Apoptosis-defective φ mutants display attenuated virulence.

<p>ND4 Swiss Webster mice were inoculated intracranially with 50 PFU of rsT3D or the indicated φ mutant. (A) Mice (18–20) were monitored for survival over 21 days. *, <i>P</i><0.05 as determined by logrank test in comparison to rsT3D. (B) Brains from infected mice were resected at the times shown and homogenized by freeze-thaw and sonication. Viral titers in brain homogenates were determined by plaque assay. Results are expressed as the mean viral titer (for 6–12 animals). Error bars indicate SD.</p

K594D exhibits altered capacity for membrane penetration.

<p>(A) A 3% v/v solution of bovine erythrocytes was incubated with 5.4×10¹⁰ ISVPs of rsT3D or the indicated φ mutant at 37°C for 1 h. Hemolysis was quantified by determining absorbance of the supernatant at 415 nm. Hemolysis following treatment of an equal number of cells with virion-storage buffer or virion-storage buffer containing 1% TX-100 was considered to be 0 or 100%, respectively. Results are expressed as mean percent hemolysis for triplicate samples. Error bars indicate SD. *, <i>P</i><0.05 as determined by Student's <i>t</i>-test in comparison to rsT3D. (B) L929 cells preincubated with ⁵¹Cr-labeled sodium chromate were adsorbed with 10⁵ ISVPs/cell of rsT3D or the indicated φ mutant at 4°C for 1 h and incubated at 37°C for 4 h following addition of complete medium. The amount of ⁵¹Cr released into the medium was determined by liquid scintillation. ⁵¹Cr release following treatment of an equal number of cells with virion-storage buffer or by addition of 4% TX-100 to the medium was considered to be 0 or 100%, respectively. Results are expressed as mean percent lysis for triplicate samples. Error bars indicate SD. *, <i>P</i><0.05 as determined by Student's <i>t</i>-test in comparison to rsT3D. (C) HeLa cells starved of cysteine and methionine were adsorbed with 10⁶ ISVPs/cell of rsT3D or the indicated φ mutant at 4°C for 1 h. Infection was initiated in medium containing ³⁵S-labeled cysteine and methionine in the presence or absence of α-sarcin. Cells were lysed following incubation at 37°C for 1 h. Proteins were precipitated with TCA, and acid-precipitable radioactivity was quantified by scintillation counting. Results are expressed as mean ³⁵S incorporated for triplicate samples. Error bars indicate SD. *, <i>P</i><0.05 as determined by Student's <i>t</i>-test in comparison to ³⁵S incorporated in the absence of α-sarcin. (D) CsCl-treated ISVPs of rsT3D or the indicated μ1 mutant were incubated with trypsin at 4°C for the intervals shown. Samples were resolved by SDS-PAGE and immunoblotted using a MAb specific for μ1. The position of the δ band is shown. (E) ISVPs of rsT3D or the indicated μ1 mutant were incubated at the temperatures shown for 15 min. Residual infectivity was assessed by plaque assay. Results are expressed as mean residual titer for triplicate samples. Error bars indicate SD.</p

φ mutant viruses produce less histopathologic injury than wild-type virus.

<p>ND4 Swiss Webster mice were inoculated intracranially with 50 PFU of rsT3D or the indicated φ mutant. (A) Brains from infected mice were resected at 10 d post-inoculation, fixed, and embedded in paraffin. Brain sections were stained with H&E, polyclonal reovirus-specific antiserum, or activated caspase-3-specific antiserum. Consecutive sections from the hippocampal region of representative brains of rsT3D-, K594D-, or I595K-infected mice are shown. Scale bars, 500 µm. (B) Higher magnification images of H&E-stained sections of the dentate gyrus and CA4 region of the hippocampus of mice infected with each virus strain are shown. Scale bars, 100 µm.</p

φ mutant viruses are apoptosis-defective in vivo.

<p>ND4 Swiss Webster mice were inoculated intracranially with 50 PFU of rsT3D or the indicated φ mutant. Brains from infected mice were resected at the times shown and homogenized by freeze-thaw and sonication. (A) Brain homogenates were resolved by SDS-PAGE and immunoblotted using an antiserum specific for activated caspase-3 (C-3) (upper panel). As a control for protein concentration, the blots were stripped and reprobed with an antibody specific for actin (lower panel). (B) Brain homogenates from five animals infected with each virus strain for the times shown were resolved by SDS-PAGE and immunoblotted using antisera specific for either activated caspase-3 or actin. Pixel intensities of activated caspase-3 and actin bands were quantified by densitometry. Results are expressed as the mean ratio of activated caspase-3 band intensity to that of actin. Error bars indicate SEM. *, <i>P</i><0.05 as determined by Student's <i>t</i> test in comparison to rsT3D at each time point. (C) For each brain homogenate, the cleaved caspase-3/actin ratio obtained by immunoblot analysis was divided by the virus titer. Results are expressed as the mean activated caspase-3 concentration/PFU. Error bars indicate SEM. *, <i>P</i><0.05 as determined by Student's <i>t</i> test in comparison to rsT3D at each time point.</p

K594D and I595K are apoptosis-defective.

<p>(A) HeLa cells were infected with rsT3D or the indicated φ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of apoptotic cells was determined after staining with acridine orange. Results are expressed as the mean percentage of apoptotic cells for triplicate samples. Error bars indicate SD. *, <i>P</i><0.05 as determined by Student's <i>t</i>-test in comparison to rsT3D. (B) HeLa cells were infected with rsT3D or the indicated φ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of cells with less than G1 DNA content (sub-G1) was quantified by staining with propidium iodide. Results are expressed as the mean percentage of sub-G1 cells for triplicate samples. Error bars indicate SD. *, <i>P</i><0.05 as determined by Student's <i>t</i>-test in comparison to rsT3D. (C) 293T cells were co-transfected with pNF-κB-Luc and pRenilla-Luc 24 h prior to adsorption with rsT3D or the indicated φ mutant at an MOI of 100 PFU/cell. Luciferase activity in cell lysates was determined at 24 h post-infection. Results are presented as the ratio of normalized luciferase activity from infected cell lysates to that from mock-infected lysates for triplicate samples. Error bars indicate SD. *, <i>P</i><0.05 as determined by Student's <i>t</i>-test in comparison to rsT3D.</p

Infectivity and growth of μ1 φ mutants.

<p>(A) L929 cells were adsorbed with rsT3D or the indicated φ mutant at an MOI of 10⁴ particles/cell. After incubation at 37°C for 18 h, cells were fixed using methanol and visualized by immunostaining. Reovirus-infected cells were quantified by counting fluorescent cells. Results are expressed as mean fluorescent focus units (FFU) per field for triplicate samples. Error bars indicate SD. (B) L929 cells were adsorbed with rsT3D or the indicated φ mutant at an MOI of 2 PFU/cell. Titers of virus in cell lysates at the indicated intervals post-infection were determined by plaque assay. Results are expressed as viral yield for triplicate samples. Error bars indicate SD.</p

MT4-MMP is expressed in regions of the hypothalamus that regulate thirst.

<p>(<b>A</b>) β-gal staining of MT4-MMP null mouse brain reveals intense blue staining. No staining is present in the wildtype controls. (<b>B</b>) β-gal staining is mainly within the cortex of the MT4-MMP null mice (12.5X). (<b>C</b>) β-gal staining is present in scattered neurons within the hypothalamus (100X), including the anterior hypothalamic area and lateral hypothalamic area, which are known to regulate thirst. (D) Inset shows high power view (400X) of the hypothalamus revealing blue staining within neurons.</p

MT4-MMP localization in the mouse kidney.

<p>(<b>A–B</b>) β-galactosidase staining kidneys from 5week old MT4-MMP null mice shows staining in the cortex and medulla (A, 50X DIC) with the most intense staining seen in the papilla (B, 200X DIC). (<b>C–F</b>) Staining with a specific antibody directed against MT4-MMP in wildtype mice shows expression throughout the metanephric mesenchyme and ureteric bud at E12.5 (C, 100X). MT4-MMP expression is abundant in the cortical regions during embryonic development (E18.5, D, 50X), but by 2weeks of age, most of the expression is localized to the papilla regions (E–F, 50X and 200X, respectively). (<b>G–H</b>) Staining with a specific antibody directed against MT4-MMP in MT4-MMP null and wildtype mice, demonstrating specificity of the antibody for MT4-MMP (200X). (<b>I–J</b>) Immunoblot of inner medullary collecting duct cells derived from wild type mice (IMCD), MT4-MMP null mice (Null) or IMCD cells from MT4-MMP null mice reconstituted with MT4-MMP (Recon), illustrating specificity of the antibody for MT4-MMP (I). Ponceau staining was performed on the nitrocellulose of the immunoblot to demonstrate equal loading of the lysates (J).</p

MT4-MMP null mice do not have abnormalities in aquaporin 1, 2 or ENaC expression.

<p>Immunostaining was performed for (<b>A–B</b>) Aquaporin I, (<b>C–D</b>) Aquaporin 2, and (<b>E–F</b>) ENaCβ (50X).</p

MT4-MMP null mice have increased urine osmolarity compared to control mice.

<p>(<b>A</b>) Urine osmolarity obtained at baseline (0) and following 24 hr water deprivation (24) reveals that MT4-MMP null mice have higher baseline urine osmolarity (*p = 0.0164) vs C57/BL6 time point 0. Both groups of animals appropriately concentrate their urines following water deprivation. (<b>B</b>) Plasma osmolarity, (<b>C</b>) Plasma sodium and (<b>D</b>) Blood urea nitrogen were the same in both groups.</p