Mutations in human parainfluenza virus type 3 hemagglutinin-neuraminidase causing increased receptor binding activity and resistance to the transition state sialic acid analog 4-GU-DANA (Zanamivir)

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANA-resistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations. The present study using cells that express the individual mutations of ZM1 HN shows that one of these mutations is responsible for the increases in receptor binding and neuraminidase activities as well as the diminished sensitivity of both activities to the inhibitory effect of 4-GU-DANA. To examine the hypothesis that increased receptor binding avidity underlies 4-GU-DANA resistance, parallel studies were carried out on the high-affinity HN variant virus C22 and cells expressing the C22 variant HN. This variant also exhibited reduced sensitivity to 4-GU-DANA in terms of receptor binding and infectivity but without concomitant changes in the neuraminidase activity of HN. Another high-affinity HN variant, C0, was not resistant in terms of infectivity; however, a small increase in the receptor binding activity of C0 HN and a partial resistance of this activity to 4-GU-DANA were revealed by sensitive methods that we developed. In each virus variant, one mutation in HN accounted for both increased receptor binding avidity and 4-GU-DANA resistance; the higher affinity for the receptor overcomes the inhibitory effect of 4-GU-DANA. Thus, in contrast to influenza viruses for which 4-GU-DANA escape variants include hemagglutinin mutants with decreased receptor binding avidity that promotes virion release, for HPF3, HN mutants with increased receptor binding avidity are those that can escape the growth inhibitory effect of 4-GU-DANA.     

The 3' end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.     

An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.     

Mammalian mitochondrial initiation factor 2 supports yeast mitochondrial translation without formylated initiator tRNA

Initiation of protein synthesis in mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl-tRNA (fMet-tRNAfMet) in a process involving initiation factor 2 (IF2). However, yeast strains disrupted at the FMT1 locus, encoding mitochondrial methionyl-tRNA formyltransferase, lack detectable fMet-tRNAfMet but exhibit normal mitochondrial function as evidenced by normal growth on non-fermentable carbon sources. Here we show that mitochondrial translation products in Saccharomyces cerevisiae were synthesized in the absence of formylated initiator tRNA. ifm1 mutants, lacking the mitochondrial initiation factor 2 (mIF2), are unable to respire, indicative of defective mitochondrial protein synthesis, but their respiratory defect could be complemented by plasmid-borne copies of either the yeast IFM1 gene or a cDNA encoding bovine mIF2. Moreover, the bovine mIF2 sustained normal respiration in ifm1 fmt1 double mutants. Bovine mIF2 supported the same pattern of mitochondrial translation products as yeast mIF2, and the pattern did not change in cells lacking formylated Met-tRNAfMet. Mutant yeast lacking any mIF2 retained the ability to synthesize low levels of a subset of mitochondrially encoded proteins. The ifm1 null mutant was used to analyze the domain structure of yeast mIF2. Contrary to a previous report, the C terminus of yeast mIF2 is required for its function in vivo, whereas the N-terminal domain could be deleted. Our results indicate that formylation of initiator methionyl-tRNA is not required for mitochondrial protein synthesis. The ability of bovine mIF2 to support mitochondrial translation in the yeast fmt1 mutant suggests that this phenomenon may extend to mammalian mitochondria as well.     

O-glycan sialylation and the structure of the stalk-like region of the T cell co-receptor CD8

Studies of mucins suggest that the structural effects of O-glycans are restricted to steric interactions between peptide-linked GalNAc residues and adjacent polypeptide residues. It has been proposed, however, that differential O-glycan sialylation alters the structure of the stalk-like region of the T cell co-receptor, CD8, and that this, in turn, modulates ligand binding (Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051-1061; Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501-512). We characterize the glycosylation of soluble, chimeric forms of the alphaalpha- and alphabeta-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8alphaalpha, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec3.2.8.1 cells (82 versus approximately 6%, respectively). Sedimentation analysis indicates that the Perrin functions, Pexp, which reflect overall molecular shape, are very similar (1.61 versus 1.54), whereas the sedimentation coefficients (s) of the CHO K1- and Lec3.2.8.1-derived proteins differ considerably (3.73 versus 3.13 S). The hydrodynamic properties of molecular models also strongly imply that the sialylated and non-sialylated forms of the chimera have parallel, equally highly extended stalks ( approximately 2.6 A/residue). Our analysis indicates that, as in the case of mucins, the overall structure of O-glycosylated stalk-like peptides is sialylation-independent and that the functional effects of differential CD8 O-glycan sialylation need careful interpretation.     

Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha activity, leading to "superactivation" of the enzyme, apparently through an interaction with its catalytic domain. We now have performed yeast two-hybrid and in vitro binding assays to determine the interaction sites between 4V and PKC alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKC alpha, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this interaction. Further assays identified a novel interaction site in the C terminus of the catalytic domain of PKC alpha (amino acid sequence 513-672). This encompasses the autophosphorylation sites, which are implicated in activation and stability. Yeast two-hybrid data were confirmed by in vitro binding and coimmunoprecipitation assays. The interaction of syndecan-4 with PKC alpha appears unique since PKC delta and epsilon did not interact with 4V in yeast two-hybrid assays or coimmunoprecipitate with syndecan-4. Finally, overexpression of syndecan-4 in rat embryo fibroblast cells, but not expression of the YF mutant, increased PKC alpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase.     

A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels

Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca2+-binding proteins termed KChIPs with pore-forming Kv4 alpha subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). Here we show that KChIPs profoundly affect the intracellular trafficking and molecular properties of Kv4.2 alpha subunits. Co-expression of KChIPs1-3 causes a dramatic redistribution of Kv4.2, releasing intrinsic endoplasmic reticulum retention and allowing for trafficking to the cell surface. KChIP co-expression also causes fundamental changes in Kv4.2 steady-state expression levels, phosphorylation, detergent solubility, and stability that reconstitute the molecular properties of Kv4.2 in native cells. Interestingly, the KChIP4a isoform, which exhibits unique effects on Kv4 channel gating, does not exert these effects on Kv4.2 and negatively influences the impact of other KChIPs. We provide evidence that these KChIP effects occur through the masking of an N-terminal Kv4.2 hydrophobic domain. These studies point to an essential role for KChIPs in determining both the biophysical and molecular characteristics of Kv4 channels and provide a molecular basis for the dramatic phenotype of KChIP knockout mice.     

Mammalian 8-oxoguanine DNA glycosylase 1 incises 8-oxoadenine opposite cytosine in nuclei and mitochondria,while a different glycosylase incises 8-oxoadenine opposite guanine in nuclei

Cells are continuously exposed to oxidative species, which cause several types of oxidative DNA lesions. Repair of some of these lesions has been well characterized but little is known about the repair of many DNA lesions. The oxidized adenine base, 7,8-dihydro-8-oxoadenine (8-oxoA), is a relatively common DNA lesion, which is believed to be mutagenic in mammalian cells. This study investigates repair of 8-oxoA in nuclear and mitochondrial mammalian extracts. In nuclei, 8-oxoA:C and 8-oxoA:G base pairs are recognized and cleaved; in contrast, only 8-oxoA:C base pairs are cleaved in mitochondria. High stability of the DNA helix increased the efficiency of incision of 8-oxoA, and the efficiency decreased at DNA bends and condensed regions of the helix. Using liver extracts from mice knocked out for 8-oxoguanine DNA glycosylase 1 (OGG1), we demonstrated that OGG1 is the only glycosylase that incises 8-oxoA, when base-paired with cytosine in mitochondria and nuclei, but a different enzyme incises 8-oxoA when base-paired with guanine in the nucleus. Consistent with this result, a covalent DNA-protein complex was trapped using purified human OGG1 or human nuclear or mitochondrial extracts with a DNA substrate containing an 8-oxoA:C base pair.     

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.