15N NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions.
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Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH. (+info)
Up-regulation of beta-chemokines and down-modulation of CCR5 co-receptors inhibit simian immunodeficiency virus transmission in non-human primates.
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A non-cognate mechanism of protection against human immunodeficiency virus-1 (HIV-1) infection involves up-regulation of beta-chemokines, which bind and may down-modulate the CCR5 co-receptors, thereby preventing transmission of M-tropic HIV-1. The objective of this investigation was to evaluate this mechanism in vivo in non-human primates. Rhesus macaques were immunized by a modified targeted lymph nodes (TLN) route with recombinant simian immunodeficiency virus (SIV) glycoprotein 120 (gp120) and p27 in alum, and adsorbed recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) with either interleukin (IL)-2 or IL-4. Immunization induced significant increases in the concentrations of CD8 cell-derived suppressor factor (CD8-SF), regulated on activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, and down-modulation of the proportion of cells expressing CCR5 (r = 0.737, P<0.05). The macaques were then challenged with SIVmac 220 by the rectal mucosal route. The plasma SIVmac RNA showed a significant inverse correlation with the CD8-SF or the concentration of the three beta-chemokines (r = 0.831 and 0.824, P<0.01), but a positive correlation between the proportion of CCR5+ cells and SIVmac RNA (r = 0.613, P = 0.05). These results demonstrate for the first time in vivo that immunization up-regulates beta-chemokines, which may down-modulate CCR5 co-receptors, and both functions are significantly correlated with the viral load. Hence, the non-cognate beta-chemokine-CCR5 mechanism should be considered as complementary to specific immunity in vaccination against HIV. (+info)
Oligomerization, secretion, and biological function of an anchor-free parainfluenza virus type 2 (PI2) fusion protein.
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A number of studies indicate that the transmembrane domain, the cytoplasmic domain, or both regions of viral surface glycoproteins are involved in quaternary structure formation. In this report, the transmembrane domain and cytoplasmic tail coding sequence of the fusion (F) glycoprotein gene from parainfluenza type 2 virus was truncated by PCR and the resulting gene (PI2F') was expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Pulse-chase experiments indicated that the anchor-free PI2F' was expressed and processed into F(1) and F(2) subunits. Both the processed and the unprocessed anchor-free PI2F' proteins were found to be efficiently secreted into the culture medium. Examination of the oligomeric form of the anchor-free PI2F' by chemical cross-linking demonstrated that it assembles posttranslationally into dimers and trimers with a pattern similar to that of the wild-type PI2F protein. In an effort to better understand the biological properties of the truncated form of PI2F', we anchored PI2F' by a glycosyl-phosphatidylinositol (GPI) linkage. The GPI-anchored PI2F' protein, when coexpressed with PI2HN, did not induce cell fusion seen as syncytium formation, but was found to initiate lipid mixing (hemifusion) as observed by transfer of R-18 rhodamine from red blood cells to the GPI-PI2F'/PI2HN cotransfected cells. The results therefore indicate that the extracellular domain of the PI2 fusion protein contains not only the structural information sufficient to direct assembly into higher oligomers, but also is competent to initiate membrane fusion, suggesting that the anchor-free PI2F' may be useful for further structural studies. (+info)
The bovine papillomavirus E2 transactivator is stimulated by the E1 initiator through the E2 activation domain.
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Bovine papillomavirus type 1 (BPV-1) encodes two regulatory proteins, E1 and E2, that are essential for viral replication and transcription. E1, an ATP-dependent helicase, binds to the viral ori and is essential for viral replication, while the viral transcriptional activator, E2, plays cis-dominant roles in both viral replication and transcription. At low reporter concentrations, E1 stimulates E2 enhancer function, while at high reporter concentrations, repression results. An analysis of cis requirements revealed that neither replication nor specific E1-binding sites are required for the initiators' effect on E2 transactivator function. Though no dependence on E1-binding sites was found, analysis of E1 DNA binding and ATPase mutants revealed that both domains are required for E1 modulation of E2. Through the use of E2 fusion-gene constructs we showed that a heterologous DNA-binding domain could be substituted for the E2 DNA-binding domain and this recombinant protein remained responsive to E1. Furthermore, E1 could rescue activation domain mutants of E2 defective for transactivation. These data suggest that E1 stimulation of E2 involves interactions between E1 and the E2 activation domain on DNA. We speculate that E1 may allosterically interact with the E2 activation domain, perhaps stabilizing a particular structure, which increases the enhancer function of E2. (+info)
Structure-function analysis of the Sendai virus F and HN cytoplasmic domain: different role for the two proteins in the production of virus particle.
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The role of the cytoplasmic domain (cytd) of the Sendai virus HN and F glycoproteins in the process of virus assembly and budding are evaluated. Recombinant Sendai virus (rSeV) mutants are generated carrying modifications in the cytd of each of the glycoprotein separately. The modifications include increasing truncations and/or amino acid sequence substitutions. Following steady-state (35)[S]methionine/cysteine labeling of the infected cells, the virus particle production is estimated. The radioactive virions in the cell supernatants are measured relative to the extent of the infection, assessed by the intracellular N protein signal. For both the F and HN cytd truncation mutants, the largest cytd deletions lead to a 20- to 50-fold reduction in virion production. This reduction cannot be explained by a reduction of the cell surface expression of the glycoproteins. For the F protein mutants, the virions produced in reduced amount always exhibit a normal F protein composition. It is then concluded that a threshold level of F is required for SeV assembly and budding. The rate or the efficiency with which this threshold is reached up appears to depend on the nature of the F cytd. A minimal cytd length is required as well as a specific sequence. The analysis of HN protein mutants brings to light an apparent paradox. The larger cytd truncations result in significant reduction of virion production. On the other hand, a normal virion production can take place with an underrepresentation of or, even, an undetectable HN in the particles. The HN uptake in virion is confirmed to depend on the previously proposed cytd SYWST signal (T. Takimoto, T. Bousse, E. C. Coronel, R. A Scroggs, and A. Portner. 1998. J. Virol. 72, 9747-9754.). (+info)
Cleavage of hepatitis C virus nonstructural protein 5A by a caspase-like protease(s) in mammalian cells.
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Nonstructural 5A protein (NS5A) of hepatitis C virus (HCV) is localized in the cytoplasm although it has a functional nuclear localization signal. To clarify the determinant of NS5A cytoplasmic localization, various N- or C-terminal deleted NS5A mutants were generated and their subcellular localization was analyzed in cell lines after transient expression. N-terminal deleted forms of NS5A were localized in the nucleus, and the sequence of the N-terminal 27 amino acids of NS5A had sufficient function to cause retention of a normally nuclear protein in the cytoplasm. These observations indicated that cytoplasmic localization of NS5A is determined primarily by the N-terminal region of the molecule. In addition, we found proteolytic processing of NS5A in transiently expressing cells. In these cells, cleavage occurred at a few sites located in the N- and C-terminal regions of NS5A. This cleavage in cells was enhanced by apoptotic stimuli and was inhibited by the caspase inhibitor Z-VAD-FMK, suggesting that a caspase-like protease(s) contributes to the cleavages of NS5A. Based on the results of mutational analysis of NS5A, we predicted one cleaved form, which had lost both the N- and the C-terminal portions of NS5A, to be composed of amino acid residues 155 to 389. Peptide containing the same amino acid sequence as this cleaved product was localized in the nucleus. Furthermore, we found that a fusion protein consisting of Gal4 DNA-binding domain fused with this cleaved form showed transcriptional activity only when the alpha-catalytic subunit of protein kinase A (PKA) was coproduced, suggesting that the transcriptional activity of this product was regulated by PKA. These results suggested that the cleavage product of NS5A by a caspase-like protease(s) plays a role in transcriptional regulation of the host cell gene(s) in HCV-infected cells. (+info)
A single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion.
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The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F(1) portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation. (+info)
Temperature dependence of fusion by sendai virus.
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Studies of the temperature dependence of liposome fusion by Sendai virus indicate that fusion occurs maximally at 55 degrees C. The fusion capacity of the virus is also inactivated maximally by preincubation at this temperature and, under the same conditions, the F glycoprotein becomes resistant to proteolysis. By analogy with the activation at elevated temperatures of fusion by influenza virus our results suggest that temperature is also a variable in the activation of fusion by paramyxoviruses and possibly in the activation of other members of the group of viruses that includes myxo-, paramyxo-, retro-, and filoviruses, which all contain cleaved, trimeric fusion glycoproteins. (+info)