Loss of the N-linked glycan at residue 173 of human parainfluenza virus type 1 hemagglutinin-neuraminidase exposes a second receptor-binding site.

BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

Probucol therapy overcomes the reproductive defect in CTP: phosphocholine cytidylyltransferase beta2 knockout mice.

The synthesis of phosphatidylcholine (PtdCho), the major phospholipid in mammalian cells, is regulated by the CTP:phosphocholine cytidylyltransferase (CCT). Loss of the CCTbeta2 isoform expression in mice results in gonadal dysfunction. CCTbeta2(-/-) females exhibit ovarian tissue disorganization with progressive loss of follicle formation and oocyte maturation. Ultrastructure revealed a disrupted association between ova and granulosa cells and disorganized Golgi apparati in oocytes of CCTbeta2(-/-) mice. Probucol is a cholesterol-lowering agent that stimulates the uptake and retention of lipids carried by lipoproteins in peripheral tissues. Probucol therapy significantly lowered both serum cholesterol and PtdCho levels. Probucol therapy increased fertility in the CCTbeta2(-/-) females 100%, although it did not completely correct the phenotype, the morphological abnormalities in the knockout ovaries or itself stimulate CCT activity directly. These data indicated that a deficiency in de novo PtdCho synthesis could be complemented by altering the metabolism of serum lipoproteins, an alternative source for cellular phospholipid.

Identification of a mammalian mitochondrial porphyrin transporter.

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.

Influenza virus neuraminidase contributes to secondary bacterial pneumonia.

Secondary bacterial pneumonia is a common cause of death during influenza epidemics. We hypothesized that virus-specific factors could contribute to differences in annual excess mortality. Recombinant influenza viruses with neuraminidases from representative strains from the past 50 years were created and characterized. The specific level of their neuraminidase activity correlated with their ability to support secondary bacterial pneumonia. Recombinant viruses with neuraminidases from 1957 and 1997 influenza strains had the highest level of activity, whereas a virus with the neuraminidase from a 1968 strain had the lowest level of activity. The high level of activity of the neuraminidase from the 1957 strain, compared with that of other neuraminidases, more strongly supported the adherence of Streptococcus pneumoniae and the development of secondary bacterial pneumonia in a mouse model. These data lend support to our hypothesis that the influenza virus neuraminidase contributes to secondary bacterial pneumonia and subsequent excess mortality.

Interferon induces the interaction of prothymosin-alpha with STAT3 and results in the nuclear translocation of the complex.

Interferons (IFNs) play critical roles in host defense by modulating the expression of various genes via tyrosine phosphorylation of STAT transcription factors. Many cytokines including IFNs induce tyrosine phosphorylation of the STAT3 transcription factor, which regulates acute phase gene expression. Using the yeast two-hybrid interaction trap, in which a tyrosine kinase is introduced into the yeast to allow tyrosine phosphorylation of bait proteins, prothymosin-alpha (ProTalpha) was identified to interact with the amino terminal half of tyrosine-phosphorylated STAT3. ProTalpha is a small, acidic, extremely abundant, and essential protein that may play a role in chromatin remodeling, and has been implicated in regulating the growth and survival of mammalian cells. Besides the interaction of tyrosine-phosphorylated STAT3 with ProTalpha in yeast cells, IFN induced the interaction of ProTalpha with STAT3 in mammalian cells, and this interaction was dependent on the tyrosine phosphorylation of STAT3. Moreover, IFNalpha induces the translocation of STAT3 and ProTalpha from the cytoplasm to the nucleus where these proteins colocalize. Since ProTalpha has an extremely strong nuclear localization and STAT proteins apparently lack any nuclear localization signals, the association of STAT3 with ProTalpha may provide a mechanism to result in STAT localization in the nucleus.

PPARalpha controls the intracellular coenzyme A concentration via regulation of PANK1alpha gene expression.

Pantothenate kinase (PanK) is thought to catalyze the first rate-limiting step in CoA biosynthesis. The full-length cDNA encoding the human PanK1alpha protein was isolated, and the complete human PANK1 gene structure was determined. Bezafibrate (BF), a hypolipidemic drug and a peroxisome proliferator activator receptor-alpha (PPARalpha) agonist, specifically increased hPANK1alpha mRNA expression in human hepatoblastoma (HepG2) cells as a function of time and dose of the drug, compared with hPANK1beta, hPANK2, and hPANK3, which did not significantly increase. Four putative PPARalpha response elements were identified in the PANKIalpha promoter, and BF stimulated hPANK1alpha promoter activity but did not alter the mRNA half-life. Increased hPANK1alpha mRNA resulted in higher hPanK1 protein, localized in the cytoplasm, and elevated PanK enzyme activity. The enhanced hPANK1alpha gene expression translated into increased activity of the CoA biosynthetic pathway and established a higher steady-state CoA level in HepG2 cells. These data are consistent with a key role for PanK1alpha in the control of cellular CoA content and point to the PPARalpha transcription factor as a major factor governing hepatic CoA levels by specific modulation of PANK1alpha gene expression.

A release-competent influenza A virus mutant lacking the coding capacity for the neuraminidase active site.

Both influenza A virus surface glycoproteins, the haemagglutinin (HA) and neuraminidase (NA), interact with neuraminic acid-containing receptors. The influenza virus A/Charlottesville/31/95 (H1N1) has shown a substantially reduced sensitivity to NA inhibitor compared with the A/WSN/33 (H1N1) isolate by plaque-reduction assays in Madin-Darby canine kidney (MDCK) cells. However, there was no difference in drug sensitivity in an NA inhibition assay. The replacement of the HA gene of A/WSN/33 with the HA gene of A/Charlottesville/31/95 led to a drastic reduction in sensitivity of A/WSN/33 to NA inhibitor in MDCK cells. Passage of A/Charlottesville/31/95 in cell culture in the presence of an NA inhibitor resulted in the emergence of mutant viruses (delNA) whose genomes lacked the coding capacity for the NA active site. The delNA mutants were plaque-to-plaque purified and further characterized. The delNA-31 mutant produced appreciable yields ( approximately 10(6) p.f.u./ml) in MDCK cell culture supernatants in the absence of viral or bacterial NA activity. Sequence analysis of the delNA mutant genome revealed no compensatory substitutions in the HA or other genes compared with the wild-type. Our data indicate that sialylation of the oligosaccharide chains in the vicinity of the HA receptor-binding site of A/Charlottesville/31/95 virus reduces the HA binding efficiency and thus serves as a compensatory mechanism for the loss of NA activity. Hyperglycosylation of HA is common in influenza A viruses circulating in humans and has the potential to reduce virus sensitivity to NA inhibitors.

Predominant nuclear localization of mammalian target of rapamycin in normal and malignant cells in culture.

Mammalian target of rapamycin (mTOR) controls initiation of translation through regulation of ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein (4E-BP). mTOR is considered to be located predominantly in cytosolic or membrane fractions and may shuttle between the cytoplasm and nucleus. In most previous studies a single cell line, E1A-immortalized human embryonic kidney cells (HEK293), has been used. Here we show that in human malignant cell lines, human fibroblasts, and murine myoblasts mTOR is predominantly nuclear. In contrast, mTOR is largely excluded from the nucleus in HEK293 cells. Hybrids between HEK293 and Rh30 rhabdomyosarcoma cells generated cells co-expressing markers unique to HEK293 (E1A) and Rh30 (MyoD). mTOR distribution was mainly nuclear with detectable levels in the cytoplasm. mTOR isolated from Rh30 nuclei phosphorylated recombinant GST-4E-BP1 (Thr-46) in vitro and thus has kinase activity. We next investigated the cellular distribution of mTOR substrates 4E-BP, S6K1, and eIF4E. 4E-BP was exclusively detected in cytoplasmic fractions in all cell lines. S6K1 was localized in the cytoplasm in colon carcinoma, HEK293 cells, and IMR90 fibroblasts. S6K1 was readily detected in all cellular fractions derived from rhabdomyosarcoma cells. eIF4E was detected in all fractions derived from rhabdomyosarcoma cells but was not detectable in nuclear fractions from colon carcinoma HEK293 or IMR90 cells.