Addition Of Exogenous Protease Research Articles

Effects of exogenous protease addition on fermentation and nutritive value of rehydrated corn and sorghum grains silages

The study objective was to evaluate the effects of the addition of exogenous protease on the fermentation and nutritive value of rehydrated corn and sorghum grain silages during various storage periods. Treatments were applied using a 2 × 6 × 3 factorial combination, with 2 types of rehydrated grains (corn and sorghum), 6 doses of the enzyme (0, 0.3, 0.6, 0.9, 1.2, and 1.5%, based on natural matter) and 3 fermentation periods (0, 60, and 90 days) in a completely randomized design, with 4 replications. The protease aspergilopepsin I, of fungal origin, produced by Aspergillus niger, was used. The lactic acid concentration increased linearly as the enzyme dose increased in corn (CG) and sorghum (SG) grain silages, at 60 and 90 days of fermentation. There was an increase in the concentrations of ammonia nitrogen and soluble protein, as well as the in situ starch digestibility in rehydrated CG and SG silages, compared to the treatment without the addition of protease. The addition of 0.3% exogenous protease at the moment of CG ensiling and 0.5% in rehydrated SG increased the proteolytic activity during fermentation, providing an increase in in situ starch digestibility in a shorter storage time.

Poultry By-Product Meal as a Potential Protein Source in Broiler Diets with Exogenous Protease Supplementation

The objective of this study was to investigate the effect of exogenous protease supplementation in diets formulated with poultry by-product meal on growth performance, small intestine magnitudes, and meat physiochemical characters in broiler chickens from 21 to 35 days post-hatch. A total of 120, one-day-old "Arbor Acres Plus" broiler chickens (male: female - 50:50) were allocated one of two dietary treatments to give six replicates and ten birds per cage. Two dietary treatments included a control diet (CON) and a diet supplemented with protease (CON+Pro). At day 35, body weight and feed intakes were measured to calculate the feed conversion ratio for the entire experiment period. Two birds from each pen were euthanized to measure the relative lengths and relative weights of three small intestine sections and meat samples were collected for physiochemical characteristic analyses at 35 days post-hatch. Exogenous protease supplementation did not influence (<i>P</i>&gt;0.05) growth performance but showed a tendency to improve FCR (<i>P</i>=0.082). Protease supplementation showed a tendency to reduce proximal small intestine length (<i>P</i>=0.091). Broilers offered dietary treatments influenced minced meat color where protease supplementation resulted in lower CIE b^* (<i>P</i>&lt;0.001) colorimetric value for yellowness and showed a significant trend (<i>P</i>=0.059) on reducing meat redness CIE a^*. In conclusion, the addition of exogenous protease to a broiler diet formulated with poultry by-product meal did not affect the growth performance, small intestine magnitudes, and meat physiochemical parameters (except CIE b^*) in broiler chickens.

Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus.

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

Contribution of individual broilers to variation in amino acid digestibility in soybean meal and the efficacy of an exogenous monocomponent protease

A total of 72 male Ross 308 broilers were reared to day 34 on a standard wheat and soy-based diet and then offered one of the four semisynthetic experimental diets, comprising two different soybean meal sources either without or with exogenous protease (treatments therefore offered in a 2*2 factorial arrangement). Each experimental diet was fed to 18 individually housed birds from 34 to 37 D after which ileal digesta were collected and digestibility coefficients were calculated. The two soybean meal sources were found to be nutritionally divergent (P < 0.01), with one having the apparent ileal amino acid digestibility coefficient of 0.80 and the other 0.71. Exogenous protease increased (P < 0.01) apparent ileal amino acid digestibility coefficients from 0.74 to 0.77. There were no interactions between soybean meal origin and protease effect. On an individual bird level, there were substantial differences in the capacity to digest amino acids with the mean total amino acid digestibility coefficients from 0.54 to 0.80 for one of the soybean meal samples. Exogenous protease addition reduced the coefficient of variation for total amino acids from 11.4 to 9.1% in one soybean meal and from 7.7 to 6.3% in the other. It can be concluded that soybean meal digestibility varies and that some of this variance is associated with heterogeneity in the digestive capacity of broilers.

A mono-component microbial protease improves performance, net energy, and digestibility of amino acids and starch, and upregulates jejunal expression of genes responsible for peptide transport in broilers fed corn/wheat-based diets supplemented with xylanase and phytase

ABSTRACTA total of 90 male Ross 308 broiler chicks were used in a digestibility and performance bioassay to explore the effect of reduction in dietary protein and digestible amino acids and inclusion of an exogenous mono-component protease on amino acid digestibility, net energy, jejunal gene expression, and bird performance. Four dietary treatments were created by the supplementation, or not, of 2 control diets with a mono-component exogenous protease. The control diets were corn/wheat/soybean meal-based and were formulated to be either nutritionally adequate or reduced in protein and amino acids (around 3%). The 2 control diets were supplemented with xylanase and phytase (2000 FYT). Treatments were therefore arranged as a 2 × 2 factorial design. The reduction in diet nutrient density had no significant effect on various experimental outcomes (including bird performance, amino acid digestibility, and net energy [NE]) that were measured with the exception of a reduction in the expression of aminopeptidase N and glucose transporter 2. However, the addition of exogenous protease resulted in an increase in weight gain and a reduction in feed conversion ratio (around 4%; P < 0.05) and an increase in the digestibility of several amino acids (P < 0.05) and starch (P = 0.06). Protease addition also resulted in an increase in both apparent metabolizable energy (AME) (+73 kcal/kg; P < 0.05) and NE (+107 kcal/kg; P < 0.05). The addition of exogenous protease to the diet also increased the jejunal expression of genes responsible for peptide transport (PepT2; P < 0.01) and starch digestion (sucrase isomaltase; P = 0.06). These results confirm the efficacy of exogenous protease in broiler diets that contain both xylanase and phytase and suggest substantial beneficial effects that extend beyond protein and amino acid nutrition. The effect of exogenous protease on energy partitioning, starch digestibility and the efficiency of nitrogen cycling is an area for further study.

Direct determination of amino acids in brewery worts produced by different processes by capillary zone electrophoresis.

The direct and simultaneous determination of cysteine, histidine, phenylalanine, lysine, tryptophan and arginine in brewery worts by capillary zone electrophoresis (CZE) was applied to evaluate the effects of temperature control and protease supplementation during mashing on the changes of these amino acids (AAs) wort composition. A cation exchange resin was used for AAs extraction from wort samples prior to CZE determination. The separation was achieved using a 50mmol/L phosphate buffer at pH12.5, containing 0.4mmol/L cetyltrimethylammonium bromide (CTAB), as background electrolyte (BGE) solution; -20kV; 20°C and hydrodynamic injection time of 15s, at 50mbar. Recovery evaluation using worts led to values between 83.1 and 96.2%, demonstrating the method feasibility, which was successfully applied in the quantification of AAs in wort samples. This study showed that temperature control and addition of exogenous proteases in the mashing may increase the AAs concentration in wort, improving the final product quality (beer). The present method is a good alternative for monitoring specific AAs in worts and their determination can allow the brewing process optimization.

Does dietary insect meal affect the fish immune system? The case of mealworm, Tenebrio molitor on European sea bass, Dicentrarchus labrax

Feeding small European sea bass, Dicentrarchus labrax, for 6 weeks with Tenebrio molitor larval meal showed significant anti-inflammatory responses (ceruloplasmin, myeloperoxidase and nitric oxide). Serum bacteriolytic activity against a Gram negative bacterium was not significantly affected by dietary Tenebrio, while both lysozyme antibacterial activity and serum trypsin inhibition usually linked to the anti-parasite activity of the fish, were significantly enhanced. The latter may be due to the similarities in the composition of the exoskeleton of parasites and insects that may therefore act as an immunostimulant potentially increasing the anti-parasitic activity. The addition of exogenous proteases significantly decreased both trypsin-inhibition and serum bacteriolytic activity probably through direct inhibition of the proteins responsible for these immune functions. Further investigation involving bacterial or parasitic challenges will be necessary to assess if the effects of dietary mealworm meal on the immune system observed in the present study are translated into an improved resistance to diseases.

The effect of a mono-component exogenous protease and graded concentrations of ascorbic acid on the performance, nutrient digestibility and intestinal architecture of broiler chickens

A total of 640 male Ross 308 broilers were used in a growth performance and digestibility study to explore the interactive effects of a mono-component exogenous protease and ascorbic acid. A total of 7 dietary treatments were generated by formulation of a nutritionally-marginal wheat/soy-based ‘negative control’ diet and supplementation of the same with three concentrations of ascorbic acid, without or with exogenous protease addition. A further ‘positive control’ diet was fed as a reference and was formulated to be nutritionally adequate. Diets were fed ad libitum from d1-35 post-hatch and water and environmental controls were as per breeder recommendations. At the termination of the experiment ileal digesta was collected for assessment of digestibility and the ileum and jejunum were excised for morphological measurements. There were no interactions (P>0.05) between ascorbic acid and exogenous protease for any of the measurements taken during the study. Overall (d1-35) addition of protease resulted in a significantly lower FCR compared with birds fed the negative control diet. There was no effect of ascorbic acid on performance of the birds with the exception of a small reduction in FCR at the highest inclusion concentration only. Ascorbic acid addition significantly increased gut integrity and exogenous protease reduced (P<0.05) the presence of sialic acid in ileal digesta. Exogenous protease significantly increased the ileal digestibility of nitrogen and all amino acids but had no effect (P>0.05) on dry matter digestibility. Ascorbic acid addition had only modest effects on ileal nutrient digestibility but this was confirmed as significant for several amino acids including threonine and cysteine. Finally, addition of ascorbic acid linearly decreased crypt depth in the jejunum and exogenous protease increased villus height, reduced epithelial thickness and goblet cell number in the jejunum. It can be concluded that ascorbic acid and exogenous protease improve intestinal integrity in growing broiler chickens and offer potential as a strategic dietary intervention to enhance performance, intestinal resilience and nutrient recovery.

Effect of ensiling time and exogenous protease addition to whole-plant corn silage of various hybrids, maturities, and chop lengths on nitrogen fractions and ruminal in vitro starch digestibility

The objective of this study was to evaluate the effects of ensiling time and exogenous protease addition on soluble CP (% of CP), ammonia-N (% of N), and ruminal in vitro starch digestibility (ivSD) of whole-plant corn silage (WPCS) from 3 hybrids, 2 maturities, and 2 chop lengths. Samples from 3 nonisogenic hybrids [brown midrib containing the bm3 gene mutation (BM3), dual-purpose (DP), or floury-leafy (LFY)] at 2 harvest maturities [2/3 kernel milk line (early) or 7d later (late)] with 2 theoretical lengths of cut settings (0.64 or 1.95cm) on a forage harvester were collected at harvest, treated with or without exogenous protease, and ensiled in triplicate in vacuum heat-sealed plastic bags for 0, 30, 60, 120, and 240d. Thus, the experiment consisted of 120 treatments (3 hybrids × 2 maturities × 2 chop lengths × 2 protease treatments × 5 time points) and 360 mini-silos (3 replications per treatment). Vitreousness, measured by dissection on unfermented kernels on the day of harvest, averaged 66.8, 65.0, and 59.0% for BM3, DP, and LFY, respectively. A protease × maturity interaction was observed with protease increasing ivSD in late but not early maturity. Ensiling time × hybrid interactions were observed for ammonia-N and soluble CP concentrations with greater values for FLY than other hybrids only after 120d of ensiling. Ensiling time × hybrid or protease × hybrid interactions were not observed for ivSD. Measurements of ivSD were greatest for FLY and lowest for BM3. Length of the ensiling period did not attenuate negative effects of kernel vitreousness or maturity on ivSD in WPCS. Results suggest that the dosage of exogenous protease addition used in the present study may reduce but not overcome the negative effects of maturity on ivSD in WPCS. No interactions between chop length and ensiling time or exogenous protease addition were observed for ivSD.

Influence of ensiling, exogenous protease addition, and bacterial inoculation on fermentation profile, nitrogen fractions, and ruminal in vitro starch digestibility in rehydrated and high-moisture corn

Exogenous protease addition may be an option to increase proteolysis of zein proteins and thus starch digestibility in rehydrated and high-moisture corn (HMC) ensiled for short periods. In addition, microbial inoculation may accelerate fermentation and increase acid production and thus increase solubilization of zein proteins. Four experiments were performed to evaluate the effect on fermentation profile, N fractions, and ruminal in vitro starch digestibility (ivSD) of the following: (1) rehydration and ensiling of dry ground corn; (2) exogenous protease addition to rehydrated un-ensiled and ensiled corn; (3) exogenous protease addition or inoculation in rehydrated ensiled corn; and (4) exogenous protease addition or inoculation in HMC. Experiments 1, 2, and 3 were performed with 7 treatments: dry ground corn (DGC); DGC rehydrated to a targeted dry matter content of 70% (REH); REH treated with exogenous protease (REH+); REH ensiled for 30 d (ENS); ENS treated with exogenous protease (ENS+); ENS treated with a microbial inoculant containing Lactobacillus plantarum, Lactobacillus casei, Enterococcus faecium, and Pediococcus sp. (ENSI); and ENS treated with exogenous protease and microbial inoculant (ENSI+). Experiment 1 compared DGC, REH, and ENS with ivSD being greater for ENS (64.9%) than DGC and REH (51.7% on average). Experiment 2 compared REH and ENS without or with exogenous protease addition (REH+ and ENS+, respectively). Ensiling and exogenous protease addition increased ivSD, but exogenous protease addition was more effective in ENS than REH (6.4 vs. 2.6 percentage unit increase). Experiment 3 compared the effects of exogenous protease addition and inoculation in ENS corn (ENS, ENS+, ENSI, and ENSI+). The addition of protease, but not inoculant, increased ivSD. Inoculation reduced pH and acetate, propionate, and ethanol concentrations, and increased lactate and total acid concentrations. In experiment 4, 8 treatments were a combination of HMC noninoculated or inoculated with 1 of 3 microbial inoculants and with or without exogenous protease addition. The inoculant treatments contained (1) Lactobacillus buchneri 40788 and Pediococcus pentosaceus, (2) L. buchneri 40788, and (3) a mixture of P. pentosaceus and Propionibacterium freudenreichii. Protease, but not inoculation, increased ivSD by 7.5 percentage units (44.4 vs. 51.9%). Protease addition increased ivSD in rehydrated corn and HMC. Microbial inoculation improved fermentation profiles but did not affect ivSD.

Proteolytic enzymes in embryonated chicken eggs sustain the replication of egg-grown low-pathogenicity avian influenza viruses in cells in the absence of exogenous proteases

Low pathogenic influenza viruses grow readily in embryonated chicken eggs but require the addition of exogenous proteases to grow in MDCK cell culture. In this study, we found that the influenza viruses propagated previously in eggs, can grow for up to two passages in cell culture without the addition of exogenous proteolytic enzymes. These results indicate that the reason for virus propagation in cells during the first two passages may be due to proteases from egg allantoic fluid carried over from egg culture. The ability of influenza viruses to grow in cells in the absence of trypsin is currently considered as a hallmark of highly pathogenic influenza viruses. Our data indicate that differentiating between high and low pathogenicity using cell culture only is not appropriate and other indicators such as sequence analysis and in vitro pathogenicity index should be performed.

Effect of exogenous protease enzymes on the fermentation and nutritive value of corn silage

The objective of this study was to evaluate the effects of adding experimental formulations of exogenous protease enzymes on the fermentation and nutritive value of whole-plant corn ensiled in laboratory silos. Chopped and processed whole-plant corn (36.8% DM) was ensiled without enzymes or treated with 1 of 2 experimental proteases (E85 or E86; AB Vista, Wiltshire, UK) at 20 or 2,000 mg/kg (wet-weight basis). Forages were packed in vacuumed and heat-sealed bags and ensiled for 45 and 150 d at 23±1°C. When compared with untreated silage, addition of proteases and length of ensiling time had no effect on silage pH or concentration of crude protein. The results were similar for the concentrations of acid detergent fiber, neutral detergent fiber, and starch, although protease × time interactions were observed for these components, which were biologically minor. When compared with untreated silages, only treatment with the 2,000-mg/kg application amount of E 0425 resulted in lower neutral detergent fiber after 45 d of ensiling. Proteases did not affect NDF digestibility after 150 d of ensiling when compared with untreated silage. Similarly, treatment with enzymes did not affect the concentrations of lactic and acetic acids or ethanol when compared with untreated silage. Concentrations of NH3-N and soluble protein (% of crude protein) increased with storage time compared with concentrations at harvest and were greater for the 2,000-mg/kg doses of proteases when compared with untreated silage at both 45 and 150 d. In vitro ruminal digestibility of starch after 7 h of incubation was 66.3% for freshly chopped corn plants. After 45 d of ensiling, starch digestion was greater for E 0430 applied at the 2,000-mg/kg dose (80.6%) than in all other treatments, with the exception that it was similar to the 2,000-mg/kg dose of E85. After 150 d of ensiling, the 20-mg/kg dose of E 0425 (81.9%), the 2,000-mg/kg dose of E 0425 (82.9%), and the 2,000-mg/kg dose of E 0430 (88.6%) had greater starch digestibility than untreated silage (74.0%). To the best of our knowledge, this is the first study to show that addition of exogenous proteases added to corn forage at the time of harvest can increase in vitro ruminal starch digestibility during silage fermentation. Data suggests that adding exogenous sources of protease enzymes at ensiling may be a method to obtain a high degree of ruminal starch digestibility in corn silage that would normally require longer periods of time to obtain from prolonged storage.

Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag

We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.

Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

See page 1406Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

Changes in pork and shark (Rhizopriondon terraenovae) protein emulsions due to exogenous and endogenous proteolytic activity

Protein unfolding is the main emulsifying mechanism in meat products. However, myofibrillar protein depletion resulting from endogenous or exogenous protease activity affects the emulsifying properties. The objective of this work was to study the effect of endogenous and exogenous proteases on protein depletion, emulsifying and viscosity properties in a model meat emulsion. Pork and shark meat were treated with a Pseudomonas fluorescens protease extract; endogenous proteolytic activity was studied in non-treated meat; samples were stored at 4 and 15 °C up to 15 days and emulsions formulated with extracted myofibrillar proteins. Endogenous proteolytic activity of shark meat extracts was significantly higher as compared to pork, promoting more extended protein degradation. This resulted in lower viscosity of protein extracts and emulsions. However, viscosity increased in both species throughout the study time, probably due to protein unfolding and molecular interaction. Emulsion capacity and stability increased in pork but decreased in shark, due to exogenous protease addition.

Enzimas digestivas exógenas na alimentação de juvenis de tambaqui

O objetivo deste trabalho foi avaliar o efeito da adição na ração das enzimas digestivas exógenas amilase, lipase e protease sobre o desempenho de tambaqui, em três experimentos independentes. Em cada experimento foi avaliado o efeito da inclusão de uma enzima na ração em diferentes porcentagens: 0,0, 0,05, 0,1 e 0,2%, cada uma com quatro repetições, com 28 dias de duração. Foram realizadas biometrias do peso inicial, para homogeneização da amostra, e do peso final a fim de verificar o efeito dos tratamentos sobre o desempenho dos peixes. Em cada unidade experimental foram estocados dez peixes, alimentados duas vezes ao dia (8 e 14h) até a saciedade aparente, com ração comercial extrusada com 28% de proteína bruta, triturada, adicionada de enzima e depois peletizada. Ao final do experimento, o ganho de peso, a conversão alimentar aparente e o crescimento específico dos peixes, apresentaram diferenças significativas entre os tratamentos. As enzimas exógenas amilase a 0,05% e lipase a 0,2% na ração influenciaram o desempenho zootécnico de juvenis de tambaqui. A adição de protease exógena não apresenta a mesma influência.

Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F(0), which is proteolytically processed to the mature form, F(1) + F(2). Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca(2+), and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXPhi. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.

Addition of exogenous protease facilitates reovirus infection in many restrictive cells.

Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.

Determinants of pantropism of the F1-R mutant of Sendai virus: specific mutations involved are in the F and M genes.

Mutations in the fusion, F, protein of Sendai virus resulting in increased cleavability by ubiquitous host protease(s), and mutations in the matrix, M, protein resulting in bipolar budding, are both important determinants for the systemic infection in mice caused by the protease activating pantropic mutant, F1-R. Several mutants of Sendai virus (BY, BF, and KD-M) with phenotypes of bipolar budding and/or increased cleavability of F protein were isolated. Genomic RNA sequence analysis of the F and M genes of the mutants revealed that several deduced amino acids in the F and M proteins were different from those of F1-R, T-5 (a revertant of F1-R), and wild-type viruses. The BF and KD-M mutants that budded bipolarly and were also activated by ubiquitous proteases were examined for replication in tissue culture cells and in mice. All of the mutants exhibited multiple-step replication in MDCK, MDBK, and LLC-MK2 cells without trypsin, but formed plaques only in MDCK cells. One of the mutants, designated KD-52M, was similar to F1-R in that it formed plaques in all three cell lines without addition of exogenous protease. However, none of the mutants viruses, including KD-52M, caused a systemic infection in mice. The mutated M protein of F1-R enhances the disruption of microtubles. However, none of the mutants with a bipolar budding phenotype (BY, BF, and KD-M), disrupted the microtubules to the same extent as F1-R. All of these mutants had mutations in the M protein that were different from those found in F1-R. Taken together, these results suggest that mutations at Ser115 to Pro in the F protein and at Asp 128 to Gly and Ile210 to Thr in the M protein of F1-R are the mutations specifically required for the systemic infection caused by F1-R.

Membrane resealing in cultured rat septal neurons after neurite transection: evidence for enhancement by Ca(2+)-triggered protease activity and cytoskeletal disassembly

Neurites of cultured septal neurons were transected with a laser under sterile conditions, and the subsequent membrane resealing was assayed using a dye exclusion method. In agreement with findings in other preparations, Ca2+ enhanced resealing: in normal culture medium the percentage of lesioned neurons that resealed within 20-30 min after transection increased with increasing bath [Ca2+] over the range 10(-7) to 2 x 10(-3) M; about 75% of cells resealed in 2 mM Ca2+. Mn2+ and Sr2+ also enhanced resealing, but Mg2+ inhibited it. The percentage of resealing neurons was sensitive to agents known to alter the stability of cytoskeletal components. Agents that tend to disassemble microtubules and/or neurofilaments (e.g., colchicine, low-ionic-strength media) strongly promoted resealing, whereas treatments that tend to stabilize microtubules (taxol, Mg2+) inhibited resealing. Addition of exogenous proteases (papain, trypsin, or dispase) enhanced resealing, whereas inhibitors of cysteine proteases (including a specific inhibitor of calpain, a Ca-activated neutral protease) strongly inhibited resealing. Calmodulin inhibitors inhibited resealing, consistent with reports that calmodulin facilitates calpain-mediated proteolysis of fodrin, a component of the cortical cytoskeleton. Based on these results, we hypothesize that one of the major mechanisms involved in resealing is activation of endogenous proteases by Ca2+ entry into the injured neurite. The resulting changes in the cellular cytoskeleton might promote fusion and resealing of the cut ends of the plasma membrane by enhancing membrane mobility and/or by removing structures that normally prevent membrane-membrane contact.