Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.     

Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography.

The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [alpha-2-O-methyl-, 4-O-acetyl-alpha-2-O-methyl-, 9-amino-9-deoxy-alpha-2-O-methyl-, and alpha-2-O-(4'-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (alpha 2-3)sialyllactose, and suggest the location of an alpha-glycosidic chain (4'-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with alpha-2-O-methylsialic acid contains the mutation Gly-135-->Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.     

CTNNBL1 facilitates the association of CWC15 with CDC5L and is required to maintain the abundance of the Prp19 spliceosomal complex

In order to catalyse the splicing of messenger RNA, multiple proteins and RNA components associate and dissociate in a dynamic highly choreographed process. The Prp19 complex is a conserved essential part of the splicing machinery thought to facilitate the conformational changes the spliceosome undergoes during catalysis. Dynamic protein interactions often involve highly disordered regions that are difficult to study by structural methods. Using amine crosslinking and hydrogen–deuterium exchange coupled to mass spectrometry, we describe the architecture of the Prp19 sub-complex that contains CTNNBL1. Deficiency in CTNNBL1 leads to delayed initiation of cell division and embryonic lethality. Here we show that in vitro CTNNBL1 enhances the association of CWC15 and CDC5L, both core Prp19 complex proteins and identify an overlap in the region of CDC5L that binds either CTNNBL1 or CWC15 suggesting the two proteins might exchange places in the complex. Furthermore, in vivo, CTNNBL1 is required to maintain normal levels of the Prp19 complex and to facilitate the interaction of CWC15 with CDC5L. Our results identify a chaperone function for CTNNBL1 within the essential Prp19 complex, a function required to maintain the integrity of the complex and to support efficient splicing.     

The ALS8-associated mutant VAPB(P56S) is resistant to proteolysis in neurons

VAMP/synaptobrevin associated proteins A and B (VAPA and VAPB), are type IV membrane proteins enriched on ER and Golgi membranes. Both VAPA and B interact with cytoplasmic lipid transport proteins and cytoskeletal elements to maintain the structure and composition of ER and Golgi membranes. Truncated forms of both proteins are present in some tissues but the functional significance of this is not clear. In rodents processing of VAPA occurs in most tissues, however, truncated forms of VAPB have only been reported in brain tissue. It is demonstrated here that the extent of VAPB processing in rat increases during postnatal development and that it is restricted to neurons. The C-terminal polypeptide generated by this cleavage reaction remains associated with cell membranes, but its subcellular distribution is distinct from the full-length protein. A mutant form of VAPB is associated with a familial form of neurodegenerative disease, amyotrophic lateral sclerosis type 8. The mutant protein, VAPB(P56S) , is resistant to truncation in primary neuronal cultures, although remains sensitive to some form of proteolysis when over-expressed in HEK293 cells. These data suggest that neuronal cells have a particular requirement for VAPB proteolysis and that reduced levels of processed polypeptides may contribute to the neurodegeneration associated with amyotrophic lateral sclerosis type 8.     

Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis

The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.     

An overview of influenza haemagglutinin and neuraminidase

This is an overview of the structures of influenza virus haemagglutinin and neuraminidase membrane glycoproteins with particular reference to antibody recognition and antigenic variation.     

H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K_M values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K_I) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.