Baculoviruses provide alternatives to chemicals for controlling insect pests and can be applied by spraying. Baculoviruses have a limited host range, but work relatively slowly. They are dissolved in the midgut of insect larvae to release infectious virions which enter gut epithelial cells and begin to replicate. Replication in other organs causes extensive tissue damage and eventually death. This process can take 4-5 days, but in the field may last for more than a week, allowing the larvae to feed for longer and thereby damaging the host plant. Baculovirus expression vectors expressing foreign genes, such as those for insect-specific toxins, hormones or enzymes, might alleviate this problem. We have now constructed a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus containing an insect-specific neurotoxin from the venom of the North African (Algerian) scorpion, Androctonus australis Hector. The neurotoxin acts by causing specific modifications to the Na+ conductance of neurons, producing a presynaptic excitatory effect leading to paralysis and death; it has no effect in mice. Expression of the neurotoxin by the virus causes a reduction in the time required to kill the host insect.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH), folate, dihydrofolate, and the inhibitors trimethoprim and methotrexate bind rapidly and reversibly to both dihydrofolate reductase isoenzymes isolated from Escherichia coli RT500. The coenzyme and substrates appear to bind to only one of the mixture of two forms of the isoenzyme present at equilibrium, while the inhibitors bind to both forms. The proportions of the two forms are different for the two isoenzymes and are pH dependent in each case. The measured association rate constants for substrates and inhibitors lie in the range (1--2) x 10(-7) M-1 s-1 at 25 degrees C but are unlikely to be diffusion controlled. The rate constant for NADPH binding is 2 x 10(6) M-1 s-1. The formation of binary complexes takes place through a multistep mechanism. A minimum of three steps is required to explain the kinetic results. An equilibrium between two or more forms of the enzyme--ligand complex governs the overall dissociation. The stability of this equilibrium is largely responsible for the tighter binding of inhibitors relative to substrate or coenzyme and also for the different binding strengths of inhibitors to the isoenzymes.

The enzyme (2-5A synthetase) which synthesizes ppp(A2'p)nA where n=2 to 4 (collectively referred to as 2-5A) is widely distributed in a variety of cells and tissues in amounts which increase response to interferon and vary with growth and hormone status. 2-5A activates a nuclease which inhibits protein synthesis. The non-phosphorylated 'core' of 2-5A ((A2'p)nA, n=2 to 4) can inhibit DNA synthesis and cell growth. Here we describe convenient and sensitive radioimmune (RI) and radiobinding (RB) assays for core and 2-5A. In combination with more satisfactory high performance liquid chromatography (HPCL) methods using reverse-phase C18 columns, these assays have been used to detect core and 2-5A in crude extracts from interferon-treated cells. The novel 2-5A synthetase products NAD2'p5' A2'p5'A and A5'p45'A2'p5'A2'p5'A (ref. 13), which can also be detected using the RB assay, were not found in significant amounts. The natural occurrence of core has not been described previously.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.

IMPROVEMENT of biological pesticides through genetic modification has enormous potential and the insect baculoviruses are particularly amenable to this approach1,2. A key aim of genetic engineering is to increase their speed of kill, primarily by the incorporation of genes which encode arthropod or bacterially derived insect-selective toxins3–11, insect hormones12,13 or enzymes14,15. We report here the first, to our knowledge, field trial of a genetically improved nuclear polyhedrosis virus of the alfalfa looper, Autogmpha californica (AcNPV) that expresses an insectselective toxin gene (AaHIT) derived from the venom of the scorpion Androclonus australisl6–18. Previous laboratory assays with the cabbage looper, Trichoplusia ni, demonstrated a 25% reduction in time to death compared to the wild-type virus, but unaltered pathogenicity6 and host range19. In the field, the modified baculovirus killed faster, resulting in reduced crop damage and it appeared to reduce the secondary cycle of infection compared to the wild-type v

HeLa cells have an unusually high level of ppp(A2′p)nA synthetase(n = 2 to ≥4) even in the absence of interferon treatment. In accord with this ppp(A2′p)nA and ppp(A2′p)nA‐mediated ribosomal RNA cleavage occur naturally in response to encephalomyocarditis virus infection in control as well as in intcrleron‐treated cells. Despite this, in the absence of interferon treatment, encephalomyocarditis virus grows well In these cells. A possible explanation for this paradox is that the ppp(A2′p)nA dependent RNase is lost or inactivated at later times post‐infection in control but not in interferon‐treated cells. It appears, therefore, to bc the prevention by interferon of the virus‐mediated inhibition of the ppp(A2′p)n‐dependent nuclease rather than the absolute level or induction of the ppp(A2′p)nA synthetase which is crucial for the activity of the ppp(A2′p)nA system in HeLa cells. These results provide evidence for a further level of control in the ppp(A2′p)nA system and show that limited ppp(A2′p)nA‐mediated ribosomal RNA cleavage alone is not sufficient to cause;in inhibition of virus growth.

High doses (100-1000 reference unitsjml) of CI or fl interferons are required to inhibit the growth of herpes simplex virus types I and I1 (HSV-I and HSV-11) in human Chang cells. In contrast, much lower doses (10-100 reference unitsjml) of interferon inhibit replication of encephalomyocarditis virus (EMCV) in these cells. In the HSV-infected cells these high doses did not prevent the virus-induced shut off of host protein synthesis. The interferons were more effective in reducing the virus yield of HSV-I than of HSV-11. At the above concentrations they inhibited HSV-I protein synthesis but had little apparent effect on that of HSV-11. Similar amounts of (2'-5')oligo(adenylate)s were synthesised in response to HSV-I, HSV-I1 and EMCV infection of Chang cells after treatment with a or fl interferons. No (i.e. < 1 nM) (2'-5')oligo(adenylate)s were found in control cells or on virus infection alone. Only low levels of ppp(A2'p),A-specific rRNA cleavage were observed in the interferon-treated HSV-infected cells. In contrast, high levels were found in response to EMCV, despite the fact that ppp(A2'p),A accumulated to similar levels with each of the three viruses in these cells. High-performance liquid chromatographic analysis of material from interferon-treated Chang cells 18 h after infection with HSV-I or HSV-11, combined with radiobinding, radioimmune and rRNA cleavage assays, confirmed the presence of ppp(A2'p),A and ppp(A2'p),A at greater than nanomolar concentration. In addition, apparently equivalent amounts of two other putative (2'-5')oligo(adenylate) derivatives which compete in the radiobinding and radioimmune assays, were present. These compounds were only weak activators of the p~p(A2'p)~A-dependent RNase and under appropriate conditions were capable of inhibiting the activation of this RNase by authentic ppp(A2'p)"A. The presence of these potentially inhibitory compounds provides a possible explanation for the relatively low levels of activation of the p~p(A2'p)~A-dependent RNase in interferon-treated, HSV-infected Chang cells.

Higher Oligomers of ppp(A2′p)n together with (A2′p)nA, (A2′p)2A3′OCH3, (A2′p)2A2′,3′CH2, (A2′p)2dA, (dA2′p)2dA, their 5′‐monophosphates and 5′‐S‐methylphorothioates have been investigated for relative stability and biological activity in mouse and human cells and mouse, human, and rabbit cell‐free systems. The oligomers from trimer to heptamer inhibited protein and DNA synthesis when introduced into intace mouse cells and activated the ppp(A2′p)n A‐dependent RNase at below nanomolar concentrations in mouse cell extracts. The 5′‐diphosphates pp(A2′p)2A and corresponding analogues were active both in cell‐free systems and on introduction into intact cells. The exception to this was the all 3′‐deoxyadenosine analogue pp(dA2′p)2 dA which failed to activate the ppp(A2′p)nA‐dependent nuclease in the mouse L and human (Daudi and HeLa) cell extracts tested. Of the active analogues the 3′‐OCH3 appeared to be the most stable in the cells and systems employed. On the other hand the non‐phosphorylated ‘core’ (A2′p)2A and its 3′‐sunstituted analogues were inactive in mouse L and Ehrlich acites tumours cell‐free systems and had to effect on intact (non‐permeabilised) 3T3 cells. The 5′‐monophoshate, p(A2′p)2A, can act as an inhibitor of ppp(A2′p)nA in mouse cell‐free systems [Torrence, Imai and Johnston (1981) Proc. Natl Acad. Sci. USA, 78, 5993‐59997]. Here in intact mouse L cells or extracts from interferon‐treated human (Daudi) cells, however, it mimicked the action of ppp(A2′p)2A, possibly through conversion to the 5′‐diphosphate or 5′‐S‐methylphosphorothioate derivatives of the 3′‐substituted analogues are both more stable to exonucleolytic cleavage and unlikely to be converted to the 5′‐diphosphated or 5′‐triphosphates. They are analogue inhibitors of ppp(A2′p)nA in mouse L cell extracts. How widely they will be effective in a variety of cell‐free systems and intact cells remains to be established. The 5′‐diphosphate pp(A2′p)2A and corresponding analogues were not equally active, nor was the CH3Sp(A2′p)2A2′,3′CH2 equally effective as an analogue inhibitor, in different cell‐free systems. This emphasises the apparent differences in the properdties of the ppp(A2′p)nA‐dependent RNases from different sources. Accordingly, in looking for a generally effective analogue inhibitor of ppp(A2′p)2A its activity in a variety of extracts should be tested and in any search for further analogues for potential clincial use human cells and extracts should be employed.