Mutations in the GDD motif of rubella virus putative RNA-dependent RNA polymerase affect virus replication.

Rubella virus (RV) nonstructural proteins are translated as a p200 polyprotein that undergoes proteolytic cleavage into p150 and p90. From conserved amino acid sequence motifs in polypeptides, p90 has been proposed to be the RV RNA-dependent RNA polymerase (RdRp). To test whether the conserved GDD motif is involved in RdRp catalytic activity, three different alanine substitutions were introduced into it. Substitution of glycine by alanine (G1966A) resulted in impaired virus infectivity. Alteration of either aspartate residue completely abolished virus replication. A fully infectious variant was isolated from the G1966A mutant. Sequencing analysis showed that the alanine residue substituted in G1966A mutant had reverted to glycine in this variant. Complementation experiments were carried out to rescue the replication-defective RNA carrying G1966A, D1967A, or D1968A mutations. The defective RNA with G1966A mutation in p90 replicated efficiently when the helper genome that supplied a wild-type p90 was provided in trans. However, the replication-defective RNA with D1967A or D1968A was not rescued by supplementation of p90 in trans. Our studies support the idea that the GDD motif is critical for RV replication and p90 function as RV RdRp.

Rubella virus RNA replication is cis-preferential and synthesis of negative- and positive-strand RNAs is regulated by the processing of nonstructural protein.

Rubella virus (RV) genome encodes nonstructural protein (NSP) in a large open reading frame at its 5' end. It is translated into p200 and further processed into p150 and p90. The NSPs are responsible for viral RNA replication, during which a full-length negative-strand RNA serves as the intermediate for the replication of positive-strand genomic RNA and the transcription of subgenomic RNA. Using complementation experiments, we demonstrated that RV negative-strand RNA is synthesized preferentially in cis while positive-strand RNAs can be synthesized both in cis and in trans but with higher efficiency in cis. During virus infection, negative-strand RNA accumulates until 10 hours postinfection (hpi) and remains nearly constant thereafter. In contrast, positive-strand RNAs (both genomic and subgenomic RNA) do not increase much before 10 hpi and accumulate rapidly thereafter. Previously we demonstrated that p200 synthesizes negative- but not positive-strand RNA, whereas cleavage products p150/p90 are required for efficient production of positive-strand RNAs. In this study, we present evidence demonstrating that a higher concentration of p150/p90 is associated with lower production of negative-strand RNA. Our data support the hypothesis that p200 is the principal replicase for negative-strand RNA, as is p150/p90 for positive-strand RNA. The switch from the synthesis of negative- to positive-strand RNA is thus regulated by NSP processing, which not only activates the efficient production of positive-strand RNA, but also disables negative-strand RNA synthesis. A mechanism for NSP translation, processing, and regulation of RV RNA synthesis is proposed.

Primary care. Lock, stock-take and barrel.

An analysis of primary care groups' investment plans revealed their main concerns were premises, IT and workforce issues. About a quarter of plans did not address out-of-hours care or community nursing. The plans lacked detail of the process guiding resource allocation. The plans showed little indication of services being organised around the priorities of the local health improvement programme.

The national health service plan: further reform of British health care?

Less than three years after initiating a series of health service reforms, the Blair government has launched another plan for the U.K. National Health Service. This article considers the origins and contents of the plan. A major investment program is designed to bring health care spending up to European averages over the next five years. In return, the government seeks to challenge the existing settlement between organized medicine and the state through tighter regulatory control, altered contractual frameworks, and a new public-private concordat. The plan does not represent a radical change in government policy but rather reaffirms existing approaches to increasing access to health services, integrating health and social care, and empowering users. Notwithstanding arrangements to increase the autonomy of health service organizations, the plan increases central control through a range of new bodies and regulatory frameworks. It represents an incremental adjustment of the existing tax-funded system. Should this reinvigoration of the state monopoly fail, alternative sources of funding will no doubt have to be reconsidered.

Cross-reactive rubella virus and glutamic acid decarboxylase (65 and 67) protein determinants recognised by T cells of patients with type I diabetes mellitus.

AIMS/HYPOTHESIS: To examine the cross-reaction between viral and beta-cell protein determinants and to further understand the potential role of this mechanism in Type I (insuline-dependent) diabetes mellitus. METHODS: Immune responses to a panel of 28 viral and beta-cell protein peptides representing selected sequences of rubella virus (RV), Coxsackie virus, human 38 KDa31G and glutamic acid decarboxylase (GAD 65 and 67) proteins in proliferation or cytotoxicity assays have been studied using uncloned and cloned T-cell cohorts from a group of 60 Type I diabetic patients. RESULTS: Peptide GAD65(252-266) induced the responses of patients with recent onset diabetes in proliferation assays at the highest frequency (77%), whereas GAD67(212-226) stimulated the cellular responses at the highest rate (61%) in patients with late-onset diabetes. RVE1(157-176) was recognised by all groups of patients at the highest frequency and the largest amplitude among the viral peptides tested. T-cell clones specific to GAD65(252-266), GAD65(274-286) or GAD67(212-226) were tested in cytotoxicity assays for their responses to rubella virus peptides. Each of these T-cell clones cross-reacted with two to four rubella virus peptides, including RVE1(157-176) and RVE2(87-107). Analysis of the sequences of cross-reactive viral and glutamic acid decarboxylase antigens showed that these epitopes shared similar peptide binding motifs to HLA DR3/DR4. There is a statistically significant correlation between the response amplitude of patient's peripheral blood mononuclear cells to RVE1(157-176), RVE2(87-107) and GAD65(274-286) in patients with recent onset diabetes, and to RVE1(157-176) and GAD67(212-226) in patients with late onset diabetes. CONCLUSION/INTERPRETATION: Cross-reactive glutamic acid decarboxylase and rubella virus determinants identified by T-cell clones were also recognised at high frequencies by general T-cell populations of Type I diabetic patients.

Mutations in the E1 hydrophobic domain of rubella virus impair virus infectivity but not virus assembly.

Rubella virus (RV) virions contain three structural proteins, a capsid protein that interacts with viral genomic RNA to form a nucleocapsid and two membrane glycoproteins, E2 and E1. We found that substitution of either an aspartic acid residue at Gly93 (G93D) or a glycine residue at Pro104 (P104G) in the internal hydrophobic domain of E1 affected virus infectivity but not virus assembly. Viruses carrying G93D and P104G mutations had impaired infectivity, reduced 1,000-fold and 10-fold, respectively. A revertant was isolated from the G93D mutant. Sequencing analysis showed that the substituted aspartic acid residue in G93D mutant had reverted to the original glycine residue, suggesting the involvement of Gly93 in membrane fusion during viral entry.

Mutational analysis of the rubella virus nonstructural polyprotein and its cleavage products in virus replication and RNA synthesis.

Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.

Rubella virus nonstructural protein protease domains involved in trans- and cis-cleavage activities.

Rubella virus (RV) genomic RNA contains two large open reading frames (ORFs): a 5'-proximal ORF encoding nonstructural proteins (NSPs) that function primarily in viral RNA replication and a 3'-proximal ORF encoding the viral structural proteins. Proteolytic processing of the RV NSP ORF translation product p200 is essential for viral replication. Processing of p200 to two mature products (p150 and p90) in the order NH(2)-p150-p90-COOH is carried out by an RV-encoded protease residing in the C-terminal region of p150. The RV nonstructural protease (NS-pro) belongs to a viral papain-like protease family that cleaves the polyprotein both in trans and in cis. A conserved X domain of unknown function was found from previous sequence analysis to be associated with NS-pro. To define the domains responsible for cis- and trans-cleavage activities and the function of the X domain in terms of protease activity, an in vitro translation system was employed. We demonstrated that the NSP region from residue 920 to 1296 is necessary for trans-cleavage activity. The domain from residue 920 to 1020 is not required for cis-cleavage activity. The X domain located between residues 834 and 940, outside the regions responsible for both cis- and trans-cleavage activities of NS-pro, was found to be important for NS-pro trans-cleavage activity but not for cis-cleavage activity. Analysis of sequence homology and secondary structure of the RV NS-pro catalytic region reveals a folding structure similar to that of papain.

A single-amino-acid substitution of a tyrosine residue in the rubella virus E1 cytoplasmic domain blocks virus release.

Rubella virus particles, consisting of a nucleocapsid surrounded by a lipid envelope in which two virus-encoded glycoproteins E1 and E2 are embedded, assemble on intracellular membranes and are secreted from cells, possibly via the cellular secretory pathway. We have recently demonstrated that the cytoplasmic domain of E1 (residues 469 to 481, KCLYYLRGAIAPR) is required for virus release. Alteration of cysteine 470 to alanine did not affect virus release, whereas mutation of leucine 471 to alanine reduced virus production by 90%. In the present study, substitutions of remaining amino acids in the E1 cytoplasmic domain were made in order to investigate the role of each amino acid in regulating rubella virus release. Generated mutants were analyzed in the context of infectious full-length cDNA clone and virus-like particles using combined genetic, biochemical, and electron microscopic approaches. Substitution of a single residue of tyrosine 472 to alanine or tyrosine 473 to serine resulted in a block in virus release without affecting protein transport and virus budding into the lumen of the Golgi complexes. Infectious RNA transcripts bearing these mutations were incapable of forming plaques. Mutants with substitutions at the amino-terminal region (leucine 474, arginine 475, and glycine 476) in the E1 cytoplasmic domain had reduced virus release and small-plaque phenotype, while mutants with substitutions at the carboxy-terminal region (alanine 477, isoleucine 478, alanine 479, proline 480, and arginine 481) had only marginal defects in virus release. Plaque-forming revertants could be isolated from mutants Y472A and Y473S. Sequencing analysis revealed that the substituted serine residue in mutant Y473S reverted to the original tyrosine residue, whereas the substituted alanine residue in mutant Y472A was retained. These results indicate that the E1 cytoplasmic domain modulates virus release in a sequence-dependent manner and that the tyrosine residues are critical for this function. We postulate that residues YYLRG constitute a domain in the E1 tail that may interact with other proteins and this interaction is involved in regulating virus release.

HImPs (health improvement programmes). Trusting to luck.

An analysis of 36 health authorities' health improvement programmes found they reflected national priorities well. The average number of priorities was 13, with coronary heart disease/stroke, mental health and cancer being the most common. One plan set no priorities. Most programmes included some consultation with the public. Only seven HImPs included measurable targets. Many plans were written in a way which would make them difficult for the public to understand. Only 13 provided a glossary.

Doctors' pay. What seems to be the trouble?

The long-standing method of paying GPs through a complex system of fees and allowances is under review. The proposed expansion of personal medical services pilots and an increase in the number of salaried GPs could destabilise the current system and lead to perverse outcomes. The introduction of a weighted capitation system allocating resources to health authorities and primary care trusts according to need might ensure equitable distribution of funds.

Health-related quality of life in type 2 diabetes (TARDIS-2).

BACKGROUND: Type 2 diabetes is now recognized as a major public health concern but its burden on society is under-researched. METHODS: T(2)ARDIS was a postal survey of 1578 people with type 2 diabetes across four UK centres, incorporating measures of resource use, treatment satisfaction and health-related quality of life (HrQoL). The findings included data on the EQ-5D that enabled the HrQoL burden of the disease to be established by comparison with equivalent data for the general population and the diabetic population as a whole from the 1996 Health Survey of England. RESULTS: The results indicate a significant deficit experienced by people with type 2 diabetes vs. their age group peers in the general population. The proportion of T(2)ARDIS respondents reporting problems increases in relation to the presence of complications, and microvascular complications appear to have more impact than macrovascular complications. CONCLUSIONS: This confirms the need for treatment policies to focus on reducing the risk of such complications and hence improve patients' HrQoL.