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

Contribution of integrated farm management (IFM) to Defra objectives

A farming system comprises a complex of interrelated and interacting factors. Any study of an isolated part of the system will not provide adequate understanding of the behaviour of the entire system and interactions may be equally or more important than individual components. There is therefore a requirement for the development of integrated approaches and practices to help farming systems adapt to, eliminate or reduce the negative impacts of production on the environment. This must be achieved whilst maintaining the economic viability of the farm enterprise. Our analysis has confirmed that IFM techniques generally have far more beneficial than adverse effects on current Defra policy objectives. However, there are some notable ‘conflicts’ where a technique that has a large beneficial effect in one policy area has a large negative effect in another. Carbon footprinting is used to quantify the impact of some integrated farming practices

Coolant side heat transfer with rotation: User manual for 3D-TEACH with rotation

This program solves the governing transport equations in Reynolds average form for the flow of a 3-D, steady state, viscous, heat conducting, multiple species, single phase, Newtonian fluid with combustion. The governing partial differential equations are solved in physical variables in either a Cartesian or cylindrical coordinate system. The effects of rotation on the momentum and enthalpy calculations modeled in Cartesian coordinates are examined. The flow of the fluid should be confined and subsonic with a maximum Mach number no larger than 0.5. This manual describes the operating procedures and input details for executing a 3D-TEACH computation

Labor Market Competition among Youths, White Women, and Others

We estimate substitution possibilities among a set of age-race-sex groups in the labor force. The estimates are based on cross-section data from SMSAs in 1969,and they allow us to consider how substitutable adult women are for young women or young men. The estimates are used, along with assumptions about the extent of wage rigidity and elasticities of labor supply, to simulate the direct and indirect effects of the growth of the female labor force on job opportunities for youth, assuming rigid wages for young workers, and on the wage rates of adult males, assuming these wages are flexible.

Photocatalytic production of organic compounds from CO and H2O in a simulated Martian atmosphere

[14C]CO2 and [14C]organic compounds are formed when a mixture of [14C]CO and water vapor diluted in [12C]CO2 or N2 is irradiated with ultraviolet light in the presence of soil or pulverized vycor substratum. The [14C]CO2 is recoverable from the gas phase, the [14C]organic products from the substratum. Three organic products have been tentatively identified as formaldehyde, acetaldehyde, and glycolic acid. The relative yields of [14C]CO2 and [14C]organics are wavelength- and surface-dependent. Conversion of CO to CO2 occurs primarily at wavelengths shorter than 2000 angstrom, apparently involves the photolysis of water, and is inhibited by increasing amounts of vycor substratum. Organic formation occurs over a broad spectral range below 3000 angstrom and increases with increasing amounts of substratum. It is suggested that organic synthesis results from adsorption of CO and H2O on surfaces, with excitation of one or both molecules occurring at wavelengths longer than those absorbed by the free gases. This process may occur on Mars and may have been important on the primitive earth

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