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...

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wildtype strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm )1 region were measured for films of the intact and in situ trypsindegraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm )1 ), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference (ÔwetÕ minus ÔdryÕ) spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.

Replication of tobacco mosaic virus (TMV) is connected with endoplasmic reticulum (ER)-

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21 fi Thr and Asp66 fi Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N¢ (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N¢ hypersensitive response suppression is discussed.Keywords: coat protein; hypersensitive response; tobacco mosaic virus; tritium planigraphy.Systems in which defined modifications in a pathogen structure cause specific changes in a host response are highly useful in studies of molecular mechanisms of pathogenesis. One of a small number of plant virology models which satisfy this criterion is the combination of tobacco mosaic virus (TMV) with plants bearing the host resistance gene N¢. In hosts with N¢ many TMV strains and coat protein (CP) mutants induce formation of necroses (small necrotic lesions) on infected leaves [hypersensitive response (HR)], whereas wild-type (strain U1) TMV avoids the induction of plant defence necrotic response machinery and produces systemic infection (yellow-green mosaic) [1][2][3]. With the help of spontaneous and site-directed mutagenesis it has been shown that defined single substitutions in U1 CP deprive the virus of the ability to escape the N¢ plant defence system and result in necroses formation on plants of, for example, Nicotiana sylvestris N¢ [4,5].Several years ago we isolated and characterized a moderately thermosensitive TMV CP mutant, ts21-66, which induces HR in N. sylvestris [6][7][8]. This mutant contains two well-known substitutions, Ile21 fi Thr and Asp66 fi Gly, in its CP molecule, each of which induces HR in N¢ hosts [9,10]. Whereas the positions of amino acid substitutions in ts21-66 are distant in the protein sequence, they are close in the known TMV CP tertiary structure, both being located at a distance of about 70 Å from the virion (disk) axis [10][11][12]. Many other classical temperaturesensitive and N¢ necrotic mutations in TMV CP are also located in this region: residues 19-21 (TMV CP mutants Ni103, Ni118, Ni511, Ni696...

Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.