The M1 matrix protein controls the filamentous phenotype of influenza A virus.

We show that most isolates of influenza A induce filamentous changes in infected cells in contrast to A/WSN/33 and A/PR8/34 strains which have undergone extensive laboratory passage and are mouse-adapted. Using reverse genetics, we created recombinant viruses in the naturally filamentous genetic background of A/Victoria/3/75 and established that this property is regulated by the M1 protein sequence, but that the phenotype is complex and several residues are involved. The filamentous phenotype was lost when the amino acid at position 41 was switched from A to V, at the same time, this recombinant virus also became insensitive to the antibody 14C2. On the other hand, the filamentous phenotype could be fully transferred to a virus containing RNA segment 7 of the A/WSN/33 virus by a combination of three mutations in both the amino and carboxy regions of the M1 protein. This observation suggests that an interaction among these regions of M1 may occur during assembly.

Pollen-stigma interactions in Brassica.

The pollen grain coating of Brassica oleracea contains a polymorphic family of highly charged small proteins (PCP-A, pollen coat protein, class A) related to the defensin class of seed proteins. On pollination these proteins are released from the grain and in vitro data suggest that at least one member of the family (PCP-A1) interacts specifically with elements of the stigmatically-expressed S(self-incompatibility) receptor complex. A new in vivo bioassay has demonstrated the male determinant of the self incompatibility system to be contained within the pollen coating, and this determinant to be a low molecular mass protein. A combination of data from interspecific studies and molecular analysis of PCP-A proteins indicates that the primary interaction between PCP-A1 and the receptor complex may be involved in establishing compatibility, while other molecular interactions, perhaps involving other PCP-A class proteins, are responsible for regulating S-specific rejection of self grains. The evolution of the self incompatibility system on the dry sigma of Brassica is discussed in the context of these data.

Identification of pollen components regulating pollination-specific responses in the stigmatic papillae of Brassica oleracea.

When components of the mature pollen grain of Brassica oleracea are applied to the cuticularised surfaces of the stigmatic papilla, a number of changes can take place in the architecture of the subjacent cell wall. Treatment of the papillar surface with isolated pollen coating evokes a rapid and extensive expansion of the outer of the two stigmatic wall layers. This response occurs within 4 h but is restricted to those regions where no callose is formed and where the coating exhibits a characteristic increase in electron opacity. This study establishes that the coating alone is responsible for initiating the expansion of the outer wall, which is considered to be an essential step preceding penetration of the stigma surface by the pollen tube. Vesicle-like inclusions, some staining intensely, occasionally occur in regions of the wall expanded by isolated coating, sometimes fusing to form a subcuticular 'boundary layer'. These structures are not observed under compatible grains in vivo and their presence is regarded as artefaetual. However, close examination of the plasma membrane at sites beneath areas of expanded cell wall reveals membrane-bound structures resembling the vesicles being generated by the cytoplasm and moving into the apoplast. These data indicate that signals from the coating initiate the loosening of the wall matrix by stimulating an unusual form of localized secretion which appears to be an essential prerequisite for stigmatic penetration. Isolated pollen coating contaminated with fragments of the grain itself engender a very different response, including the formation of densely-staining vesicles accompanied by extensive accumulations of callose; wall expansion never occurs under these circumstances. Self-pollinations in B. oleracea are frequently accompanied by the synthesis of stigmatic callose, the presence of which has, in the past, been interpreted as forming a structural barrier to incompatible pollen tubes. However, callose elicited both by self-pollinations and coating supplemented by killed grains is independent of protein synthesis and occurs in the presence of the phosphatase inhibitor, okadaic acid. Since both these inhibitors also overcome self-incompatibility (SI) in B. oleracea, these data strongly suggest that callose synthesis is unrelated to the operation of SI, and it is proposed that callose, when formed, is elicited by molecules released from the necrotic pollen protoplast. Interestingly, isolated pollen coating of self- and cross-genotypes does not readily elicit callose. The significance of these changes in the stigmatic wall is discussed in the perspective of current views on pollination and self-incompatibility in Brassica.

Pollination in species with dry stigmas: the nature of the early stigmatic response and the pathway taken by pollen tubes.

Interaction between the pollen grain, pollen tube and the stigma surface has been studied in five species regarded as possessing dry stigma surfaces; Brassica oleracea L., Arabidopsis thaliana (L.), Heynh, Papaver rhoeas L., Cosmos bipinnatus Cav. and Helianthus annuus L. In B. oleracea and A. thaliana, stigmatic response to pollination includes events in the papillar cytoplasm and changes to the stigmatic surface beneath the grain. In particular a specialized outer element of the cell wall expands prior to pollen tube penetration. The pollen tube, which enters through a 'foot' of pollen coating, grows in a space generated between an inner and the outer element of the wall and extends to the base of the papilla where it enters the middle lamellae of the subjacent cell layer. However, in A. thaliana tubes frequently were seen to penetrate all components of the stigmatic cell wall, an event only previously recorded in immature stigmas of B. oleracea. In self pollinations of self-incompatible B. oleracea involving strong S (incompatibility) alleles no changes take place in the papillar cell wall. In P. rhoeas the stigmatic surface responds to self and cross pollination by the secretion of electron-lucent material beneath the cuticle, causing it to become detached from the outer surface of the stigmatic cell wall. The pollen tubes then penetrate the cuticle and grow towards the base of the papilla in the space thus generated. The tubes continue to grow intercellularly in the transmitting tissue which lies horizontally in rays beneath the papillae. In members of the Compositae, C. bipinnatus and H. annuus, both stigma and pollen respond to pollination by producing copious quantities of an electron-opaque matrix, which frequently causes individual papillae to adhere together. Pollen tubes, which are formed following both compatible and incompatible intraspecific pollinations, grow into this matrix and toward the base of the papillae. There, in common with the other plants studied, they grow intercellularly and enter the transmitting tissue of the style. These findings are discussed in the light of current views of the mechanisms operating during angiosperm pollination and of the significance of the stigmatic response to the functioning of self-incompatibility mechanisms.

Control of pollen hydration in Brassica requires continued protein synthesis, and glycosylation in necessary for intraspecific incompatibility.

Pollen hydration and self-incompatibility (SI) in Brassica have been studied by using a combination of in vivo video-microscopy and experiments with metabolic inhibitors. Experiments with cycloheximide confirm earlier observations that pollen hydration is regulated through protein synthesis. No protein or glycoprotein has positively been identified with this event; however, it is unlikely that the total pool of any particular glycoprotein is involved, but rather a newly synthesized or otherwise activated fraction. Micromanipulation of pollen on the stigmatic papillae suggests that access to this hydration regulation system is limited to members of the Brassicaceae: pollen grains of other species-even those possessing dry stigmas-fail to hydrate. It is proposed that an interaction between enzymes of the stigma surface and the superficial layer of the pollen grain coating creates continuity between the content of the papillar wall and the grain protoplast. Inhibition of protein synthesis also overcomes SI, and since the advent of regulated hydration and synthesis of the so-called S-gene glycoproteins coincide with the acquisition of the SI system, there is strong circumstantial evidence that the same molecular species is involved in both processes. Experiments with tunicamycin, which prevents glycosylation of glycoproteins, indicate that the glycosyl groups of the S-gene glycoprotein are required for the operation of the SI system but not for the regulation of hydration. Further experiments suggest that pollen is positively inhibited on incompatible papillae but that this inhibition is biostatic. Recovery from the effects of the SI system appears to involve the metabolism of an inhibitor by the pollen. SI in Brassica thus emerges as a sophisticated process under dynamic control in both the female and male partners. The evolutionary advantages of such a system are discussed.

Pollen-stigma interactions in Brassica. IV. Structural reorganization in the pollen grains during hydration.

With the aid of osmium tetroxide vapour, dry pollen and pollen at various stages of hydration has been fixed anhydrously for examination with the transmission electron microscope (TEM). In addition to establishing features characteristic of grains at different states of hydration, this technique has enabled the detection of a superficial layer investing both the exine and the pollen coating. This layer, some 10 nm in depth, binds both lanthanum and Alcian Blue and is shown to be the first component of the pollen grain to make contact with the stigmatic pellicle. The use of vapour fixation has also rendered it possible to chart the passage of water into the pollen grains with great accuracy, for each level of hydration displays a strikingly different cytoplasmic organization. For example, dry pollen is characterized by the presence of unusual structures at the protoplast surface and large numbers of spherical fibrillar bodies, whilst the protoplast of hydrating pollen is conspicuously stratified and contains a peripheral layer of membranous cisternae, subjacent to which is a fibrillar matrix derived from the spherical bodies found in the dry grains. Vapour-fixed, fully hydrated pollen resembles conventionally fixed grains. The pollen coating appears electron-translucent after anhydrous fixation and contains discrete, slightly rounded bodies some 50 nm in diameter. The uptake of water by grains on the stigma is accompanied by conspicuous structural changes in this layer for, after a short period in contact with the papillar surface, the spherical bodies rapidly disappear and the coat becomes electron-opaque. Close examination of this 'converted' coating reveals the presence of membranous vesicles and other structural components.

ConA induced suppressor cells in scrapie-infected mice.

Lymphocyte suspensions prepared from the spleens of mice (LACG/COM) clinically affected with scrapie (ME7) were cultured for 48 hrs in the presence of Concanavalin (ConA) in order to induce suppressor cells. These cells behaved exactly as similarly prepared cells from normal age-matched mice in suppressing the response of fresh normal splenic lymphocytes to phytohaemagglutinin (PHA).

Attempts to establish the scrapie agent in cell lines.

Attempts were made to establish a persistent infection with scrapie agent in four murine cell lines. Of the four lines tested, viz. P388D1, P3-NS1-Ag-1 (NS1), L cells and an NS1 spleen cell hybrid, only the NS1 cell line showed any evidence of agent replication. Ten per cent dimethylsulphoxide (DMSO) was included in the culture media of L cells inoculated with scrapie agent. This treatment raised the initial levels of scrapie agent associating with the L cells but did not result in a persistently infected cell line. An effect of DMSO in the inoculum was observed in mice inoculated intraperitoneally, the incubation period of the disease was considerably shortened.

Defective replication of porcine transmissible gastroenteritis virus in a continuous cell line.

During a search for established cell lines to produce large quantities of porcine transmissible gastroenteritis virus (TGEV), we observed bright immunofluorescent staining 6- 12h after infection of pig kidney derived LLC-PK1 line. Infectious virus yield was, however, 2 log10 lower than that from secondary adult pig thyroid (APT/2) cell cultures, although small plaques were visible by three days in cultures maintained under agarose, suggesting limited replication. Attempts to adapt TGEV to the LLC-PK1 cell line by 10 serial 20h passes were unsuccessful. Procedures to purify virions from infected LLC-PK1 cells produced less than 1% of the particles isolated from parallel APT/2 cultures. Examination of intracellular viral RNA in actinomycin-D treated cells revealed similar amounts of genomic RNA and the 4 major subgenomic species in both cell types, suggesting that there was no defect in viral RNA replication. In vitro translation of polyadenylated RNA from infected APT/2 and LLC-PK1 cells, followed by immune precipitation of the products, showed similar profiles of precursors to structural polypeptides, confirming the functional integrity of the viral messengers in the restrictive cell. Comparison of the viral polypeptides synthesised following infection of the two cell types showed that similar species were synthesised in both, corresponding to a group of 28-30,000 mol. wt. envelope glycopolypeptides, a 47,000 mol. wt. nucleoprotein and peplomer glycopolypeptides of about 200,000 mol. wt. The rate of viral polypeptide synthesis in LLC-PK1 cells was reproducibly higher than in APT/2, resulting in the earlier detection of bands and greater incorporation of isotope. Tunicamycin at 1 microgram/ml had a similar effect in both cells, preventing glycosylation of the 26,000 mol. wt. precursor of the envelope glycopolypeptides and synthesis of the 200,000 peplomer glycoprotein. Degradation of the nucleoprotein from 47,000 to 42,000 mol. wt. although detectable in both cells was more marked in the LLC-PK1 cultures. Phosphorylation of these proteins was readily demonstrated in both cells, although phosphorylation of host proteins and, to some extent, viral envelope proteins was considerably greater in the LLC-PK1. The significance of this finding with respect to virus maturation is being investigated.