Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

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Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ϳ6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ϳ5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pHdependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. IMPORTANCEInfluenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coat of protein inside the viral surface reacts to the changes in acid that come soon after internalization. We found that acid makes the molecules of the protein coat push each other while they are still stuck to the virus, so that they would like to rip the membrane apart. This ripping force is known to promote membrane fusion, the process by which infection actually occurs. While it is becoming clearer that multiple processes unite to form a pathway for the entry of the influenza virus after its uptake into endosomes, it is not yet clear how and to what extent the pH-driven changes in the viral matrix contribute to that pathway. Influenza virus is an enveloped negative-strand RNA virus of the Orthomyxoviridae family (1). Its outer envelope consists of a host cell-derived bilayer lipid membrane (BLM) with the incorporated glycoproteins hemagglutinin (HA) and neuraminidase along with proton channel M2. The inner envelope of the virion is a membrane-associated scaffold of matrix protein M1, w...

We found that a 2-h incubation of potato virus X (PVX) virions in 10 mM Tris-HCl buffer pH 7.5 at -20 degrees C results in a strong but reversible drop in virion stability. Under these conditions, the PVX virions are completely disrupted by low (starting from 50 mM) concentrations of LiCl and CaCl(2) but not of NaCl. Incubation of PVX samples with 0.05-2 M LiCl at +4 degrees C did not result in virion disassembly and the virions were not disrupted upon incubation at -20 degrees C in 10 mM Tris-HCl buffer pH 7.5 without LiCl. We suggest that a 2-h incubation of the PVX virions at -20 degrees C in 10 mM Tris-HCl pH 7.5 results in a structural transition in the virus particles. A revised model of the three-dimensional organization of coat protein subunits in the PVX virions is proposed. This two-domain model explains better the high plasticity of the PVX CP structure.

1. The reaction of site-specific cleavage of tRNA at a 7-methylguanine residue, including subsequent treatment with sodium borohydride and aniline [Wintermeyer, W. and Zachau, H. G. (1975) FEBS Lett. 58,306-3091, was shown to work only within a certain range of tRNA concentrations (higher than 30 pM). The Escherichia coli 16s rRNA, which contained a unique m7G (position 527), could not be split by this method when taken at any concentration.2. It was found that the presence of statistically methylated carrier RNA in the reaction mixture at the borohydride stage significantly stimulates site-specific fragmentation of 16s rRNA and 32P-labeled tRNAs.3. Direct sequencing proved that 16s rRNA and tRNAs are cleaved by this procedure successfully at the m7G residue.4. The E. coli 16s rRNA was preparatively cleaved by the described procedure into two fragments. The 5'-terminal fragment (1 -526) and the 3'-terminal fragment (528 -1542) were isolated in the pure form and their secondary structure investigated by the circular dichroism method. The results of this study showed that the secondary and tertiary structures of the 5'-terminal one-third of the 16s rRNA are at least as ordered as those of intact 16s rRNA or its 3'4erminal two-thirds.The site-specific fragmentation of a polynucleotide chain is widely used for the study of the structure and functions of RNA and ribonucleoprotein complexes. Apart from the enzymatic approaches El-41, the chemical methods of splitting native RNAs at modified bases hold promise. Previously Zachau and co-workers described the procedure for splitting the yeast tRNAPhe at some minor nucleotides [5-71. The procedure of tRNA cleavage at a 7-methylguanine residue was based on reducing this base with sodium borohydride and subsequently splitting the polynucleotide chain at the reduced base with aniline.However, we have not been successful in our attempts to make direct use of this method for splitting the Escherichiu coli 16s rRNA at the unique 7-methylguanine residue or the 3'-terminally labeled yeast [32P]tRNAPhe. A closer study of the reaction has shown that the addition of the methylated RNA-carrier (methyl-RNA) at the borohydride reduction stage considerably enhances the efficiency of the reaction and makes it possible to cleave site-specifically 16s rRNA or tRNA with a 50-70% yield. Using this method we could cleave relatively large amounts of E. coli 16s rRNA at the unique residue of 7-methylguanine at position 527. A 526-nucleotide-long 5'-terminal fragment and a 1 01 5-nucleotidelong 3'-terminal fragment have been isolated and their secondary structure investigated by the circular dichroism (CD) method. MATERIALS AND METHODSThe 16s rRNA was isolated by phenol extraction from the 30s ribosomal subunits of Escherichia coli (strain MRE600).Abbreviations. Methyl-RNA, carrier RNA statistically methylated with dimethylsulfate; CD, circular dichroism.The phenylalanine and valine tRNA from yeast were kindly provided by Dr T.V. Venkstern. The RNase H preparation from E. coli was furnished by...

Function of brain amino acids as neurotransmitters or their precursors implies changes in the amino acid levels and/or metabolism in response to physiological and environmental challenges. Modelling such challenges by pregnancy and/or hypoxia, we characterize the amino acid pool in the rat cerebellum, quantifying the levels and correlations of 15 amino acids and activity of 2-oxoglutarate dehydrogenase complex (OGDHC). The parameters are systemic indicators of metabolism because OGDHC limits the flux through mitochondrial TCA cycle, where amino acids are degraded and their precursors synthesized. Compared to non-pregnant state, pregnancy increases the cerebellar content of glutamate and tryptophan, decreasing interdependence between the quantified components of amino acid metabolism. In response to hypoxia, the dependence of cerebellar amino acid pool on OGDHC and the average levels of arginine, glutamate, lysine, methionine, serine, phenylalanine, and tryptophan increase in non-pregnant rats only. This is accompanied by a higher hypoxic resistance of the non-pregnant vs. pregnant rats, pointing to adaptive significance of the hypoxia-induced changes in the cerebellar amino acid metabolism. These adaptive mechanisms are not effective in the pregnancy-changed metabolic network. Thus, the cerebellar amino acid levels and OGDHC activity provide sensitive markers of the physiology-dependent organization of metabolic network and its stress adaptations.

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