Detailed analysis of the requirements of hepatitis A virus internal ribosome entry segment for the eukaryotic initiation factor complex eIF4F.

The hepatitis A virus (HAV) internal ribosome entry segment (IRES) is unique among the picornavirus IRESs in that it is inactive in the presence of either the entero- and rhinovirus 2A or aphthovirus Lb proteinases. Since these proteinases both cleave eukaryotic initiation factor 4G (eIF4G) and HAV IRES activity could be rescued in vitro by addition of eIF4F to proteinase-treated extracts, it was concluded that the HAV IRES requires eIF4F containing intact eIF4G. Here, we show that the inability of the HAV IRES to function with cleaved eIF4G cannot be attributed to inefficient binding of the cleaved form of eIF4G by the HAV IRES. Indeed, the binding of both intact eIF4F and the C-terminal cleavage product of eIF4G to the HAV IRES was virtually indistinguishable from their binding to the encephalomyocarditis virus IRES, as assessed by UV cross-linking and filter retention assays. Rather, we show that HAV IRES activity requires, either directly or indirectly, components of the eIF4F complex which interact with the N-terminal fragment of eIF4G. Effectively, HAV IRES activity, but not that of the human rhinovirus IRES, was sensitive to the rotavirus nonstructural protein NSP3 [which displaces poly(A)-binding protein from the eIF4F complex], to recombinant eIF4E-binding protein (which prevents the association of the cap binding protein eIF4E with eIF4G), and to cap analogue.

Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation.

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5' m(7)GpppN cap and the 3' poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3' end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3'-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.

Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to synergistically stimulate translation initiation in vivo. We recently described mammalian cytoplasmic extracts which, following ultracentrifugation to partially deplete them of ribosomes and associated initiation factors, reproduce cap-poly(A) synergy in vitro. Using these systems, we demonstrate that synergy requires interaction between the poly(A)-binding protein (PABP) and the eukaryotic initiation factor (eIF) 4F holoenzyme complex, which recognises the 5' cap. Here we further characterise the requirements and constraints of cap-poly(A) synergy in reticulocyte lysates by evaluating the effects of different parameters on synergy. The extent of extract depletion and the amounts of different initiation factors in depleted extracts were examined, as well as the effects of varying the concentrations of KCl, MgCl(2) and programming mRNA and of adding a cap analogue. The results presented demonstrate that maximal cap-poly(A) synergy requires: (i) limiting concentrations of ribosome-associated initiation factors; (ii) precise ratios of mRNA to translation machinery (low concentrations of ribosome-associated initiation factors and low, non-saturating mRNA concentrations); (iii) physiological concentrations of added KCl and MgCl(2). Additionally, we show that the eIF4G-PABP interaction on mRNAs which are capped and polyadenylated significantly increases the affinity of eIF4E for the 5' cap.

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.

Intact eukaryotic initiation factor 4G is required for hepatitis A virus internal initiation of translation.

The requirements for optimal activity of the hepatitis A virus (HAV) internal ribosome entry segment (IRES) differ substantially from those of other picornavirus IRESes. One such difference is that, to date, the HAV IRES is the only one whose efficiency is severely inhibited in the presence of the picornaviral 2A proteinase. Here we describe experiments designed to dissect the mechanism of proteinase-mediated inhibition of HAV translation. Using dicistronic mRNAs translated in vitro, we show that the HAV IRES is inhibited by the foot-and-mouth disease virus Lb proteinase, as well as by the human rhinovirus 2A proteinase. Furthermore, using mutant Lb proteinase, we demonstrate that proteolytic activity is required for inhibition of HAV IRES activity. Translation inhibition correlated closely with the extent of cleavage of the one identified common cellular target for the 2A and Lb proteinases, eukaryotic initiation factor (eIF) 4G, a component of the eIF4F cap-binding protein complex. Total rescue of HAV IRES activity was possible if purified eIF4F was added to translation extracts. In contrast, if the added eIF4F contained cleaved eIF4G, no rescue of HAV IRES activity was evidenced. Thus the HAV IRES requires intact eIF4G for activity. This is unique among the picornavirus IRESes studied to date and may help explain why HAV does not inhibit host cell translation during viral infection.

Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins.

We recently compared the efficiency of six picornaviral internal ribosome entry segments (IRESes) and the hepatitis C virus (HCV) IRES for their ability to drive internal initiation of translationin vitro. Here we present the results of a similar comparison performed in six different cultured cell lines infected with a recombinant vaccinia virus expressing the T7 polymerase and transfected with dicistronic plasmids. The IRESes could be divided into three groups: (i) the cardiovirus and aphthovirus IRESes (and the HCV element) direct internal initiation efficiently in all cell lines tested; (ii) the enterovirus and rhinovirus IRESes are at least equally efficient in several cell lines, but are extremely inefficient in certain cell types; and (iii) the hepatitis A virus IRES is incapable of directing efficient internal initiation in any of the cell lines used (including human hepatocytes). These are the same three groups found when IRESes were classified according to their activitiesin vitro, or according to sequence homologies. In a mouse neuronal cell line, the poliovirus and other type I IRESes were not functional in an artificial bicistronic context. However, infectious poliovirions were produced efficiently after transfection of these cells with a genomic length RNA. Furthermore, activity of the type I IRESes was dramatically increased upon co-expression of the poliovirus 2A proteinase, demonstrating that while IRES efficiency may vary considerably from one cell type to another, at least in some cases viral proteins are capable of overcoming cell-specific translational defects.

elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs.

Rhinovirus 2A and foot-and-mouth disease virus Lb proteinases stimulate the translation of uncapped messages and those carrying the rhinovirus and enterovirus Internal Ribosome Entry Segments (IRESes) by a mechanism involving the cleavage of host cell proteins. Here, we investigate this mechanism using an artificial dicistronic RNA containing the human rhinovirus IRES as intercistronic spacer. Because both proteinases cleave eukaryotic initiation factor 4G (eIF4G), we examined whether the cleavage products of eIF4G could stimulate uncapped or IRES-driven translation. Addition of intact eIF4F to translation extracts inhibited IRES-driven translation and reduced the translation stimulation observed in reactions pre-treated with Lb proteinase. Prolonged incubation of translation extracts with Lb proteinase removed all endogenous eIF4G and a substantial amount of the primary C- and N-terminal cleavage products. The translation of all mRNAs was reduced in such extracts. Capped mRNA translation was rescued by the addition of intact eIF4F. In contrast, addition of pre-cleaved eIF4F stimulated translation of uncapped or IRES-bearing messages to the levels seen upon proteinase addition. Furthermore, fractions containing the C-terminal, but not N-terminal, cleavage product of eIF4G stimulated translation moderately. These results demonstrate that the Lb and 2A proteinases stimulate translation of uncapped RNAs and those carrying IRESes by the production of cleavage products of eIF4G that enhance translation and by the removal of intact eIF4G that interferes with this stimulation.

Effects of P2 cleavage site mutations on poliovirus polyprotein processing.

The poliovirus genome comprises a single open reading frame which is translated to give one large polyprotein. The proteolytic cascade involved in the processing of this polyprotein is not yet understood in full detail,particularly concerning the processing of P2-P3, the precursor to the viral nonstructural polypeptides, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. To investigate the possibility that the cleavage events within P2 and at the 2C/3A junction occur in an ordered fashion, we used oligonucleotide-directed mutagenesis of poliovirus cDNA to modify the 3C(prn)-mediated cleavage sites. The Gin residue of the Gin-Gly sequence at the 2A/2B, 2B/2C, and 2C/3A junctions in the poliovirus polyprotein was replaced by Asn, Glu, Asp, or Lys. The effects of each of these substitutions were studied in vivo after transfection onto HeLa cells and in vitro in a cell-free translation assay, using full-length mutated RNA transcripts. Only the mutant with the Glu-Gly sequence at the 2C/3A junction was viable. Analysis of the in vitro processing profiles showed that the efficiency of the 3C protease cleavage at any of the sites in P2 was in the following order: Gin-Gly > Glu-Gly > Asn-Gly. No cleavage could be detected with the Asp-Gly or Lys-Gly sequence at any junction. Lack of 2A/2B or 2B/2C cleavage had no consequences on the cleavage efficiency at other Gin-Gly sites in the polyprotein. Abolition of cleavage at the 2C/3A junction did not prevent the generation of the 2A, 2B, and 3CD polypeptides. Thus, these polypeptides. Thus, these polypeptides could be produced independently of the generation of the P2 and P3 precursors.

Natural isolates of ECHO virus type 25 with extensive variations in IRES sequences and different translational efficiencies.

The complete nucleotide sequence of the 5'-untranslated region (5'-UTR) of the genome of three ECHO virus type 25 strains (the JV4 reference strain and two wild isolates) was determined. The 5'-UTR of the two isolates shared 85 and 82% nucleotide identity with the reference strain. The overall folding of the predicted secondary structure of the ECHO virus 5'-UTRs showed significant conservation with that of the poliovirus. Most significant differences were observed in specific domains of the predicted IRES of the wild isolates. The efficiency with which ECHO virus type 25 5'-UTRs promoted internal initiation of translation was examined in an in vitro translation system. At low doses of input RNA, all three 5'-UTRs were similarly efficient in promoting internal ribosome entry and behaved similarly to the poliovirus IRES in that downstream cistron translation was markedly stimulated by the addition of HeLa cell extracts to standard reticulocyte lysates. At high RNA doses, the JV4 5'-UTR was much more efficient than the other two ECHO virus 5'-UTRs. This difference was much less marked when reticulocyte lysates were supplemented with 20% HeLa S-10 extracts, which suggests that the two less efficient 5'-UTRs were characterized by a reduced binding capacity to at least one factor present in HeLa cells. In MRC5 cell S-10 extract, internal initiation of translation was stimulated from the IRES of all three ECHO strains, the M1262 IRES being the least stimulated.

Picornavirus 2A proteinase-mediated stimulation of internal initiation of translation is dependent on enzymatic activity and the cleavage products of cellular proteins.

Poliovirus and human rhinovirus 2A proteinases are known to stimulate translation initiation on the cognate viral Internal Ribosome Entry Segments (IRESes). The molecular mechanism of this translational transactivation was investigated in vitro using dicistronic mRNAs containing picornaviral IRESes as the intercistronic spacer and purified human rhinovirus type 2 and coxsackievirus B4 2A proteinases. The stimulation achieved on the HRV2 IRES in the presence of the cognate 2A proteinase at 1 microgram/ml was twofold; the maximum stimulation at 100 micrograms/ml was fivefold. The IRESes and proteinases from rhino- and enteroviruses were interchangeable; however, stimulation of translation initiation on a cardiovirus IRES by these proteinases was minimal. Studies using an inhibitor or a mutant 2A proteinase demonstrated that translation stimulation requires 2A-mediated enzymatic conversion of some cellular component(s). The HRV2 2A proteinase also stimulated translation initiation on full-length viral RNA, suggesting that 2A proteinase-mediated stimulation of IRES-driven translation has a physiological role.

Picornavirus internal ribosome entry segments: comparison of translation efficiency and the requirements for optimal internal initiation of translation in vitro.

On the basis of primary sequence comparisons and secondary structure predictions, picornavirus internal ribosome entry segments (IRESes) have been divided into three groups (entero- and rhinoviruses; cardio- and and aphthoviruses; and hepatitis A virus). Here, we describe a detailed comparison of the ability of IRESes from each group to direct internal initiation of translation in vitro using a single dicistronic mRNA (the only variable being the IRES inserted into the dicistronic region). We studied the influence of various parameters on the capacity of six different picornaviral IRESes, and the non-picornaviral hepatitis C virus IRES, to direct internal initiation of translation: salt concentration, the addition of HeLa cell proteins to rabbit reticulocyte lysate translation reactions, the presence of foot-and-mouth disease virus Lb or human rhinovirus 2A proteinase. On the basis of the characteristics of IRES-driven translation in vitro, the picornaviral IRESes can be classified in a similar manner to when sequence homologies are considered. IRESes from each of the three groups responded differently to all of the parameters tested, indicating that while all of these elements can direct internal ribosome entry, the functional requirements for efficient IRES activity vary dramatically. In the individual optimal conditions for translation initiation, the best IRESes were those from the cardio- and aphthoviruses, followed by those from the enteroviruses, which exhibited up to 70% of the efficiency of the EMCV element in directing internal initiation of translation.

Foot-and-mouth disease virus Lb proteinase can stimulate rhinovirus and enterovirus IRES-driven translation and cleave several proteins of cellular and viral origin.

Rhinovirus and enterovirus 2A proteinases stimulate translation initiation driven from the cognate internal ribosome entry segment (IRES) (S. J. Hambidge and P. Sarnow, Proc. Natl. Acad. Sci. USA 89:10272-10276, 1992; H.-D. Liebig, E. Ziegler, R. Yan, K. Hartmuth, H. Klump, H. Kowalski, D. Blaas, W. Sommergruber, L. Frasel, B. Lamphear, R. Rhoads, E. Kuechler, and T. Skern, Biochemistry 32:7581-7588, 1993). Given the functional similarities between the foot-and-mouth disease virus (FMDV) L proteinase and these 2A proteinases (autocatalytic excision from the nascent viral polyprotein and cleavage of eIF-4 gamma), we investigated whether the FMDV L proteinase would also be able to stimulate translation initiation. We found that purified recombinant FMDV Lb proteinase could stimulate in vitro translation driven from a rhinovirus or enterovirus IRES by 5- to 10-fold. In contrast, stimulation of translation initiation on a cardiovirus IRES by this proteinase was minimal, and stimulation of translation driven from the cognate FMDV IRES could not be evidenced. Studies using an inhibitor or a mutant Lb proteinase indicated that stimulation of IRES-driven translation is mediated via proteolysis of some cellular component(s). Our studies also demonstrated that the Lb proteinase is capable of stimulating initiation of translation on an uncapped cellular message. Unexpectedly, and in contrast to the 2A proteinases, the Lb proteinase specifically cleaved the products of the two reporter genes used in this study: Xenopus laevis cyclin B2 and influenza virus NS. Therefore, we also set out to investigate the requirements for substrate recognition by the Lb proteinase. Purified recombinant Lb proteinase recognized at least one mengovirus polypeptide and specifically cleaved human cyclin A and poliovirus replicase-related polypeptides. In the latter case, the site(s) of cleavage was located within the N-terminal part of polypeptide 3D. Sequence comparisons revealed no significant primary sequence similarities between the target proteins and the two sites already known to be recognized by the FMDV L proteinase.

A highly defective HIV-1 group O provirus: evidence for the role of local sequence determinants in G-->A hypermutation during negative-strand viral DNA synthesis.

The sequence of 2350 nucleotides in the env and IN regions of a group O HIV-1 genome which is hypermutated throughout its entirety was compared to the equivalent sequence of a nonhypermutated genome from the same isolate. Almost 30% of G residues were affected by G-->A transitions. As previously reported, transitions occurred mainly at GpA and GpG dinucleotides, with a marked preference for changes of the 5'-proximal G residues in poly(G) stretches. Inspection of the sequences around the hypermutation sites revealed no bias when the mutation was at the 5' G residue of a GpG dinucleotide. In contrast, a preferred context for hypermutation at the 3' G (or at single G residues) could be defined. In addition to a preference for A residues immediately downstream of hypermutated 3' G residues, C residues were underrepresented in these positions. The observed context fits well with a model whereby G-->A mutation occurs by a combination of dislocation mutagenesis at GpA dinucleotides and direct misincorporation of dTTP at the 5' G of GpG dinucleotides. Furthermore, both runs of six G residues present in the polypurine tracts (PPTs) had escaped hypermutation, despite the fact that 95% of runs of three G residues contained at least one G-->A transition. This finding suggests that genomes with hypermutated PPT motifs had been selected against and provides direct evidence that hypermutation occurs during negative-strand DNA synthesis.

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis.

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.

Substitution mutations at the putative catalytic triad of the poliovirus 3C protease have differential effects on cleavage at different sites.

Picornavirus 3C proteases are substrate-specific cysteine proteases, proposed to be homologous to the trypsin/chymotrypsin-like serine proteases on the basis of structural predictions. Substitutions at the putative active-site residues (Glu71 and Cys147) of the poliovirus 3C protease did not completely abolish proteolytic processing in vitro. The activity of mutated 3C proteases was in the following hierarchy: Glu71-Cys147 (wild type) > Asp71-Cys147 > Glu71-Ser147 > Gln71-Cys147 > Asp71-Ser147 > Gln71-Ser147 (inactive at all sites). Such mutations had differential effects on cleavage at different sites of the poliovirus polyprotein. Cleavage within the P1 region of the polyprotein was the most defective, at the 1ABC/VP1 junction and particularly at the VP0/VP3 junction. Cleavage at the 3AB/3CD and 2B/2C junctions was less affected by the mutations, and the P2/P3 and 2A/2BC junctions were cleaved efficiently by all mutants except Gln71-Ser147. All the 3C mutants gave negative results in infectivity and replication assays after transfection, indicating that mutation of Glu71 or Cys147 virtually abolishes viral replication, irrespective of the efficiency of processing of the nonstructural part of the polyprotein.

Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein.

The amino acid sequence of the poliovirus 2C protein contains two highly conserved stretches, GSPGTGKS136 and MDD177, which correspond to the consensus 'A' and 'B' motifs (GXXXXGKS/T and DD/E, respectively) found in nucleoside triphosphate-binding proteins. To assess the functional importance of these amino acid sequences, we changed conserved and non-conserved amino acids. The replacement of the non-conserved Thr133 residue with Ser or Ala did not markedly change the virus phenotype. Similarly, replacement of the non-conserved Pro131 residue by Ala did not abolish virus viability, but changes of this residue to Thr or Asn were not tolerated. No viable mutant could be isolated after transfection of cultured cells with transcripts mutated at the conserved Lys135, Ser136 or Asp177 residues. However, true revertants were selected from Arg135 and Ser135 mutants, from Glu177 and Gly177 mutants, and from Ala136 mutants. Thr136 mutants not only gave rise to true revertants, but also to two independent isolates of a suppressor mutant, Asn140----Tyr. All the lethal mutations resulted in severe inhibition of viral RNA synthesis in vivo, although no translational deficiency was detected in a cell-free system. This is the first direct evidence for the functional significance of the nucleoside triphosphate-binding pattern in the poliovirus 2C protein.

Sarcoplasmic reticulum from rabbit and winter flounder: temperature-dependence of protein conformation and lipid motion.

A comparative study of lipids and proteins in sarcoplasmic reticulum (SR) from rabbit and flounder has been undertaken. The protein/phospholipid ratio (w/w) was 3:1 in flounder SR (FSR) and 2.2:1 in rabbit SR (RSR). Both membranes had similar contents of PC (70%) and PI (6%). PE constituted 15% in RSR and 21% in FSR. PS and sphingomyelin were minor components of both SR (less than 4%). There were differences in the unsaturated chains of the total lipid extracts, PC, PE, and PI between FSR and RSR. RSR was high in linoleate and arachidonate while FSR contained substantial amounts of eicosapentaenoate and docosahexaenoate. FTIR spectroscopy revealed that the lipids of both membranes did not undergo a phase transition between 0 and 50 degrees C. The lipids were in the liquid-crystalline state at physiological temperatures and underwent monotonic increases in conformational disorder as the temperature was raised. CD spectra indicated higher content of alpha-helical structure of proteins in RSR than in FSR. Increasing temperature caused diminution of alpha-helix content. Relatively large decreases in ellipticity were observed between 20 degrees C and 40 degrees C for FSR and 30 degrees C and 60 degrees C for RSR. Measurements of intrinsic tryptophan fluorescence as a function of temperature gave similar results for membrane proteins in both FSR and RSR. The rate of change of tryptophan fluorescence and fluorescence lifetimes was constant over the temperature ranges studied, and no abrupt shifts in fluorescence occurred in the temperature regions where ellipticity decreased rapidly.

Intestinal absorption of fish oil in rats previously adapted to diets containing fish oil or corn oil.

A study was conducted to evaluate whether the composition of previous dietary fat affects the absorption and composition of lymph obtained after a meal of fish oil. Adult male Sprague-Dawley rats were fed diets containing either corn oil or fish oil (MaxEPA) for 2 weeks. They were then given intraduodenally a bolus of an emulsion of 0.5 ml of fish oil plus 0.5 ml of 20 mM sodium taurocholate. Intestinal lymph was collected from a cannula in the main intestinal lymph trunk for various times after oil administration. Rats proportion of the test dose fo fish oil than those fed corn oil. There was an effect of previous diet on the fatty acid composition of the lymph. Rats fed fish oil had a higher percentage of eicosapentaenoic and docosahexaenoic acids in the lymph lipids than those fed corn oil while those fed corn oil had a higher percentage of linoleic acid. These results rule out decreased intestinal absorption as a mechanism for the hypotriacylglycerolemic effect of dietary fish oils. They also indicate a significant contribution of endogenous lipids to the fatty acids in lymph.

Analysis of putative active site residues of the poliovirus 3C protease.

It was recently suggested that the picornavirus 3C proteases are homologous to the chymotrypsin-like serine proteases. The two structural models proposed differ in one of the postulated active site residues, Glu/Asp71 or Asp85. We changed Glu71 of the poliovirus type 1 protease to Asp or Gln and Asp85 to Glu by oligonucleotide-directed site-specific mutagenesis of an infectious cDNA, and attempted to recover virus after transfection. Both Glu71 changes were lethal for the virus and proteolytic activity was abolished in vitro with the exception of the primary cleavage event at the P2/P3 junction. In contrast, the Asp85----Glu virus was viable. This mutant was temperature-sensitive for growth at 39 degrees and exhibited a minute plaque phenotype at permissive temperature. This defect correlated with low levels of viral-specific RNA and protein syntheses and slow virus growth. Proteolytic processing at the COOH-terminus of 3C was impaired, reducing the production of mature 3C and the viral replicase 3D. In addition, 3C-mediated cleavage events within the P2 region of the polyprotein seemed to occur rather inefficiently. 3C-specific processing within P1 and elsewhere within P3 was unaffected. We suggest that Asp85 does not form part of the active site of 3C, but could be important for the specific recognition of cleavage sites within P2.