Differential expression of exon 5 splice variants of sodium channel alpha subunit mRNAs in the developing mouse brain.

Sodium channel alpha subunit genes expressed in the human brain, SCN1A, SCN2A, SCN3A and SCN8A, are subject to alternative splicing of coding exons 5N and 5A. In this study we examined expression of alpha subunit mRNA and exon 5 splicing in the developing mouse brain. Expression levels of Scn1a, Scn2a and Scn8a mRNAs increase postnatally, whereas Scn3a mRNA expression levels decrease. Scn1a mRNA contains only exon 5A, due to the absence of exon 5N in the mouse Scn1a gene. At birth, Scn2a is the only sodium channel alpha subunit mRNA that contains higher or equal amounts of the 5N isoform compared to the 5A isoform in most brain regions. In contrast, the predominant isoform of Scn3a and Scn8a mRNAs in the newborn mouse brain is 5A. 5N/5A ratios for each of the three mRNAs vary across brain regions, with cortex >or= hippocampus>thalamus>cerebellum. In all brain regions and for all three alpha subunits, 5N/5A ratios gradually decrease with age, levelling at a value between 0.1 and 0.2. These findings suggest potential involvement of common factors in the alternative splicing of exon 5 for all three transcripts, and that expression of these factors varies between brain regions and changes during development. Differences in the strength of exon 5N and/or exon 5A splice sites in Scn2a pre-mRNA as compared to Scn1a and Scn8a may underlie the observed differences in 5N/5A ratios in the three alpha subunit mRNAs.

Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function.

Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis.

Core protein phosphorylation modulates pregenomic RNA encapsidation to different extents in human and duck hepatitis B viruses.

To clarify the role of core protein phosphorylation in pregenomic-RNA encapsidation of human and duck hepatitis B viruses (HBV and DHBV, respectively), we have examined the phosphorylation states of different forms of intracellular HBV core protein and the phenotypic effects of mutations in the phosphorylation sites of HBV and DHBV core proteins. We show that HBV core protein is phosphorylated to similar extents in the form of protein dimers and after further assembly in pregenomic RNA-containing capsids. Individual and multiple substitutions of alanine and aspartic acid for serine in the phosphorylation sites of HBV core protein resulted in site-specific and synergistic effects on RNA encapsidation, ranging from 2-fold enhancement to more than 10-fold inhibition. Core protein variants with mutations in all phosphorylation sites exhibited dominant-negative effects on RNA encapsidation by wild-type protein. The results suggest that the presence of phosphoserine at position 162 of HBV core protein is required for pregenomic-RNA encapsidation, whereas phosphoserine at position 170 optimizes the process and serine might be preferable in position 155. Examination of the pregenomic-RNA-encapsidating capacities of DHBV core protein variants, in which four phosphorylation sites were jointly mutated to alanine or aspartic acid, suggests that phosphorylation of DHBV core protein at these sites may optimize pregenomic-RNA encapsidation but that its impact is much less profound than in the case of HBV. The possible mechanisms by which RNA encapsidation may be modulated by core protein phosphorylation are discussed in the context of the observed differences between the two viruses.

Normal phosphorylation of duck hepatitis B virus L protein is dispensable for infectivity.

A fraction of the large surface protein (L) of duck hepatitis B virus (DHBV) is phosphorylated at serine or threonine residues (E. Grgacic & D. Anderson, Journal of Virology 68, 7344-7350, 1994). We now report the identification of phosphorylation sites in DHBV L protein. Using site-directed mutagenesis, we have identified serine-118 (S118) as the major phosphorylation site, accepting approximately 64% of the total phosphate groups incorporated in L, and resulting in retarded migration of phosphorylated L in SDS-PAGE. Proline-119 is indispensable for S118 phosphorylation. Mutation of other serine/threonine residues which are followed by prolines (T79, T89, S117 and T155) together with S118 further reduced phosphorylation to around 19% of wild-type. Non-equilibrium pH gel electrophoresis (NEPHGE) and SDS-PAGE of 33P-labelled L protein revealed two phosphorylated L species, while protein with the S118 to alanine mutation was detected as only one labelled species, consistent with multiple phosphorylations in wild-type L. Together, these results demonstrate that serine 118 is the major phosphorylation site for a proline-directed kinase, and that a proportion of L molecules are additionally phosphorylated at one of a number of secondary sites. DHBV mutants encoding L proteins with minimal phosphorylation (alanine mutants) or mimicking constitutive phosphorylation (aspartic acid mutants) remained infectious both in cell culture and in ducks, demonstrating that L phosphorylation may play only a minor role in DHBV replication.

Intracellular retention of duck hepatitis B virus large surface protein is independent of preS topology.

The mechanism of intracellular retention for the large surface protein (L) of duck hepatitis B virus (DHBV) was analyzed by examination of the transmembrane topologies and secretory properties of a collection of DHBV L mutants and compared with that of human hepatitis B virus (HBV) L. Our results demonstrate that, in contrast to its HBV counterpart, intracellular retention of DHBV L does not depend on the cytosolic disposition of its preS domain. L mutants with either cytosolic or lumenal preS were mostly retained in the absence of the small surface protein (S), whereas coexpression with S resulted in efficient secretion of both topological forms. Coexpression of the wild-type DHBV L with S resulted in efficient incorporation of L into secreted S + L particles, whereas HBV L was partially excluded from secreted particles under the same conditions. We propose that HBV provides L retention even in the presence of an excess of S, by exclusion of molecules with cytosolic preS domains from secreted particles at the stage of their assembly. DHBV lacks such a retention mechanism due to the absence of topological selection in particulate assembly.

[Preparation and purification of a polypeptide containing antigenic determinants of hepatitis C core protein]. / Poluchenie i ochistka rekombinantnogo polipeptida, soderzhashchego antigennye determinanty serdtsevinnogo belka virusa gepatita C.

A method has been developed for preparing and purifying recombinant polypeptide--hepatitis C virus core protein (HCcoreAg) expressed in E. coli from pFC105-302 plasmid coding for 150 N-terminal amino acids of HCcoreAg in the hybrid from the C-terminal with beta-galactosidase. HCcoreAg was purified by ion-exchange chromatography on P-11 phosphocellulose. The bulk of protein carrying HCcoreAg antigenic determinants was found in two polypeptides: with molecular weights 26 and 136 kD. Antigenic and immunogenic properties of the resultant polypeptide were studied. The 26 kD protein can be used in enzyme immunoassay and immunoblotting as antigen for detecting antibodies to the HCV core protein. The results of immunization of rabbits indicate a high immunogenic activity of the protein. High diagnostic value of HCcoreAg preparation has been demonstrated, for the rapid variant of indirect solid-phase enzyme immunoassay among other tests.

[Expression in Escherichia coli of hepatitis B virus polymerase and its functional domains]. / Ekspressiia v Escherichia coli polimerazy virusa gepatita B i ee funktsional'nykh domenov.

The plasmids have been constructed permitting expression of hepatitis B viral polymerase and its functional domains, the catalytic and end ones, as hybrid proteins containing 12 alien amino acids in the N-terminal. Immunoblotting with the rabbit antisera to N- and C-terminals of hepatitis B polymerase amino acid sequence has demonstrated that constructed plasmids determine the synthesis of the corresponding proteins in bacterial cells. HBV polymerase is processed in Escherichia coli cells. The successful expression of HBV polymerase and its functional domains makes possible the detailed study of this protein structure and function.

The effect of the structure of the terminal regions of the hepatitis B virus gene C polypeptide on the formation of core antigen (HBcAg) particles.

A series of plasmids encoding native and modified sequences of the hepatitis B virus core antigen (HBcAg) was created. Analysis of the products generated by expression of the plasmid genomes in Escherichia coli showed that a polypeptide with primary structure identical to that deduced for native HBcAg forms particles in the bacterial cells which are indistinguishable from the native nucleocapsids in morphological and antigenic properties. Removal of the thirty-nine C-terminal amino acids which form a protamine-like domain caused insignificant impairment of the particle-forming process. Modification of the N-terminal region of the polypeptide showed that at least part of the structural determinant governing particle formation is localised between amino acid residues 3 and 11. When the plasmid genes were expressed in an E. coli cell-free transcription - translation system, polypeptides devoid of ten to twenty N-terminal amino acids were formed in addition to the full-length products. From the results obtained it is proposed that a protease digestion site situated within the region containing amino acid residues 10 - 20 plays a role in the formation of the HBe antigen.