Development of mouse hepatitis virus and SARS-CoV infectious cDNA constructs.

The genomes of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) have been generated with a novel construction strategy that allows for the assembly of very large RNA and DNA genomes from a panel of contiguous cDNA subclones. Recombinant viruses generated from these methods contained the appropriate marker mutations and replicated as efficiently as wild-type virus. The MHV cloning strategy can also be used to generate recombinant viruses that contain foreign genes or mutations at virtually any given nucleotide. MHV molecular viruses were engineered to express green fluorescent protein (GFP), demonstrating the feasibility of the systematic assembly approach to create recombinant viruses expressing foreign genes. The systematic assembly approach was used to develop an infectious clone of the newly identified human coronavirus, the serve acute respiratory syndrome virus (SARS-CoV). Our cloning and assembly strategy generated an infectious clone within 2 months of identification of the causative agent of SARS, providing a critical tool to study coronavirus pathogenesis and replication. The availability of coronavirus infectious cDNAs heralds a new era in coronavirus genetics and genomic applications, especially within the replicase proteins whose functions in replication and pathogenesis are virtually unknown.

Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes.

The coronavirus replicase gene (gene 1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene 1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopic studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not in the fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of these populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.

The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis.

The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.

Expression, purification, and activity of recombinant MHV-A59 3CLpro.

The 3C-like proteinase (3CLpro) of MHV-A59 is predicted to mediate the majority of proteolytic processing events within the gene 1 polyprotein. We have overexpressed 3CLpro in E. coli as a fusion protein with maltose binding protein (MBP). The MBP-3CLpro fusion protein was purified from contaminating E. coli proteins by amylose column chromatography, and r3CLpro was cleaved from the fusion protein by factor Xa. Recombinant 3CLpro (r3CLpro) was able to cleave a polypeptide substrate containing mutated inactive 3CLpro and portions of the flanking domains. R3CLpro cleaved substrate completely within 5 minutes and the activity of r3CLpro was sensitive to inhibition by serine and cysteine proteinase inhibitors; however, it was not inhibited by EDTA, suggesting that metal ions were not critical for 3CLpro activity.

Processing of the MHV-A59 gene 1 polyprotein by the 3C-like proteinase.

The 3C-like proteinase of mouse hepatitis virus (MHV-3CLpro) is predicted to cleave at least 10 sites in the gene 1 polyprotein, resulting in processing of proteinase, polymerase and helicase proteins from the polyprotein. We have used E. coli expressed recombinant 3CLpro (r3CLpro) to define cleavage sites in carboxy-terminal region of the ORF 1a polyprotein. Polypeptides containing one or more putative 3CLpro cleavage site were translated in vitro from subcloned regions of gene 1, and the polypeptides were incubated with r3CLpro. Analysis of the cleavage products confirmed several putative cleavage sites, as well as identifying cleavage sites not previously predicted by analysis of the MHV sequence. Antibodies directed against a portion of the ORF 1a polyprotein were used to probe virus infected cells, and detected proteins that correspond to the cleavage sites used by 3CLpro in vitro. These results suggest that MHV 3CLpro cleaves at least 7 sites in the ORF 1a polyprotein, and that the specificity of 3CLpro for cleavage site dipeptides may be broader than previously predicted.

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro.

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

'Neurosis' and the personal social environment. The effects of a time-limited course of intensive day care.

The Interview Schedule for Social Interactions (ISSI) was used to assess the social environment of 65 British inner-city patients suffering from severe neurotic disorder; all patients were offered a 12-week course of intensive day treatment with an educational and psychodynamic basis. Compared with a general population in Canberra, the neurosis sufferers had lower (morbid) scores on the ISSI for the extent and quality of their social relationships. Of the 34 subjects who completed treatment and attended for the post-treatment investigation, 21 attained a PSE score below the level for 'caseness'. Twenty-five subjects who attended for follow-up at 18-24 months had improved significantly on all four of the standard ISSI measures, although they had not done so immediately after treatment. This suggests that although symptoms may improve at the time of treatment, social relationships improve only over several months.

Trial-by-fire transformation: an interview with Globe Metallurgical's Arden C. Sims. Interview by Bruce Rayner.

Globe Metallurgical Inc., a $115 million supplier of specialty metals, is best known as the first small company to win the Baldrige Award in 1988. But there is much more to this gutsy little company than total quality. During the 1980s, Globe transformed itself from a rust-belt has-been on the verge of bankruptcy into a high-technology, high-quality industry leader. Along the way, the company went private in a management-led leveraged buyout, embraced flexible work teams, adopted a high-value-added, niche marketing strategy, and took its business global. Leading the way in Globe's reinvention was Chief Executive Arden C. Sims, the slow-talking son of a West Virginian coal miner. When he joined the company in 1984, Sims had no experience in the new managerial techniques. He was a product of the old school of management: cut costs and trim operations to regain competitiveness. But he soon discovered that old-style management was not enough to battle offshore competitors, an unproductive work force, rising costs, and outdated production technology. He was forced to go looking for new ideas and practices. In a succession of learning experiences, Sims attended a seminar on total quality in 1985, paving the way for the company's quality program; he discovered the power of flexible work teams when management was forced to run the furnaces during a year-long strike; he organized an LBO, allowing him to change the work order even more dramatically; and he took the company global and into highly profitable niche markets by severing a long-standing relationship with Globe's sales and marketing representative. As a result of these and other changes, Globe leads the specialty metals industry in virtually all performance measures.

Symptoms and beliefs.

Religious belief and psychiatric symptoms are distinct and separate phenomena. They occur in different realms of experience. However, they may occur individually or together and it may be difficult to decide which is which. When a person describes his own experience as religious and another observes this as mental illness they are unlikely to be commenting on precisely the same phenomena. The first person is describing an internal experience whilst the latter is inferring from observed behaviour. It is important for psychiatrists and mental health professionals to recognize that there is a spiritual dimension for their patients (and for themselves), and for pastoral theologians to form a concept of mental illness. The approach of phenomenological psychopathology is useful in making the distinction.

The repertory grid as a measure of change and predictor of outcome in the treatment of alcoholism.

Fifty male in-patients receiving treatment for alcohol dependence were studied on admission using the repertory grid and the grids analysed using the principal component technique (INGRIDA). Three self-elements were examined: self-when-sober, self-when-drunk, and ideal self. Sober-ideal self distance was below average in 90 per cent and drunk-ideal above average in 83 per cent, suggesting unrealistically favourable perceptions of sobriety and unrealistically unfavourable perceptions of drunkenness. The percentage of total variation accounted for by the first two components (VAR1 + VAR2) was generally high, suggesting tightness of construing. The first grid did not predict treatment completion. Twenty-eight subjects completed treatment and were given a second grid test on discharge. Consistent changes in grid measures with treatment were not apparent. Forty-nine subjects were followed up six months after discharge. For the 28 who had completed treatment, tightening of construing during treatment (i.e. a higher value of VAR1 + VAR2 in the second grid than in the first) was associated with drinking at follow-up. Measures derived from the first grid alone or from the second grid alone were not associated with status at follow-up.

Age, unemployment and parasuicide in Leeds.

The relationship between unemployment and parasuicide was examined amongst males and females in the City of Leeds between 1978 and 1982. The association between unemployment and parasuicide was found to be positive and significant in middle aged men. However, there was a negative and significant relationship between unemployment and parasuicide in older men aged between 55 and 59 years. For females between the ages of 20 and 25 years there was a significant negative relationship between unemployment and parasuicide. The findings which are discussed are considered to provide evidence for the hypothesis that unemployment may exert a differing vulnerability effect on proneness to parasuicide in different age groups and between the two sexes.