The evidence presented shows that the M2 protein of influenza A viruses exists in infected cells as a homotetramer composed of two disulfide-linked dimers held together by noncovalent interactions. The amphiphilic nature of the transmembrane alpha-helical domain is consistent with the protein forming a transmembrane channel with which amantadine, the specific anti-influenza A drug, interacts. Together these features provide a structural basis for the hypothesis that M2 has a proton translocation function capable of regulating the pH of vesicles of the trans-Golgi network, a role important in promoting the correct maturation of the hemagglutinin glycoprotein.

Amantadine hydrochloride specifically blocks the release of virus particles from H7 influenza virus infected cells. This appears to be the direct consequence of an amantadine induced change in the haemagglutinin (HA) to its low pH conformation. The effect is indirect and mediated via interaction of the drug with the M2 protein since mutants altered in this component alone are insensitive to amantadine. The tining of drug action, some 15-20 min after synthesis, and its coincidence with proteolytic cleavage indicates that the modifications to HA occur late during transport but prior to insertion into the plasma membrane. Reversal by mM concentrations of amines and 0.1 /M monensin indicates that amantadine action causes a reduction in intravesicular pH which triggers the conformational change in HA. We conclude, therefore, that the function of MN inhibited by amantadine is involved in counteracting the acidity of vesicular compartments of the exocytic pathway in infected cells and is important in protecting the structural integrity of the acid-sensitive glycoprotein.

The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA.

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In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/ MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of ␤-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy. Molecular & Cellular Proteomics 9:1829 -1848, 2010. Respiratory syncytial virus (RSV)1 belongs to the paramyxovirus group of viruses, and it is the most important respiratory virus causing lower respiratory tract infection in young children and neonates. The mature RSV particle comprises a ribonucleoparticle (RNP) core formed by the interaction between the viral genomic RNA (vRNA), the nucleocapsid (N) protein (42 kDa), the phospho (P) protein (35 kDa), and the large (L) protein (250 kDa). The RNP core is visualized by electron microscopy as a strand of repeating N protein subunits that form a herringbone-like structure of ϳ10 -20 nm in diameter (1). Although the minimal functional polymerase activity requires an association between the N, P, and L proteins and the virus genome vRNA (2-4), additional viral proteins called the M2-1 protein (22 kDa), M2-2 protein, and M protein (28 kDa) regulate the activity of the polymerase (5-8). The virus is surrounded by a lipid envelope that is formed from the host cell during the budding process in which the three virus membrane proteins are inserted. The G protein (90 kDa) mediates attachment of...