Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis

Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344-->Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala-->Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus

The HCV Core Protein Acts as a Positive Regulator of Fas-Mediated Apoptosis in a Human Lymphoblastoid T Cell Line

AbstractHepatitis C virus (HCV) is a major human pathogen causing mild to severe liver disease worldwide and is remarkably efficient at establishing persistent infections. Previously, we have shown that the core protein has an immunomodulatory function including the suppression of T lymphocyte responses to viral infection. To investigate the underlying mechanism for the role of core protein in immune modulation, we examined the effect of core on the sensitivity of the human T cell line, Jurkat, to Fas-mediated apoptosis. The transient and stable expression of core protein in Jurkat cells increased the sensitivity of cells to Fas-mediated apoptosis when compared to control cells expressing vector DNA alone. In addition, we demonstrated that the core protein binds to the cytoplasmic domain of Fas which may enhance the downstream signaling event of Fas-mediated apoptosis. The expression of core protein did not alter the cell surface expression of Fas, indicating that the increased sensitivity of core-expressing cells to Fas ligand was not due to upregulation of Fas. Furthermore, we observed the augmentation of caspase-3 activity in core-expressing cells. These results suggest that the core protein may promote the apoptosis of immune cells during HCV infection via the Fas signaling pathway, thus facilitating HCV persistence

Radiative Corrections to Fixed Target Moller Scattering Including Hard Bremsstrahlung Effects

We present a calculation of the complete $O(\alpha)$ electroweak radiative corrections to the Moller scattering process e^-e^- -> e^-e^-, including hard bremsstrahlung contributions. We study the effects of these corrections on both the total cross section and polarization asymmetry measured in low energy fixed target experiments. Numerical results are presented for the experimental cuts relevant for E-158, a fixed target e^-e^- experiment being performed at SLAC; the effect of hard bremsstrahlung is to shift the measured polarization asymmetry by approximately +4%. We briefly discuss the remaining theoretical uncertainty in the prediction for the low energy Moller scattering polarization asymmetry.Comment: 22 pgs; minor clarifications added and typos fixe

The Higgs sector of the complex MSSM at two-loop order: QCD contributions

Results are presented for the leading two-loop contributions of O(alpha_t alpha_s) to the masses and mixing effects in the Higgs sector of the MSSM with complex parameters. They are obtained in the Feynman-diagrammatic approach using on-shell renormalization. The full dependence on all complex phases is taken into account. The renormalization of the appropriate contributions of the Higgs-boson sector and the scalar top and bottom sector is discussed. Our numerical analysis for the lightest MSSM Higgs-boson mass is based on the new two-loop corrections, supplemented by the full one-loop result. The corrections induced by the phase variation in the scalar top sector are enhanced by the two-loop contributions. We find that the corresponding shift in M_h1 can amount to 5 GeV.Comment: 15 pages, 7 figures; minor changes; published versio

Higgs Signal for h to aa at Hadron Colliders

We assess the prospect of observing a neutral Higgs boson at hadron colliders in its decay to two spin-zero states, a, for a Higgs mass of 90-130 GeV, when produced in association with a W or Z boson. Such a decay is allowed in extensions of the MSSM with CP-violating interactions and in the NMSSM, and can dominate Higgs boson final states, thereby evading the LEP constraints on standard Higgs boson production. The light spin-zero state decays primarily via a to bb and tau+tau-, so this signal channel retains features distinct from the main backgrounds. Our study shows that at the Tevatron, there may be potential to observe a few events in the bb tau+tau- or bbbb channels with relatively small background, although this observation would be statistically limited. At the LHC, the background problem is more severe, but with cross sections and integrated luminosities orders of magnitude larger than at the Tevatron, the observation of a Higgs boson in this decay mode would be possible. The channel h to aa to bbbb would provide a large statistical significance, with a signal-to-background ratio on the order of 1:2. In these searches, the main challenge would be to retain the adequate tagging efficiency of b's and tau's in the low p_T region.Comment: Version to be published in JHEP. 20 pages, 5 figure

CP-odd A^0 production at e^+e^- colliders in MSSM with CP violating phases

We study the production of a heavy CP-odd $A^0$ boson in association with a photon $e^+e^-\to A^0\gamma$ and a Z boson $e^+e^-\to A^0 Z$ as well as the single production of $A^0$ via $e^+e^- \to \nu_e\bar{\nu}_e A^0$ in the MSSM with CP violating phases. In the case of $e^+e^-\to A^0\gamma / A^0 Z$, we show that the squark contribution, which vanishes in the MSSM with real parameters, turns out to be sizeable in presence of CP violating phases in the soft SUSY parameters. For $e^+e^- \to \nu_e \bar{\nu}_e A^0$ in both the 2HDM and MSSM with real parameters, the cross section does not reach observable rates at a NLC. It is found that with a large CP violating phase for $A_t$, cross sections of the order 0.1 fb are attainable for all the processes $e^+e^-\to A^0\gamma$, $e^+e^-\to A^0 Z$ and $e^+e^- \to \nu_e \bar{\nu}_e A^0$.Comment: 12 pages, latex, 7 eps figures. One new figure, new discussion arroud it. Version to appear in Phys. Rev.

Testing SUSY

If SUSY provides a solution to the hierarchy problem then supersymmetric states should not be too heavy. This requirement is quantified by a fine tuning measure that provides a quantitative test of SUSY as a solution to the hierarchy problem. The measure is useful in correlating the impact of the various experimental measurements relevant to the search for supersymmetry and also in identifying the most sensitive measurements for testing SUSY. In this paper we apply the measure to the CMSSM, computing it to two-loop order and taking account of current experimental limits and the constraint on dark matter abundance. Using this we determine the present limits on the CMSSM parameter space and identify the measurements at the LHC that are most significant in covering the remaining parameter space. Without imposing the LEP Higgs mass bound we show that the smallest fine tuning (1:13) consistent with a relic density within the WMAP bound corresponds to a Higgs mass of 114$\pm$2 GeV. Fine tuning rises rapidly for heavier Higgs.Comment: 12 pages, 7 figures; references added, figures updated for extended parameter space sca

Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets