Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant

The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10(-6) relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.     

Retroviruses have differing requirements for proteasome function in the budding process

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined. We found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane assembly, PPPY L domain) and Mason-Pfizer monkey virus (cytoplasmic assembly, PPPY L domain), similar to the reduction observed for HIV-1. Thus, proteasome inhibitors can affect the budding of a virus that assembles within the cytoplasm. However, the budding of mouse mammary tumor virus (MMTV; cytoplasmic assembly, unknown L domain) was unaffected by proteasome inhibitors, similar to the proteasome-independent budding previously observed for equine infectious anemia virus (plasma membrane assembly, YPDL L domain). Examination of MMTV particles detected Gag-ubiquitin conjugates, demonstrating that an interaction with the ubiquitination system occurs during assembly, as previously found for other retroviruses. For all of the cell lines tested, the inhibitor treatment effectively inactivated proteasomes, as measured by the accumulation of polyubiquitinated proteins. The ubiquitination system was also inhibited, as evidenced by the loss of monoubiquitinated histones from treated cells. These results and those from other viruses show that proteasome inhibitors reduce the budding of viruses that utilize either a PPPY- or PTAP-based L domain and that this effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion.     

Partial AUC estimation and regression

Accurate diagnosis of disease is a critical part of health care. New diagnostic and screening tests must be evaluated based on their abilities to discriminate diseased from nondiseased states. The partial area under the receiver operating characteristic (ROC) curve is a measure of diagnostic test accuracy. We present an interpretation of the partial area under the curve (AUC), which gives rise to a nonparametric estimator. This estimator is more robust than existing estimators, which make parametric assumptions. We show that the robustness is gained with only a moderate loss in efficiency. We describe a regression modeling framework for making inference about covariate effects on the partial AUC. Such models can refine knowledge about test accuracy. Model parameters can be estimated using binary regression methods. We use the regression framework to compare two prostate-specific antigen biomarkers and to evaluate the dependence of biomarker accuracy on the time prior to clinical diagnosis of prostate cancer.     

Insulin secretory deficiency and glucose intolerance in Rab3A null mice

Insulin secretory dysfunction of the pancreatic beta-cell in type-2 diabetes is thought to be due to defective nutrient sensing and/or deficiencies in the mechanism of insulin exocytosis. Previous studies have indicated that the GTP-binding protein, Rab3A, plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A(-/-) mice develop fasting hyperglycemia and upon a glucose challenge show significant glucose intolerance coupled to ablated first-phase insulin release and consequential insufficient insulin secretion in vivo, without insulin resistance. The in vivo insulin secretory response to arginine was similar in Rab3A(-/-) mice as Rab3A(+/+) control animals, indicating a phenotype reminiscent of insulin secretory dysfunction found in type-2 diabetes. However, when a second arginine dose was given 10 min after, there was a negligible insulin secretory response in Rab3A(-/-) mice, compared with that in Rab3A(+/+) animals, that was markedly increased above that to the first arginine stimulus. There was no difference in beta-cell mass or insulin production between Rab3A(-/-) and Rab3A(+/+) mice. However, in isolated islets, secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or fatty acid) was approximately 60-70% lower in Rab3A(-/-) islets compared with Rab3A(+/+) controls. Nonetheless, there was a similar rate of glucose oxidation and glucose-induced rise in cytosolic [Ca(2+)](i) flux between Rab3A(-/-) and Rab3A(+/+) islet beta-cells, indicating the mechanistic role of Rab3A lies downstream of generating secondary signals that trigger insulin release, at the level of secretory granule transport and/or exocytosis. Thus, Rab3A plays an important in vivo role facilitating the efficiency of insulin exocytosis, most likely at the level of replenishing the ready releasable pool of beta-granules. Also, this study indicates, for the first time, that the in vivo insulin secretory dysfunction found in type-2 diabetes can lie solely at the level of defective insulin exocytosis.     

Zonal changes in the three-dimensional morphology of the chondron under compression: the relationship among cellular, pericellular, and extracellular deformation in articular cartilage

The pericellular matrix (PCM) is a narrow region of tissue that completely surrounds chondrocytes in articular cartilage. Previous theoretical models of the "chondron" (the PCM with enclosed cells) suggest that the structure and properties of the PCM may significantly influence the mechanical environment of the chondrocyte. The objective of this study was to quantify changes in the three-dimensional (3D) morphology of the chondron in situ at different magnitudes of compression applied to the cartilage extracellular matrix. Fluorescence immunolabeling for type-VI collagen was used to identify the boundaries of the cell and PCM, and confocal microscopy was used to form 3D images of chondrons from superficial, middle, and deep zone cartilage in explants compressed to 0%, 10%, 30%, and 50% surface-to-surface strain. Lagrangian tissue strain, determined locally using texture correlation, was highly inhomogeneous and revealed depth-dependent compressive stiffness and Poisson's ratio of the extracellular matrix. Compression significantly decreased cell and chondron height and volume, depending on the zone and magnitude of compression. In the superficial zone, cellular-level strains were always lower than tissue-level strains. In the middle and deep zones, however, tissue strains below 25% were amplified at the cellular level, while tissue strains above 25% were decreased at the cellular level. These findings are consistent with previous theoretical models of the chondron, suggesting that the PCM can serve as either a protective layer for the chondrocyte or a transducer that amplifies strain, such that cellular-level strains are more homogenous throughout the tissue depth despite large inhomogeneities in local ECM strains.