Nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, RNA templates, and polymerase activity

Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside ～50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+)RNA viruses at various membranes. To better understand replication complex assembly, we studied the mechanisms of FHV spherule formation. FHV has two genomic RNAs; RNA1 encodes multifunctional RNA replication protein A and RNA interference suppressor protein B2, while RNA2 encodes the capsid proteins. Expressing genomic RNA1 without RNA2 induced mitochondrial spherules indistinguishable from those in FHV infection. RNA1 mutation showed that protein B2 was dispensable and that protein A was the only FHV protein required for spherule formation. However, expressing protein A alone only "zippered" together the surfaces of adjacent mitochondria, without inducing spherules. Thus, protein A is necessary but not sufficient for spherule formation. Coexpressing protein A plus a replication-competent FHV RNA template induced RNA replication in trans and membrane spherules. Moreover, spherules were not formed when replicatable FHV RNA templates were expressed with protein A bearing a single, polymerase-inactivating amino acid change or when wild-type protein A was expressed with a nonreplicatable FHV RNA template. Thus, unlike many (+)RNA viruses, the membrane-bounded compartments in which FHV RNA replication occurs are not induced solely by viral protein(s) but require viral RNA synthesis. In addition to replication complex assembly, the results have implications for nodavirus interaction with cell RNA silencing pathways and other aspects of virus control.     

Human T-cell leukemia virus type II Rex binding and activity require an intact splice donor site and a specific RNA secondary structure.

The human T-cell leukemia virus type II (HTLV-II) regulatory protein Rex augments cytoplasmic levels of unspliced gag-pol mRNA by acting through a Rex-responsive element (RxRE) in the long terminal repeat. Purified Rex protein binds to long terminal repeat mRNA. Here, using an immunobinding assay to measure the binding of Rex protein to mutated RxRE RNAs, we show that efficient Rex binding requires a stem-bulge-loop RNA secondary structure (nucleotides [nt] 465 to 500) and specific sequences both within the stem-bulge (nt 470 to 476) and within a conserved upstream splice donor site (nt 449 to 455). Rex function in a transient transfection expression system correlates with Rex protein-RxRE RNA binding. The ability of HTLV-II Rex to interact directly with the HTLV-II splice donor site suggests that HTLV-II Rex may increase expression of unspliced gag-pol mRNA, in part, by inhibiting splicing.     

N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan β1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.     

Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4(IRAK-1-K239S)). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4(IRAK-1-K239S) cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate.     

A novel tetraspanin fusion protein, peripherin-2, requires a region upstream of the fusion domain for activity

Peripherin-2 (also known as peripherin/rds), a photoreceptor specific tetraspanin protein, is required to maintain normal cell structure through its role in renewal processes requiring membrane fusion. It is the first tetraspanin fusogen and has been shown to directly mediate fusion between disk membranes and opposing membranes to maintain the highly ordered structure of rod outer segments. Localized to the C terminus of human, bovine, and murine peripherin-2 is an amphiphilic fusion peptide domain (residues 312-326) and a highly conserved region upstream of this domain that we hypothesize is essential for fusogenic function. Our previous studies indicated that substitution of a threonine for a proline at position 296 within this highly conserved region enhanced fusion activity. In this study we wanted to determine whether this proline is essential with the introduction of three additional substitutions of proline with alanine, leucine, and glutamic acid. Wild type, P296T, P296A, P296L, and P296E mutants of peripherin-2 were expressed as His6-tagged full-length proteins in Madin-Darby canine kidney (MDCK) cells. All of the proteins were localized to intracellular membranes and detected as 42-kDa monomers by Western blot analysis. The wild type, P296A, and P296L assembled into core tetramers; in contrast the P296T and P296E formed higher order oligomers. Fusogenic activity of full-length protein expressed in MDCK membranes and purified protein reconstituted in model membrane liposomes was determined using fluorescence quenching techniques. Fusion activity was decreased in the P296L, P296A, and P296E mutants both in endogenous MDCK membranes and in model liposomes. Collectively, these results suggest that the proline at position 296 is necessary for optimal function.     

The cytotoxic activity of human natural killer cells requires an intact secretory apparatus

We have analyzed the effect of various inhibitors of cellular secretion and motility on the cytolytic activity of human natural killer (NK) cells. As effector cells we used highly purified peripheral blood lymphocytes consisting of 75–85% large granular lymphocytes (LGL) that have previously been shown to be responsible for the NK activity in man. Treatment of the effector cells with a carboxylic ionophore monensin inhibited irreversibly the NK-cell-mediated killing. This drug is known to interrupt the vesicular traffic of Golgi-derived vesicles and thus the results strongly suggested that secretory processes are required in the cytolytic activity of human NK cells. In the monensin-treated effector cells large amounts of glycoprotein accumulated in the Golgi area within 24 hr of incubation. The lytic activity did not require intact microtubules since effector cells in which vinblastine-induced tubulin-containing paracrystals were demonstrated still mediated normal NK activity. Energy was required in the human NK-cell-mediated cytolysis. The lethal hit stage of the cytolytic activity was preceded by formation of intimate contacts between effector and target cells and required active cell movement and divalent cations.     

Vesicular stomatitis virus mRNA capping machinery requires specific cis-acting signals in the RNA

Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5' end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.     

Characterization of the spermidine-dependent, sequence-specific endoribonuclease that requires transfer RNA for its activity.

The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) in mouse FM3A cells consists of protein and transfer RNA lacking its 3' terminus. In vitro properties of this enzyme were characterized using partially purified enzyme. The RNase 65 activity requires spermidine, which is not replaceable with spermine or Mg++. The enzyme cleaves an RNA substrate on the 3' side of the phosphodiester bond. The cleavage reaction has a temperature optimum around 50 degrees C and a pH optimum around 7.0. The optimum KCl concentration for the activity is around 10 mM. Relative cleavage efficiency of two differently folded RNA substrates with the common target sequence was analyzed at 37 degrees C and 50 degrees C. The results of this analysis suggest that unfolding of the target sequence is critical for recognition by RNase 65. Furthermore, in experiments using several point-mutated RNA substrates designed to form basically the same secondary structure as the wild type, one to three nucleotide substitutions in the target sequence all reduced cleavage efficiency. The RNase 65 activity is found only in cytosolic extracts, not in nuclear ones. Gel filtration analysis suggests that the native size of the endoribonuclease is approximately 150 kDa.     

Efficient and specific initiation of subgenomic RNA synthesis by cucumber mosaic virus replicase in vitro requires an upstream RNA stem-loop

We defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (CMV) subgenomic RNA4. The necessary sequence maps to positions -28 to +15 relative to the initiation cytidylate used to initiate RNA synthesis in vivo. Positions -28 to -5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. Considerable changes in the stem and the loop are tolerated for RNA synthesis, including replacement with a different stem-loop. In a template competition assay, the stem-loop and the initiation cytidylate are sufficient to interact with the CMV replicase. Thus, the mechanism of core promoter recognition by the CMV replicase appears to be less specific in comparison to the minimal subgenomic core promoter of the closely related brome mosaic virus.