Comparison of direct and indirect estimates of apparent total tract digestibility in swine with effort to reduce variation by pooling of multiple day fecal samples.

The intent of this study was to establish a fecal sampling procedure for the indicator method (IM) to provide digestibility values similar to those obtained by the total collection (TC) method. A total of 24 pigs (52.6 ± 1.5 kg) were fed 1 of 4 diets with a 2 × 2 factorial arrangement of virginiamycin and phytase (PHY) added to a corn-soybean meal diet with no inorganic P supplement. Pigs were housed in metabolism crates for a 5-d TC period after 7 d of adaptation. Immediately after the TC, a fecal collection period followed, using the IM by including 0.25% of Cr2O3 in the feed for 10 d. Fecal collection for the IM started the day after diets containing Cr2O3 were first fed, and continued for 9 consecutive days with a single grab sample per day. Similar portions of feces from d 5 to 9 were also composited into 4 samples to evaluate multi-day pooling combinations. Highly variable means and CV among samples for apparent total tract digestibility (ATTD) were observed at d 1 and 2 using the IM. The mean ATTD for DM, GE, and nutrients appeared to be stabilized by d 5 or 6 in all dietary treatments. The TC data seemed to have lower CV than the IM data for many components. Based on the linear broken-line analysis, fecal Cr concentration plateaued at d 3.75 (P < 0.001) after the first feeding of Cr. Mean ATTD values by the IM were lower than those by the TC method for DM (P < 0.05), GE (P < 0.01), P (P < 0.01), and Ca (P < 0.001). The PHY supplementation improved ATTD of P (P < 0.001) and Ca (P < 0.001) in both collection methods, whereas the PHY effect on ATTD of DM was observed only for the IM (P < 0.05). Differences related to PHY effect on ATTD were detected from d 4 to 9 in a single grab sample for P and DM but the ATTD of DM had inconsistent P-values by day. Fecal sampling after 4 d of initial feeding of marker always allowed detection of treatment effects on ATTD of P but not on ATTD of DM. Results indicated that the IM results in lower digestibility values than the TC method and does not provide the same treatment difference as the TC digestibility for energy and nutrients that are not highly impacted by the dietary treatment. For the IM, ATTD values and fecal Cr concentration stabilize at least on d 5 after initial feeding of diets containing Cr2O3. At least 2-d pooling of feces for the IM appears to be needed to provide greater accuracy and lower variations than a single grab sample.

Intracellular effects of the Hepatitis C virus nucleoside polymerase inhibitor RO5855 (Mericitabine Parent) and Ribavirin in combination.

Mericitabine (RG7128) is the prodrug of a highly selective cytidine nucleoside analog inhibitor (RO5855) of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. This study evaluated the effects of combining RO5855 and ribavirin on HCV replication in the HCV subgenomic replicon by using two drug-drug interaction models. The effects of RO5855 and ribavirin on the intracellular metabolism of each compound, on interferon-stimulated gene (ISG) expression, and on the viability of hepatocyte-derived cells were also investigated. RO5855 and ribavirin had additive inhibitory activities against HCV subgenomic replicon replication in drug-drug interaction analyses. RO5855 did not affect the uptake or phosphorylation of ribavirin in primary human hepatocytes, human peripheral blood mononuclear cells, or genotype 1b (G1b) replicon cells. Similarly, ribavirin did not affect the concentrations of intracellular species derived from RO5855 in primary human hepatocytes or the formation of the triphosphorylated metabolites of RO5855. Ribavirin at concentrations of >40 µM significantly reduced the viability of primary hepatocytes but not of Huh7, the G1b replicon, or interferon-cured Huh7 cells. RO5855 alone or with ribavirin did not significantly alter the viability of Huh7 or G1b replicon cells, and it did not significantly affect the viability of primary hepatocytes when it was administered alone. The viability of primary hepatocytes was reduced when they were incubated with RO5855 and ribavirin, similar to the effects of ribavirin alone. RO5855 alone or with ribavirin had no effect on ISG mRNA levels in any of the cells tested. In conclusion, RO5855 did not show any unfavorable interactions with ribavirin in human hepatocytes or an HCV subgenomic replicon system.

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain.

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 degrees C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.

Endoprotease activities other than furin and PACE4 with a role in processing of HIV-I gp160 glycoproteins in CHO-K1 cells.

We addressed the question of whether furin is the endoprotease primarily responsible for processing the human immunodeficiency virus type I (HIV-I) envelope protein gp160 in mammalian cells. The furin-deficient Chinese hamster ovary (CHO)-K1 strain RPE.40 processed gp160 as efficiently as wild-type CHO-K1 cells in vivo. Although furin can process gp160 in vitro, this processing is probably not physiologically relevent, because it occurs with very low efficiency. PACE4, a furin homologue, allowed processing of gp160 when both were expressed in RPE.40 cells. Further, PACE4 participated in the activation of a calcium-independent protease activity in RPE.40 cells, which efficiently processed the gp160 precursor in vitro. This calcium-independent protease activity was not found in another furin-deficient cell strain, 7.P15, selected from the monkey kidney cell line COS-7.

The low-density-lipoprotein receptor-related protein (LRP) is processed by furin in vivo and in vitro.

The low-density-lipoprotein receptor-related protein (LRP) is a multifunctional receptor involved in the clearance of a large number of diverse ligands, including proteases, protease-inhibitor complexes and lipoproteins. The mature receptor is composed of a 515 kDa and a 85 kDa subunit generated by proteolytic cleavage from a 600 kDa precursor polypeptide in a trans-Golgi compartment. Proteolytic processing occurs C-terminal to the tetrabasic amino acid sequence RHRR, a consensus recognition site for precursor processing endoproteases or convertases. In this study we have identified furin, a subtilisin-type protease, to be necessary for efficient processing of LRP in cells. Furin-deficient RPE.40 cells exhibited an impaired processing of endogenous LRP and of a recombinant soluble form of the receptor containing the processing site. The processing defect in RPE.40 cells could be complemented by expression of furin from a transfected cDNA in cultured cells and by purified furin in vitro. The impaired maturation of LRP in RPE.40 cells did not affect its intracellular transport, and correlated with a slight but consistent reduction in the endocytosis of LRP-specific ligands. These data suggest that proteolytic processing of LRP by furin is not necessary for intracellular trafficking but might be required for normal receptor activity.

Furin activates Pseudomonas exotoxin A by specific cleavage in vivo and in vitro.

We have demonstrated that the native proenzymatic form of Pseudomonas exotoxin A can be cleaved at its specific activation site by furin in intact Chinese hamster ovary cells or in vitro by furin in isolated membrane fractions from these cells. We have compared the activity of furin in cell membrane fractions with that of purified, recombinant human furin. We have verified that RPE.40, a Pseudomonas toxin-resistant mutant cell strain, is mutant in the fur gene, and we have demonstrated that these cells are deficient in cleavage of the toxin. We have also determined that this cleavage of Pseudomonas toxin by furin takes place at the authentic activation site to release the 37-kDa active fragment.

Expression of mouse furin in a Chinese hamster cell resistant to Pseudomonas exotoxin A and viruses complements the genetic lesion.

RPE.40 is a strain of mutated CHO-K1 cells with elevated resistance to Pseudomonas exotoxin A, Sindbis virus, and Newcastle disease virus. Virus resistance is due to an inability to cleave precursor viral membrane glycoproteins and produce infectious virions. Transfection of RPE.40 cells with cDNA for mouse furin causes them to lose all resistance and become as sensitive as wild-type cells to the toxin and viruses. Transfection of RPE.40 cells with cDNA for the related yeast protease Kex2 reduces their resistance to the toxin and viruses, but does not completely eliminate it.

A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A is unable to process the precursor fusion glycoprotein of Newcastle disease virus.

RPE.40, a mutant strain of CHO-K1 cells isolated for resistance to Pseudomonas exotoxin A and cross-resistant to alphaviruses, is also highly resistant to virulent strains of Newcastle disease virus. The resistance of RPE.40 cells to Newcastle disease virus results from the failure to cleave the viral envelope precursor glycoprotein Fo to fusion glycoprotein F1 at the consensus sequence (Lys/Arg)-Arg-Gln-(Lys/Arg)-Arg.

Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3.

The fusion glycoprotein precursor of Newcastle disease virus is ubiquitously cleaved in the constitutive secretory pathway if it possesses an oligobasic cleavage motif (RRQR/KR), whereas the precursor is refractory to cleavage if the motif is monobasic (GR/KQGR). We examined the cleavage activity of the mammalian subtilisin-related proteinases furin/PACE, PC2, and PC1/PC3, which are thought to be responsible for proprotein processing in either the constitutive (furin/PACE) or the regulated (PC2 and PC1/PC3) secretory pathway, for the viral precursors with different cleavage motifs. Only furin/PACE was fully capable of cleaving the precursors with the oligobasic motif. PC2 and PC1/PC3 were incapable or only partially capable of cleaving at this motif. None of the proteinases cleaved the monobasic motif. These results suggest involvement of furin/PACE in viral protein processing in the constitutive secretory pathway.

Paramyxovirus tropism dependent on host proteases activating the viral fusion glycoprotein.

An essential step in paramyxovirus fusion (F) glycoprotein biosynthesis is the posttranslational endoproteolytic cleavage of the inactive precursor glycoprotein Fo by host cell proteases. When the Fo possesses a pair or a cluster of basic residues at the cleavage site, cleavage is catalyzed by a ubiquitous protease(s) and the infection is consequently pantropic. When the site is monobasic with a single arginine, cleavage is allowed to occur only by the enzyme(s) expressed in limited tissue types and the infection is localized there. We have isolated from chick embryo an example of the latter type of endoprotease specific for the single arginine motif and demonstrate its identity with the clotting factor Xa. The ectopic expression of the FXa appeared to be the sole determinant for the viral tropism in chick embryo. The latter type of protease specific for a paired or multiple basic cleavage motif have neither been identified nor characterized extensively. We show here that this cleavage can be induced by the yeast KEX2 protease, a unique subtilisin-like serine protease, responsible for pro factor processing at the paired basic sites.

An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo.

Host cell proteases responsible for activation of viral fusion glycoproteins are an important determinant for spread and tropism of various animal viruses. Exemplifying such proteases for the first time, we isolated an endoprotease from chick embryo, that activates para- and orthomyxovirus fusion glycoproteins by cleaving their precursor proteins at a specific, single arginine site. The protease is a calcium dependent serine protease consisting of two subunits, the 33 kd catalytic chain and the 23 kd chain possibly required for Ca2+ binding, and was found to be highly homologous, if not identical, to the blood clotting factor X(FX), a member of the prothrombin family. Its high efficiency and specificity in cleavage reactions was attributable to the properties characteristic of FX. Its role in vivo was strongly supported by cleavage inhibition in ovo highly selective for this virus group with a specific peptide inhibitor against FX.

Evaluation of the antiviral effect of synthetic oligopeptides whose sequences are derived from paramyxovirus F1 N termini.

We examined the antiviral effects of three oligopeptides, carbobenzoxy(Z)-D-Phe-Ile-Gly, Z-D-Leu-Ile-Gly and Z-D-Phe-Phe-Gly, which mimic the N-terminal regions of F1 glycoproteins of two Newcastle disease virus strains (Miyadera and D26) and Sendai virus, respectively. Only one of these peptides, Z-D-Phe-Phe-Gly, significantly and with a similar potency inhibited viruses of homologous and heterologous F1 N-terminal sequences, suggesting no strict sequence requirement for inhibition. Furthermore, the enveloped RNA viruses of several different families showed essentially the same sensitivity to the three peptides as the paramyxoviruses, while a non-enveloped RNA virus was not susceptible to any of them. In addition, the Z-D-Phe-Phe-Gly peptides was effective only when the virus particles had been pretreated before infection.

Newcastle disease virus evolution. I. Multiple lineages defined by sequence variability of the hemagglutinin-neuraminidase gene.

We compared the hemagglutinin-neuraminidase gene sequence among 13 strains of Newcastle disease virus (NDV) isolated over the last 50 years. Although overall homology was remarkably high, the sequence variability demonstrated the existence of at least three distinct lineages, which must have co-circulated for considerable periods. The sequence variability also appears to reflect some accumulation of mutations over time. Strictly correlating with the lineages, the translation products could be classified into three size classes. One class lacked the interchain disulfide bond, and another represented unusual precursor protein of biologically inactive form. The lineages correlated to some extent with virulence and place of isolation of the strains. However, antigenic variations, which were neither cumulative nor progressive, did not correlate with the lineages. These analyses showing multiple lineages were greatly facilitated by a precise calculation of synonymous substitutions, which had been largely free from selective pressures and had occurred frequently and evenly throughout the coding region.

Structural features unique to each of the three antigenic sites on the hemagglutinin-neuraminidase protein of Newcastle disease virus.

Antigenic variants of D26 strain of Newcastle disease virus (NDV) were selected with monoclonal antibodies directed to the three nonoverlapping antigenic sites on the hemagglutinin-neuraminidase (HN) protein, and their HN genes were sequenced to identify the amino acids important for the integrity of each site. Seven variants for site I, which is immunodominant and conserved among NDV strains, had a change of glutamic acid at position 347, mostly to lysine, and in a single case, to glycine. In the second group of two variants for site IV, a change of asparagine to aspartic acid was found at position 481. This resulted in elimination of the oligosaccharide attached to this asparagine residue of the parental virus. Together with the finding that the site IV was destroyed by treatment with endoglycosidase F, it was suggested that the oligosaccharide is important for maintaining the structure of site IV. The oligosaccharide appeared to contribute to exposing a nearby determinant by conferring hydrophilicity on it. A variant for site II had also a nonconservative mutation resulting in the change of glutamic acid to valine at position 495. The site I recognized by antibodies which inhibit neuraminidase activity with a small substrate neuraminlactose was located closer to the predicted sialic acid-binding site than to the other sites recognized by antibodies lacking the enzyme-inhibiting capacity. The sequence of the parental virus HN gene revealed that the HNo precursor for the HN protein is an extra-long protein whose C terminus is elongated by 45 amino acids, compared with the usual HN protein sequenced in parallel.

Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus.

The nucleotide sequence of the mRNA encoding the fusion (F0) protein of a virulent strain of Newcastle disease virus was determined. A single open reading frame in the sequence encodes a protein of 553 amino acids with a calculated molecular weight of 59058. The amino acid sequence predicted several structural features involving the fusion-inducing hydrophobic stretch (residues 117-142) and the cleavage-activation site (residues 112-116) to generate the disulfide-linked F1 and F2 subunits. The cleavage-activation site as well as a part of the fusion-inducing sequence were compared among a series of virulent and avirulent strains by the chain-termination method using a synthetic oligonucleotide primer. It was found that without exception, the cleavage-activation site of virulent strains consisted of two dibasic residues with an intervening glutamine, Arg-Arg-Gln-Arg-Arg, whereas the corresponding region of avirulent strains was made of a sequence with single basic residues scattered among uncharged residues, Gly-LysArg-Gln-GlySer-Arg. On the basis of these observations and the previous results showing a strict correlation between the pathogenicity and the cleavability of the fusion protein of NDV (Y. Nagai, H-D. Klenk, and R. Rott, Virology, 72, 494-508, 1976), we propose the importance of the dibasic residues for efficient proteolytic activation of the fusion protein and for the pantropic property of NDV. Some strains were found to have Leu-Ile-Gly as the N-terminus of F1, whereas others contained Phe-Ile-Gly, indicating that Phe-X-Gly is not always conserved at F1 N-terminus of paramyxovirus.