Functional significance of the interaction of hepatitis A virus RNA with glyceraldehyde 3-phosphate dehydrogenase (GAPDH): opposing effects of GAPDH and polypyrimidine tract binding protein on internal ribosome entry site function.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a cellular enzyme involved in glycolysis, binds specifically to several viral RNAs, but the functional significance of this interaction is uncertain. Both GAPDH and polypyrimidine tract binding protein (PTB) bind to overlapping sites in stem-loop IIIa of the internal ribosome entry site (IRES) of Hepatitis A virus (HAV), a picornavirus. Since the binding of GAPDH destabilizes the RNA secondary structure, we reasoned that GAPDH may suppress the ability of the IRES to direct cap-independent translation, making its effects antagonistic to the translation-enhancing activity of PTB (D. E. Schultz, C. C. Hardin, and S. M. Lemon, J. Biol. Chem. 271:14134-14142, 1996). To test this hypothesis, we constructed plasmids containing a dicistronic transcriptional unit in which the HAV IRES was placed between an upstream GAPDH-coding sequence and a downstream Renilla luciferase (RLuc) sequence. Transfection with this plasmid results in overexpression of GAPDH and in RLuc production as a measure of IRES activity. RLuc activity was compared with that from a control, null-expression plasmid that was identical except for a frameshift mutation within the 5' GAPDH coding sequence. In transfection experiments, GAPDH overexpression significantly suppressed HAV IRES activity in BSC-1 and FRhK-4 cells but not in Huh-7 cells, which have a significantly greater cytoplasmic abundance of PTB. GAPDH suppression of HAV translation was greater with the wild-type HAV IRES than with the IRES from a cell culture-adapted virus (HM175/P16) that has reproducibly higher basal translational activity in BSC-1 cells. Stem-loop IIIa RNA from the latter IRES had significantly lower affinity for GAPDH in filter binding experiments. Thus, the binding of GAPDH to the IRES of HAV suppresses cap-independent viral translation in vivo in African green monkey kidney cells. The enhanced replication capacity of cell culture-adapted HAV in such cells may be due in part to reduced affinity of the viral IRES for GAPDH.

Hepatitis A virus translation is rate-limiting for virus replication in MRC-5 cells.

Translation of hepatitis A virus (HAV) RNA is controlled by an internal ribosome entry site (IRES) located within the 5' untranslated region (UTR). In some cell types, the characteristically slow growth of HAV may be due to inefficient viral translation. We investigated whether this is true in MRC-5 cells, which are used for vaccine production. We measured the impact of two clusters of mutations in the 5' UTR on virus translation and replication: the AG group was selected during passage in African green monkey kidney cells, and the MR group was selected during subsequent passage in MRC-5 cells. The efficiency of cap-independent translation was assessed by inserting cDNA encoding an HAV IRES upstream of the chloramphenicol acetyl transferase gene and transcription was driven in vivo by a hybrid T7/vaccinia virus system. A luciferase gene was inserted upstream of the IRES to serve as an internal control. Each HAV UTR was also inserted into an infectious cDNA clone; the average rate of viral RNA accumulation was determined for each mutant virus. In MRC-5 cells, the rate of virus replication was highly correlated with the efficiency of cap-independent translation (P = 0.006). The MR but not the AG mutations significantly increased both translation and viral RNA accumulation. Reversion of just one MR mutation (687 G to A) eliminated all of the replication-stimulating and translation-enhancing effects of the MR mutations. In the control BS-C-1 cells, there was no discernible correlation between the rate of virus replication and the efficiency of cap-independent translation (P = 0.136): the AG and MR groups combined had a small impact on translation, but no detectable impact on virus replication. We conclude that in MRC-5 cells viral translation is rate-limiting for HAV replication.

Performance of the Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay in detecting Chlamydia trachomatis in endocervical and urine specimens from women and urethral and urine specimens from men attending sexually transmitted disease and family planning clinics.

The Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay (AMP CT) uses transcription-mediated amplification and hybridization protection assay procedures to qualitatively detect Chlamydia trachomatis rRNA in urine, endocervical swab, and urethral specimens. The performance of the AMP CT was compared to that of cell culture for endocervical swab and urine specimens from women and urethral and urine specimens from men. Analysis of specimens with discrepant results was performed by a combination of reculture, direct fluorescent-antibody (DFA) staining of specimen sediment, and amplification which targeted a different chlamydial rRNA. A total of 800 urine samples were tested by the AMP CT (607 from women and 193 from men), and 7. 1% were positive for C. trachomatis, with a sensitivity of 91.2% and a specificity of 99.6% upon discrepant analysis. A total of 926 swab specimens were tested by culture and AMP CT (717 endocervical swab specimens and 209 urethral swab specimens from men), and 7.7% were positive for C. trachomatis, with a sensitivity and specificity of 100% upon discrepant analysis. The AMP CT is a sensitive and specific nucleic acid hybridization assay for the detection of C. trachomatis in endocervical swab specimens from women, urethral swab specimens from men, and urine specimens from men and women.

Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization.

GB viruses A and C (GBV-A and GBV-C) are two recently described RNA viruses which appear to be members of the Flaviviridae. Although both viruses appear to contain long 5' nontranslated regions, the sites of polyprotein initiation and the presence of core-like proteins remain to be determined. Translation studies were undertaken to determine the mechanism and sites of polyprotein initiation in GBV-A and GBV-C. Rabbit reticulocyte lysates programmed with monocistronic RNAs containing 5' ends of GBV-A or GBV-C fused in-frame with the chloramphenicol acetyltransferase (CAT) open reading frame generated GBV-CAT fusion proteins in vitro. Site-specific mutagenesis and N-terminal sequencing located the sites of translation initiation immediately upstream of the putative signal sequence for the GBV E1 envelope glycoproteins. Efficient translation of the monocistronic GBV-CAT RNAs required the inclusion of GBV coding sequences. This, coupled with the presence of at least 523 nucleotides of 5' nontranslated RNA containing multiple AUG codons, suggests that translation initiation of these RNAs did not utilize a ribosome scanning mechanism. Translation of bicistronic RNAs containing 5' nontranslated sequences within the intercistronic space was consistent with the presence of a weakly active internal ribosome entry site in both GBV-A and GBV-C. Secondary structure predictions indicate that the 5' ends of these viruses assume similar complex structures distinct from those identified in the internal ribosome entry site-containing picornaviruses, pestiviruses, and hepatitis C viruses. The data indicate that GBV-A and GBV-C are unique members of the Flaviviridae that do not contain core-like proteins at the N termini of their putative polyproteins.

Specific interaction of glyceraldehyde 3-phosphate dehydrogenase with the 5'-nontranslated RNA of hepatitis A virus.

Initiation of translation of hepatitis A virus (HAV) RNA occurs by internal entry and is likely to involve the interaction of trans-acting cellular protein factors with cis-acting structural elements of an internal ribosomal entry segment (IRES) within the 5'-nontranslated RNA. To characterize interactions between African green monkey kidney (BS-C-1) cell proteins and the predicted stem-loop IIIa (nucleotides 155-235) located at the 5' border of the HAV IRES, we utilized an electrophoresis mobility shift assay (EMSA) to identify a 39-kDa RNA-binding protein (p39). Amino-terminal amino acid sequencing of highly purified p39 revealed absolute identity with human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The identity of p39 as simian GAPDH was further confirmed by antigenic and biochemical similarities between p39 and human GAPDH. Analysis of the RNA binding properties of simian GAPDH revealed that this cellular protein interacts with two additional sites in the HAV 5'-nontranslated RNA, one located between nucleotides 1-148 and the other between nucleotides 597-746. Competitive EMSAs also demonstrated that GAPDH and human polypyrimidine tract-binding protein, a putative picornavirus translation initiation factor, compete with each other for binding to stem-loop IIIa, suggesting that the relative cytoplasmic abundance of GAPDH and polypyrimidine tract-binding protein in individual cell-types may be an important determinant of viral translation activity. Human GAPDH was found to destabilize the folded structure of the stem-loop IIIa RNA based upon observed decreases in the circular dichroism spectra of this RNA following binding of the protein. This RNA helix-destabilizing activity of GAPDH could directly influence IRES-dependent translation and/or replication of picornavirus RNA.

Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli.

We have shown previously that the periplasmic domain of penicillin-binding protein 1B (PBP 1Bper; residues 90-844) from Escherichia coli is insoluble in the absence of detergents, and can be reconstituted into liposomes [Nicholas, Lamson and Schultz (1993) J. Biol. Chem. 268, 5632-5641]. These data suggested that native PBP 1B contains a membrane association site in addition to its N-terminal transmembrane anchor. We have studied the membrane topology of PBP 1B in greater detail by assessing detergent binding and solubility in the absence of detergents for PBP 1Bper and a set of proteolytic fragments of PBP 1B. PBP 1Bper was shown by three independent methods to bind to detergent micelles, which strongly suggests that the periplasmic domain interacts with the hydrophobic milieu of membrane bilayers. Digestion with high weight ratios of thrombin of purified PBP 1B containing an engineered thrombin cleavage site on the periplasmic side of the transmembrane anchor generated four fragments in addition to PBP 1Bper that varied in size from 71 to 48 kDa. In contrast to PBP 1Bper, all fragments of 67 kDa and smaller were eluted from a gel-filtration column in the absence of detergents and did not bind to detergent micelles. The N-terminal sequences of the four fragments were determined, allowing the cleavage sites to be located in the primary sequence of PBP 1B. These data localize the membrane association site of PBP 1B to a region comprising the first 163 amino acids of the periplasmic domain, which falls within the putative transglycosylase domain. Lipid modification does not appear to be the mechanism by which PBP 1Bper associates with membranes.

Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells.

Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S. P. Day, P. Murphy, E. A. Brown, and S. M. Lemon, J. Virol. 66: 6533-6540, 1992). To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors.

Expression of atrial natriuretic peptide in cultured adult cardiac ventricular muscle cells as studied by immunofluorescence microscopy.

Atrial natriuretic peptide (ANP) is known to be present predominantly in the granules of adult cardiac atrial muscle cells. The functional role of this peptide is known to be involved in diuresis, natriuresis and control of blood pressure. The present study using antibody to ANP, revealed that the cultured adult cardiac ventricular muscle cells, unlike their counterparts in vivo, expressed ANP to be as abundant as in adult atrial cells. Cultured atrial cells did not express ANP any more than their counterparts in vivo. Both cells, specifically ventricular cells, contained various forms of ANP such as granular, aggregate, ring-shape, fibrous and amorphous in different parts of the cell body. Vesicular or granular ANP was also observed beneath the cell membrane, above the cell membrane or outside the cell body, which were possibly suggestive of exocytosis of ANP by cells. The current studies were extended to the cultured embryonic ventricular and atrial cardiac myocytes, and revealed that these cells expressed considerable ANP, which were comparable to those in in vivo cells, reported by others. The high expression of ANP in cultured adult ventricular cardiac muscle cells indicates that these adult cells are capable of producing high content of ANP as observed in embryonic ventricular cells.

Kinetic parameters of the translocation of bacteriophage T4 gene 41 protein helicase on single-stranded DNA.

The bacteriophage T4 gene 41 helicase protein (gp41) carries a single-stranded DNA-dependent ATPase activity that is essential to its helicase activity. This ATPase activity can be stimulated by a wide variety of single-stranded DNA cofactors, including homo-oligomers and homopolymers 8 to approximately 10,000 nucleotide residues in length, and by natural single-stranded DNA, such as bacteriophage M13 DNA. The steady-state ATPase activity of gp41 on single-stranded homopolymeric cofactors is dependent on the length of the cofactor, in that the kinetic parameters Vmax and K(act) (or KmDNA) have a characteristic length dependence. Vmax values for different DNA lengths show a hyperbolic dependence on DNA length, while K(act) values are independent of DNA lengths exceeding approximately 20 nucleotide residues. Use of the detailed theoretical analysis developed in the preceding paper reveals that: (1) these results support the earlier proposal that gp41 translocates on single-stranded DNA in an ATP-dependent manner; (2) translocation is undirectional; (3) translocation is processive to an extent that depends on the base composition of the DNA employed, with the average distance translocated per binding event ranging from 60 to 700 nucleotide residues; and (4) the detailed translocation mechanism of gp41 includes an obligatory slow step before or after the ATP-driven translocation process. Defined lengths of natural and homopolymer single-stranded DNA have also been created as gaps of known length distribution between clusters of gene 32 protein (gp32) bound along long single-stranded DNA molecules. ATPase data obtained with cofactors of this type also show unidirectional ATP-driven translocation of gp41 on both natural and homopolymeric single-stranded DNA. Direct binding studies of gp41 to short dT oligomers reveal two further features of the interaction of gp41 to single-stranded DNA: (1) nucleoside triphosphate binding is necessary for the formation of stable gp41-ssDNA complexes; and (2) the DNA binding site size of gp41 is between 12 and 20 nucleotide residues per protein monomer. Possible translocation mechanisms for gp41 are discussed within the context of these results.

Penicillin-binding protein 1B from Escherichia coli contains a membrane association site in addition to its transmembrane anchor.

A working structural model of penicillin-binding protein 1B (PBP 1B) from Escherichia coli derived from previous data consists of a highly charged aminoterminal cytoplasmic tail, a 23-amino-acid hydrophobic transmembrane anchor, and a 758-amino-acid periplasmic domain. Using an engineered thrombin cleavage site, we have investigated the solubility properties of the periplasmic domain of PBP 1B. Twelve amino acids, comprised of the consensus thrombin cleavage site (LVPR decreases GS) and flanking glycine residues, were inserted into PBP 1B just past its putative transmembrane segment. To aid in purification, a hexa-histidine tag was also inserted at its amino terminus, and the engineered protein (PBP 1B-GT/H6) was purified and characterized. Inclusion of the thrombin cleavage site had no effect on the protein's intrinsic tryptophan fluorescence and affinity for [14C]penicillin G, indicating that the protein structure was not significantly perturbed. PBP 1B-GT/H6 was readily cleaved by thrombin at low thrombin/protein ratios to a protein with properties consistent with the removal of its cytoplasmic tail and transmembrane regions. Cleavage of the protein was dependent upon the presence of the thrombin cleavage site, and the thrombin-cleaved protein (PBP 1Bper) displayed an identical affinity for [14C] penicillin G binding as wild-type PBP 1B and uncleaved PBP 1B-GT/H6. [14C]Penicillin G-labeled PBP 1Bper eluted from a gel filtration column in the presence but not in the absence of 0.7% 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonic acid, and PBP 1Bper was found entirely in the membrane fraction of a thrombin digest of membranes containing overproduced PBP 1B-GT/H6. To further characterize this unusual solubility behavior, purified PBP 1Bper was reconstituted into lipid vesicles, which were then floated on a sucrose gradient. Floated vesicles contained > 95% of total 125I-penicillin V binding, indicating that PBP 1Bper directly associates with lipid membranes. These results strongly suggest that the periplasmic domain of PBP 1B associates with membranes independent of its amino terminal transmembrane region.

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae.

Resistance to penicillin in non-beta-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42-581) into an ATG expression vector. When the recombinant PBP 2 molecules were overexpressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2' (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2' for [3H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other beta-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.

Utility of fiberoptic bronchoscopy in nonresolving pneumonia.

Although fiberoptic bronchoscopy (FOB) has been traditionally used to evaluate nonresolving pneumonia, its efficacy is unknown. We, therefore, reviewed FOB in 35 consecutive patients who had (1) a roentgenographic infiltrate, (2) cough, (3) either temperature greater than 38.1 degrees C, leukocytosis, sputum production, (4) symptoms present for at least ten days, and antibiotic therapy for at least one week. Known lung cancer and AIDS were excluded. Fiberoptic bronchoscopy was diagnostic in 86 percent (12/14) in whom a specific cause was found. No patient had endobronchial cancer. Two patients with nondiagnostic FOB and persistent systemic symptoms had open lung biopsy specimens showing Wegener's granulomatosis and bronchiolitis obliterans with organizing pneumonia (BOOP). Twenty-one patients with nondiagnostic FOB had no final diagnoses other than community-acquired pneumonia. We conclude that FOB is extremely useful in finding a specific diagnosis for a nonresolving pneumonia when a specific diagnosis can be made. Fiberoptic bronchoscopy was most likely to yield a specific diagnosis in nonsmoking patients with multilobar infiltrates of long duration and could have been avoided in older, smoking, or otherwise compromised patients with lobar or segmental infiltrates with no decrease in diagnostic yield in our series.

Isozymes in androgenetic and gynogenetic white amur, gynogenetic carp, and carp-amur hybrids.

Gynogentic and androgenetic progeny appeared in crosses between white amur, Ctenopharyngodon idella, and carp, Cyprinus carpio. Hemoglobin, plasma general proteins, and plasma isoenzymes were studied by electrophoresis to determine inheritance patterns. Electrophoretic bands indicated that gynogenetic white amur had no paternal inheritance from carp and that adrogenetic white amur also were pure white amur. Gynogeneiss in carp was confirmed by the absence of paternal inheritance. Hemoglobins, general proteins, and esterases distinguished the two species. Within a species there were no differences in proteins between gynogenetic, adrogenetic and normal fish. Lactate dehydrogenase (LDH) differed between carp and white amur and was a good marker for detecting heterologous inheritance in adrogenesis or gynogenesis because expression of LDH alleles from white amur was inhibited by the carp genome. Alkaline phosphatase and malate dehydrogenase had similar electrophoretic mobility in the two species.