Kinetics of transmission, infectivity, and genome stability of two novel mouse norovirus isolates in breeding mice.

Murine noroviruses are a recently discovered group of viruses found within mouse research colonies in many animal facilities worldwide. In this study, we used 2 novel mouse norovirus (MNV) wildtype isolates to examine the kinetics of transmission and tissue distribution in breeding units of NOD.CB17-Prkdc(scid)/J and backcrossed NOD.CB17-Prkdc(scid)/J x NOD/ShiLtJ (N1) mice. Viral shedding in feces and dissemination to tissues of infected offspring mice were monitored by RT-PCR over a 6-wk period postpartum. Histologic sections of tissues from mice exposed to MNV were examined for lesions and their sera monitored for the presence of antibodies to MNV. Viruses shed in feces of parental and offspring mice were compared for sequence homology of the Orf2 gene. Studies showed that the wildtype viruses MNV5 and MNV6 behaved differently in terms of the kinetics of transmission and distribution to tissues of offspring mice. For MNV5, virus transmission from parents to offspring was not seen before 3 wk after birth, and neither isolate was transmitted between cages of infected and control mice. Susceptibility to infection was statistically different between the 2 mouse strains used in the study. Both immunodeficient NOD.CB17-Prkdc(scid)/J mice and NOD. CB17-Prkdc(scid)/J x NOD/ShiLtJ offspring capable of mounting an immune response shed virus in their feces throughout the 6-wk study period, but no gross or histologic lesions were present in infected tissues. Progeny viruses isolated from the feces of infected offspring showed numerous mutations in the Orf2 gene for MNV5 but not MNV6. These results confirm previous studies demonstrating that the biology of MNV in mice varies substantially with each virus isolate and mouse strain infected.

Major histocompatibility complex-linked diabetes susceptibility in NOD/Lt mice: subcongenic analysis localizes a component of Idd16 at the H2-D end of the diabetogenic H2(g7) complex.

The diabetogenic major histocompatibility complex (MHC) (H2(g7)) of NOD mice comprises contributions from several class II loci collectively designated as Idd1. Introduction of the H2(gx) haplotype from the related but diabetes-resistant cataract Shionogi (CTS) strain demonstrated an additional MHC-linked locus designated Idd16. The NOD-related alloxan resistant (ALR)/Lt strain is also characterized by the H2(gx) haplotype, which does not differ from H2(g7) from the class I H2-K(d) gene distally through the class II and into the class III region. Polymorphisms distal to the heat shock protein 70 locus (Hspa1b) include a rare H2-D(dx) rather than the H2(g7) encoded D(b) allele. Two differential-length NOD.ALR-H2(gx) congenic stocks (D.R1 and D.R2), both containing H2-D(dx), significantly suppressed diabetogenesis. This protection was lost when ALR alleles between the class III region and H2-D were removed in a shorter interval congenic (D.R3). Because no differences were observed in the ALR-derived interval extending 0.41 mB proximal to H2-K in any of these congenic stocks, a component of what was originally designated "Idd16" was sited to an interval shorter than 7.33 mB, distinguishing D.R2 from D.R3. Evidence supporting the candidacy of the ALR/CTS-shared H2-D(dx) MHC class I variant present in both diabetes-resistant stocks, but not the susceptible stock, is discussed.

EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton.

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.

Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c.

Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.

The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides.

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40(phox) PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47(phox) PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47(phox) PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47(phox) PX domain to the plasma membrane does involve 3'-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47(phox) PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40(phox) and p47(phox) PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity.