Sung Key Jang

Crystal Structure of RNA Helicase from Genotype 1b Hepatitis C Virus A FEASIBLE MECHANISM OF UNWINDING DUPLEX RNA

Crystal structure of RNA helicase domain from genotype 1b hepatitis C virus has been determined at 2.3 A resolution by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain containing two highly conserved NTP binding motifs. Another is an RNA binding domain containing a conserved RNA binding motif. The third is a helical domain that contains no beta-strand. The RNA binding domain of the molecule is distinctively separated from the other two domains forming an interdomain cleft into which single stranded RNA can be modeled. A channel is found between a pair of symmetry-related molecules which exhibit the most extensive crystal packing interactions. A stretch of single stranded RNA can be modeled with electrostatic complementarity into the interdomain cleft and continuously through the channel. These observations suggest that some form of this dimer is likely to be the functional form that unwinds double stranded RNA processively by passing one strand of RNA through the channel and passing the other strand outside of the dimer. A descending molecular see-saw model is proposed that is consistent with directionality of unwinding and other physicochemical properties of RNA helicases.

Cap-independent translation of picornavirus RNAs : structure and function of the internal ribosomal entry site

Picornaviruses are mammalian plus-strand RNA viruses whose genomes serve as mRNA. A study of the structure and function of these viral mRNAs has revealed differences among them in events leading to the initiation of protein synthesis. A large segment of the 5 nontranslated region, approximately 400 nucleotides in length, promotes internal entry of ribosomes independent of the non-capped 5 end of the mRNA. This segment, which we have called the internal ribosome entry site (IRES), maps approximately 200 nt down-stream from the 5 end and is highly structured. IRES elements of different picornaviruses, although functionally similar in vitro and in vivo, are not identical in sequence or structure. However, IRES elements of the genera entero- and rhinoviruses, on the one hand, and cardio- and aphthoviruses, on the other hand, reveal similarities corresponding to phylogenetic kinship. All IRES elements contain a conserved Yn-Xm-AUG unit (Y, pyrimidine; X, nucleotide) which appears essential for IRES function. The IRES elements of cardio-, entero- and aphthoviruses bind a cellular protein, p57. In the case of cardioviruses, the interaction between a specific stem-loop of the IREs is essential for translation in vitro. The IRES elements of entero- and cardioviruses also bind the cellular protein, p52, but the significance of this interaction remains to be shown. The function of p57 or p52 in cellular metabolism is unknown. Since picornaviral IRES elements function in vivo in the absence of any viral gene products, we speculate that IRES-like elements may also occur in specific cellular mRNAs releasing them from cap-dependent translation. IRES elements are useful tools in the construction of high yield expression vectors, or for tagging cellular genetic elements.

Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.

Picornaviral mRNAs have been shown to possess special structures in their 5 nontranslated regions (5NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.

Two rice MADS domain proteins interact with OsMADS1.

OsMADS1 is a MADS box gene controlling flower development in rice. In order to learn more about the function of OsMADS1, we searched for cellular proteins interacting with OsMADS1 employing the yeast two-hybrid system. Two novel proteins with MADS domains, which were named OsMADS14 and OsMADS15, were isolated from a rice cDNA library. OsMADS14 and -15 are highly homologous to the maize MADS box gene ZAP1 which is an orthologue of the floral homeotic gene APETALA1 (AP1). Interactions among the three MADS domain proteins were confirmed by in vitro experiments using GST-fused OsMADS1 expressed in Escherichia coli and in vitro translated proteins of OsMADS14 and -15. We determined which domains in OsMADS1, -14, and -15 were required for protein-protein interaction employing the two-hybrid system and pull-down experiments. While the K domain was essential for protein-protein interaction, a region preceded by the K domain augmented this interaction. Interestingly, the C-terminal region of OsMADS1 functioned as a transcriptional activation domain in yeast and mammalian cells, while, on the other hand, the C domains of OsMADS14 and -15 exhibited only very weak transcriptional activator functionality, if any at all.

Subcellular localization of hepatitis C viral proteins in mammalian cells

SummaryWe determined the subcellular localization of hepatitis C viral (HCV) proteins as a first step towards the understanding of the functions of these proteins in the mammalian cell (CHO-K1). We used fluorescence emitted from green fluorescent protein (GFP)-fused to the viral proteins to determine the subcellular localization of the viral proteins. We found that most of the viral proteins were excluded from the nucleus. Core exhibited a globular pattern near the nucleus. NS2 was concentrated in the perinuclear space. NS4A accumulated in the ER and the Golgi regions. NS3 was detected in the nucleus as well as the cytoplasm, when it was expressed by itself. However, NS3 became restricted to the cytoplasm, when it was produced together with NS4A. NS4B showed a spot-like pattern throughout the cytoplasm. NS5A and NS5B were distributed throughout the cytoplasm in a mesh-like pattern. These results can provide a basis for further investigations into the functions of the HCV proteins.

Polypyrimidine tract-binding protein interacts with HnRNP L

Polypyrimidine tract‐binding protein (PTB) is involved in pre‐mRNA splicing and internal ribosomal entry site (IRES)‐dependent translation. In order to identify cellular protein(s) interacting with PTB, we performed a yeast two‐hybrid screening. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as a PTB‐binding protein. The interaction between PTB and hnRNP L was confirmed in an in vitro binding assay. Both PTB and hnRNP L were found to localize in the nucleoplasm, excepting the nucleoli, in HeLa cells by the green fluorescent protein (GFP)‐fused protein detection method. The N‐terminal half of PTB (aa 1–329) and most of hnRNP L (aa 141–558) is required for the interaction between PTB and hnRNP L.

Phospholipase D1 is located and activated by protein kinase Cα in the plasma membrane in 3Y1 fibroblast cell

The subcellular location of phospholipase D1 (PLD1) and its activation by protein kinase C alpha (PKC alpha) were examined by subcellular fractionation and by microscopic observation of green fluorescent protein-fused PLD1 (GFP-PLD1) or PKC alpha (GFP-PKC alpha) in fibroblastic 3Y1 cells. Major PLD1 immunoreactivity and PKC alpha-stimulated PLD activity segregated with a plasma membrane marker, even though a significant amount was co-fractionated with markers for endoplasmic reticulum (ER) and Golgi. Upon treatment with phorbol myristate acetate (PMA), PKC alpha translocated from the cytosolic fraction to the membrane fraction to which PLD1 also localized. GFP-PLD1 was found in the plasma membrane as well as a in a perinuclear compartment consistent with ER and Golgi and in other dispersed vesicular structures in the cytoplasm. However, most of GFP-PKC alpha was translocated from the cytosol to the plasma membrane after treatment with PMA. From these results, we concluded that the plasma membrane is the major site of PLD1 activation by PKC alpha in 3Y1 cells.

La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site

Translation of internal ribosomal entry site (IRES)-dependent mRNAs is mediated by RNA-binding proteins as well as canonical translation factors. In order to elucidate the roles of RNA-binding proteins in IRES-dependent translation, the role of polypyrimidine tract-binding protein (PTB) and La protein in encephalomyocarditis virus (EMCV) IRES-dependent translation was investigated. PTB was required for efficient EMCV IRES-driven translation but, intriguingly, an excess of PTB suppressed it. Such a translational suppression by surplus PTB was relieved by addition of La protein. A possible role for La protein in IRES-dependent translation is discussed.

Identification of the protease domain in NS3 of hepatitis C virus

NS3 of hepatitis C virus (HCV) is a serine protease that carries out the proteolytic processing of the nonstructural proteins of the HCV polyprotein. Deletion analysis of the N terminus of NS2,3,4 fusion protein revealed that the N-terminal boundary of the active protease resides between amino acids 1050 and 1083. The processing patterns of internal deletion mutants of NS2,3,4 indicated that the C terminus of the enzymically active protease resides between amino acids 1115 and 1218. The N- and C-terminal boundaries of the protease were also confirmed by determining the trans-cleavage activity of internally deleted NS3,4. NS3 protease activity was inhibited by Cu2+ but was slightly enhanced by Zn2+. This report provides a possible approach for development of antiviral agents based on protease inhibitors.

Effect of frameshift mutation in the pre-C region of hepatitis B virus on the X and C genes

We have previously cloned a mutant hepatitis B virus (HBV) genome which had one thymidine addition in the pre-C region resulting in a frameshift mutation in the pre-C region and fusion of the X and C genes. We constructed plasmids containing serially deleted and/or back-mutated (authentic) pre-C regions to study the effect of the frameshift mutation. COS cells transfected with plasmids containing the frameshifted pre-C region produced a 21K C protein (P21c) but not a 22K partially processed pre-C protein (P22). On the other hand, COS cells transfected with plasmids containing the back-mutated pre-C region produced P22. This result was also observed in HepG2-K8 cells producing the mutant HBV particles. Therefore, the pre-C region of HBV is likely to be non-essential for virus replication. COS cells transfected with the plasmid containing a fused X-C open reading frame (ORF) produced a 40K X-C fusion protein. This X-C fusion protein exerted transcriptional trans-activation. These results suggest that the mutant HBV has a C gene with a defective pre-C region and a fused X-C ORF, and hence cannot synthesize 16K HBeAg (P16e).