Luis Pérez

Involvement of the vacuolar H(+)-ATPase in animal virus entry.

Semliki Forest virus (SFV) enters cells by receptor-mediated endocytosis, followed by acidification of endosomes by the action of the vacuolar H(+)-ATPase. Fusion of the viral and the endosomal membrane delivers the viral genome to the cytoplasm. Direct blockade of the vacuolar H(+)-ATPase by the selective inhibitor bafilomycin A1 (BFLA1) prevented the infection of cells by SFV, if the compound was present during the first minutes of infection. Attachment and penetration of virus particles were not the targets of the antibiotic. BFLA1 and the ionophore monensin potently blocked SFV infection even at low pH, indicating that acidic pH is not sufficient for SFV to deliver its genome to the cytoplasm, but the proper functioning of the H(+)-ATPase pump is necessary. Other enveloped RNA-containing viruses, such as vesicular stomatitis virus or influenza virus were also blocked by BFLA1, whereas no effect was observed with Sendai virus, which enters into cells by direct fusion with the plasma membrane. Enveloped DNA-containing viruses, such as herpes-viruses and vaccinia virus, infected the cells even when the vacuolar H(+)-ATPase was inhibited by BFLA1; similar behaviour was observed with poliovirus and adenovirus. Animal virus particles promote the internalization of proteins and other macromolecules during entry. BFLA1 blocked co-entry of the toxin alpha-sarcin when induced by SFV, but not when induced by Sendai virus. The inhibition of the enzyme responsible for acidification of endosomes by means of the potent inhibitor BFLA1 constitutes a selective and powerful tool to analyse the low-pH dependent mechanism(s) during virus entry and will aid in understanding the mechanisms and routes of entry of animal viruses into cells.

Involvement of membrane traffic in the replication of poliovirus genomes: Effects of brefeldin A

Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449-451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2Apro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.

Lack of direct correlation between p220 cleavage and the shut-off of host translation after poliovirus infection

Poliovirus induces a drastic inhibition of host protein synthesis soon after infection of susceptible cells. The correlation between this inhibition and the cleavage of p220, a polypeptide that forms part of protein synthesis initiation factor elF-4F, has been examined in detail. Measurements of protein synthesis at half-hourly intervals after infection with poliovirus show the lack of direct correlation between p220 cleavage and the blockade of cellular translation. Moreover, the use of inhibitors of poliovirus RNA synthesis helped to dissociate those two events more clearly. Thus, in the presence of guanidine or Ro 09-0179 when little shut-off was induced by poliovirus extensive proteolytic degradation of p220 took place. When HeLa cells infected with poliovirus are placed at 28 degrees the inhibition of host protein synthesis is prevented and cellular translation continues for at least 8 hr, albeit at a reduced level compared to cells incubated at 37 degrees. At 28 degrees, cleavage of p220 is observed and about 80% of p220 is degraded after 6 hr of incubation at that temperature. Strikingly, when cells in which more than 50% of p220 is cleaved are shifted to 37 degrees, cellular translation recuperates to 100%, in spite of the fact that no detectable p220 is present. Furthermore, if poliovirus-infected cells are incubated for 2 hr at 37 degrees to permit the cleavage of p220 and then are shifted to 28 degrees in the presence of guanidine, cellular proteins are synthesized at the same level as uninfected HeLa cells incubated at 28 degrees. These results show that translation of cellular mRNAs takes place in cells containing a cleaved p220 and indicate that this cleavage is not directly responsible for the shut-off of host translation induced by poliovirus.

Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis.

The involvement of lipid biosynthesis in the replication of Semliki Forest virus (SFV) in HeLa cells has been analyzed by the use of cerulenin, an inhibitor of lipid synthesis. The presence of this agent from the beginning of infection blocks the appearance of viral proteins. However, when the antibiotic is added at later stages of infection it has no effect on protein synthesis, the cleavage of viral proteins and their acylation by palmitic acid. Cerulenin is a powerful inhibitor of viral RNA synthesis, as analyzed by [3H]uridine incorporation, incorporation of [32P]phosphate into viral replication complexes, or Northern blot analysis of viral RNAs hybridized with minus- or plus-stranded riboprobes. Finally, analysis of phospholipids made in SFV-infected cells indicates that viral infection clearly stimulates the synthesis of phosphatidyl choline and modifies the membrane formed as analyzed by sucrose gradient centrifugation. Cerulenin blocks the synthesis of phospholipids and inhibits the formation of new membranes. These results show that, when the synthesis of lipids is blocked by cerulenin, SFV RNA replication is hampered, suggesting that the synthesis of viral RNAs needs continuous lipid synthesis and membrane formation.

Amino acid ileal digestibility of some grain legume seeds in growing chickens

The apparent and true amino acid (AA) digestibility from soya-bean meal (SBM), vetch meal (VM), field pea meal (FPM) and bitter vetch meal (BVM) were determined in the lower ileum of growing chickens force-fed on semisynthetic diets (120 g crude protein and 13·1 MJ metabolizable energy per kg dry matter) based on each meal as the sole source of protein. The average apparent and true digestibility values were 0·82, 0·73, 0·76 and 0·60 and 0·90, 0·91, 0·87 and 0·74 for diets SBM, VM, FPM and BVM, respectively. Marked differences in AA digestibility among diets were found. The apparent digestibility of methionine, the most limiting essential AA in poultry diets, was significantly higher in diet SBM than in diets FPM and BVM ( P

Effect of dietary protein quality, feed restriction and short-term fasting on protein synthesis and turnover in tissues of the growing chicken

The effect of dietary protein quality and quantity on fractional rates of protein synthesis (ks) and degradation (kd) in the skeletal muscle, liver, jejunum and skin of young growing chickens was studied. Chickens were either fasted overnight or were fed at frequent intervals, using continuous feeders, with equal amounts of a diet containing soya-bean meal as the sole protein source, unsupplemented, or supplemented with either lysine or methionine. Each of the three diets was provided at 2 or 0.9 x maintenance. On the higher intake, birds on the unsupplemented diet gained weight, lysine supplementation decreased and methionine supplementation increased body-weight gain (by -23% and +22% respectively). Birds fed at 0.9 x maintenance lost weight; supplementation with methionine or lysine did not influence this weight loss. None of the dietary regimens had significant effects on protein synthesis rates in any of the tissues, thus the mechanism whereby muscle mass increased in response to methionine supplementation appeared to be a decrease in the calculated rate of protein degradation. Similarly, on the 0.9 x maintenance diet the failure of the animals to grow appeared to be due to an increase in the rate of protein degradation rather than an effect on synthesis. Conversely, muscle ks was decreased in fasted chickens previously fed on the unsupplemented diet at 2 x maintenance, and in birds which had received the 0.9 x maintenance diet fasting resulted in a similar reduction in protein synthesis in muscle; ks in the liver and jejunum was also significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure-based design and synthesis of selective inhibitors of Trypanosoma cruzi dihydrofolate reductase

This paper describes the design and synthesis of potential inhibitors of Trypanosoma cruzi dihydrofolate reductase using a structure-based approach. A model of the structure of the T. cruzi enzyme was compared with the structure of the human enzyme. The differences were used to design modifications of methotrexate to produce compounds which should be selective for the parasite enzyme. The derivatives of methotrexate were synthesised and tested against the enzyme and intact parasites.

Evaluation of the cobas 4800 CT/NG Test for detecting Chlamydia trachomatis and Neisseria gonorrhoeae DNA in urogenital swabs and urine specimens

We have evaluated 696 samples (488 swabs and 208 urine specimens) with the cobas 4800 (c4800) CT/NG Test for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in swab and urine specimens. c4800 results were compared with those obtained from COBAS AMPLICOR (CAM) CT/NG Test. Discordant results were reanalyzed with the MultiNA system and compared with clinical data. For C. trachomatis detection by both methods, we obtained 93.8%, 100%, 100%, and 99.1% for sensitivity, specificity, and positive and negative predictive values, respectively. For urine specimens analyzed in c4800, our results were 96.6%, 100%, 100%, and 99.4%, respectively. For N. gonorrhoeae detection, swab results were:88.0%, 100%, 100%, and 99.4%. For urine specimen, results obtained were 100%, 100%, 100%, and 100%. Reanalyses were all concordant between both methods. c4800 results were comparable with those obtained with the CAM system. We had an excellent correlation between swab and urine specimens analyzed by c4800.

CERULENIN, AN INHIBITOR OF LIPID SYNTHESIS, BLOCKS VESICULAR STOMATITIS VIRUS RNA REPLICATION

The replication of genomex of animal viruses in the cytoplasm of susceptible cells is usually coupled to specialized membrane structures. The inhibitor of lipid synthesis cerulenin blocks the formation of vesicular stomatitis virus polypeptides when added to cells soon after virus entry, but has much less effect on viral translation, or the acylation of the glycoprotein G, when cerulenin is added late during infection. By contrast, cerulenin powerfully blocks viral RNA synthesis or the incorporation of glycerol into lipids when present at any time after VSV‐infection. These findings suggest that the systhesis of VSV RNA is dependent on continuous synthesis of lipids.

Δ24 (25)-sterol methenyltransferase: Intracellular localization and azasterol sensitivity in Leishmania major promastigotes overexpressing the enzyme

Trypanosomatids contain predominantly ergostane-based sterols, which differ from cholesterol, the main sterol in mammalian cells, in the presence of a methyl group in the 24 position. The methylation is initiated by S-adenosyl-L-methionine:Delta(24 (25))-sterol methenyltransferase, an enzyme present in protozoa, but absent in mammals. The importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniases. Here, we report studies concerning the intracellular distribution of sterol methenyltransferase in Leishmania major promastigotes and overexpressing cells using a specific antibody raised against highly purified recombinant protein. It was found by immunofluorescence and electron microscopy studies that in L. major wild-type cells sterol methenyltransferase was primarily associated to the endoplasmic reticulum. In addition to this location, the protein was incorporated into translucent vesicles presumably of the endocytic pathway. We also found in this study that cells overproducing the enzyme do not have increased resistance to the sterol methenyltransferase inhibitor 22, 26 azasterol.