Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins

Four complementation groups of temperature-sensitive (ts) mutants of Sindbis virus that fail to make RNA at the nonpermissive temperature are known, and we have previously shown that group F mutants have defects in nsP4. Here we map representatives of groups A, B, and G. Restriction fragments from a full-length clone of Sindbis virus, Toto1101, were replaced with the corresponding fragments from the various mutants. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious RNA transcripts, and the virus recovered was tested for temperature sensitivity. After each lesion was mapped to a specific region, cDNA clones of both mutants and revertants were sequenced in order to determine the precise nucleotide change responsible for each mutation. Synthesis of viral RNA and complementation by rescued mutants were also examined in order to study the phenotype of each mutation in a uniform genetic background. The single mutant of group B, ts11, had a defect in nsP1 (Ala-348 to Thr). All of the group A and group G mutants examined had lesions in nsP2 (Ala-517 to Thr in ts17, Cys-304 to Tyr in ts21, and Gly-736 to Ser in ts24 for three group A mutants, and Phe-509 to Leu in ts18 and Asp-522 to Asn in ts7 for two group G mutants). In addition, ts7 had a change in nsP3 (Phe-312 to Ser) which also rendered the virus temperature sensitive and RNA-. Thus, changes in any of the four nonstructural proteins can lead to failure to synthesize RNA at a nonpermissive temperature, indicating that all four are involved in RNA synthesis. From the results presented here and from previous results, several of the activities of the nonstructural proteins can be deduced. It appears that nsP1 may be involved in the initiation of minus-strand RNA synthesis. nsP2 appears to be involved in the initiation of 26S RNA synthesis, and in addition it appears to be a protease that cleaves the nonstructural polyprotein precursors. It may also be involved in shutoff of minus-strand RNA synthesis. nsP4 appears to function as the viral polymerase or elongation factor. The functions of nsP3 are as yet unresolved

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

Condensation cyclization reactions of electron deficient aromatics. 4: Tricyclic nitropropene nitronates from the reaction of phloroglucinol and cycloalkanones with sym-trinitrobenzene

Interesting similarities have been shown between the reactions of sym-trinitrobenzene with cycloalkanones, and with phloroglucinol. Previously unsuspected common intermediates have been shown to intervene. The structurally similar products in each case are tricyclic nitropropene nitronates. Protonation of these yields the corresponding nitronic acids in certain instances

Dynamics of few-body states in a medium

Strongly interacting matter such as nuclear or quark matter leads to few-body bound states and correlations of the constituents. As a consequence quantum chromodynamics has a rich phase structure with spontaneous symmetry breaking, superconductivity, condensates of different kinds. All this appears in many astrophysical scenarios. Among them is the formation of hadrns during the early stage of the Universe, the structure of a neutron star, the formation of nuclei during a supernova explosion. Some of these extreme conditions can be simulated in heavy ion colliders. To treat such a hot and dense system we use the Green function formalism of many-body theory. It turns out that a systematic Dyson expansion of the Green functions leads to modified few-body equations that are capable to describe phase transitions, condensates, cluster formation and more. These equations include self energy corrections and Pauli blocking. We apply this method to nonrelativistic and relativistic matter. The latter one is treated on the light front. Because of the medium and the inevitable truncation of space, the few-body dynamics and states depend on the thermodynamic parameters of the medium.Comment: 3 pages, 2 figures, talk presented at the 19th European Conference on Few-Body System

Well-posedness, energy and charge conservation for nonlinear wave equations in discrete space-time

We consider the problem of discretization for the U(1)-invariant nonlinear wave equations in any dimension. We show that the classical finite-difference scheme used by Strauss and Vazquez \cite{MR0503140} conserves the positive-definite discrete analog of the energy if the grid ratio is $dt/dx\le 1/\sqrt{n}$, where $dt$ and $dx$ are the mesh sizes of the time and space variables and $n$ is the spatial dimension. We also show that if the grid ratio is $dt/dx=1/\sqrt{n}$, then there is the discrete analog of the charge which is conserved. We prove the existence and uniqueness of solutions to the discrete Cauchy problem. We use the energy conservation to obtain the a priori bounds for finite energy solutions, thus showing that the Strauss -- Vazquez finite-difference scheme for the nonlinear Klein-Gordon equation with positive nonlinear term in the Hamiltonian is conditionally stable.Comment: 10 page

Wavelength Dependent PSFs and their impact on Weak Lensing Measurements

We measure and model the wavelength dependence of the PSF in the Hyper Suprime-Cam (HSC) Subaru Strategic Program (SSP) survey. We find that PSF chromaticity is present in that redder stars appear smaller than bluer stars in the $g, r,$ and $i$-bands at the 1-2 per cent level and in the $z$ and $y$-bands at the 0.1-0.2 per cent level. From the color dependence of the PSF, we fit a model between the monochromatic PSF trace radius, $R$, and wavelength of the form $R(\lambda)\propto \lambda^{b}$. We find values of $b$ between -0.2 and -0.5, depending on the epoch and filter. This is consistent with the expectations of a turbulent atmosphere with an outer scale length of $\sim 10-100$ m, indicating that the atmosphere is dominating the chromaticity. We find evidence in the best seeing data that the optical system and detector also contribute some wavelength dependence. Meyers and Burchat (2015) showed that $b$ must be measured to an accuracy of $\sim 0.02$ not to dominate the systematic error budget of the Large Synoptic Survey Telescope (LSST) weak lensing (WL) survey. Using simple image simulations, we find that $b$ can be inferred with this accuracy in the $r$ and $i$-bands for all positions in the LSST field of view, assuming a stellar density of 1 star arcmin$^{-2}$ and that the optical PSF can be accurately modeled. Therefore, it is possible to correct for most, if not all, of the bias that the wavelength-dependent PSF will introduce into an LSST-like WL survey.Comment: 14 pages, 10 figures. Submitted to MNRAS. Comments welcom

High resolution Ge/Li/ spectrometer reduces rate-dependent distortions at high counting rates

Modified spectrometer system with a low-noise preamplifier reduces rate-dependent distortions at high counting rates, 25,000 counts per second. Pole-zero cancellation minimizes pulse undershoots due to multiple time constants, baseline restoration improves resolution and prevents spectral shifts