when they acquire it from a mixedly infected plant. When extracts from donor aphids were treated with ether before injection, recipient aphids transmitted both CRLV and CMotV, although the infectivity of CMotV grown in Nicotiana clevelandii in the absence of CRLV is destroyed by ether treatment. CMotV particles acquired by aphids from mixedly infected plants therefore differed in some way from those in singly infected plants. A plausible explanation of these results, and of the dependence of CMotV on CRLV for aphid transmission, is that doubly infected plants contain some particles that consist of CMotV nucleic acid coated with CRLV protein.