Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.     

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02 µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (V_mem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only V_mem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and V_mem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.     

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.     

A Novel Method to Evaluate the Community Built Environment Using Photographs – Environmental Profile of a Community Health (EPOCH) Photo Neighbourhood Evaluation Tool

Background

Previous research has shown that environments with features that encourage walking are associated with increased physical activity. Existing methods to assess the built environment using geographical information systems (GIS) data, direct audit or large surveys of the residents face constraints, such as data availability and comparability, when used to study communities in countries in diverse parts of the world. The aim of this study was to develop a method to evaluate features of the built environment of communities using a standard set of photos. In this report we describe the method of photo collection, photo analysis instrument development and inter-rater reliability of the instrument.

Methods/Principal Findings

A minimum of 5 photos were taken per community in 86 communities in 5 countries according to a standard set of instructions from a designated central point of each community by researchers at each site. A standard pro forma derived from reviewing existing instruments to assess the built environment was developed and used to score the characteristics of each community. Photo sets from each community were assessed independently by three observers in the central research office according to the pro forma and the inter-rater reliability was compared by intra-class correlation (ICC). Overall 87% (53 of 60) items had an ICC of ≥0.70, 7% (4 of 60) had an ICC between 0.60 and 0.70 and 5% (3 of 60) items had an ICC ≤0.50.

Conclusions/Significance

Analysis of photos using a standardized protocol as described in this study offers a means to obtain reliable and reproducible information on the built environment in communities in very diverse locations around the world. The collection of the photographic data required minimal training and the analysis demonstrated high reliability for the majority of items of interest.     

Removal of the C-terminal regulatory domain of α-isopropylmalate synthase disrupts functional substrate binding

α-Isopropylmalate synthase (α-IPMS) catalyzes the metal-dependent aldol reaction between α-ketoisovalerate (α-KIV) and acetyl-coenzyme A (AcCoA) to give α-isopropylmalate (α-IPM). This reaction is the first committed step in the biosynthesis of leucine in bacteria. α-IPMS is homodimeric, with monomers consisting of (β/α)(8) barrel catalytic domains fused to a C-terminal regulatory domain, responsible for binding leucine and providing feedback regulation for leucine biosynthesis. In these studies, we demonstrate that removal of the regulatory domain from the α-IPMS enzymes of both Neisseria meningitidis (NmeIPMS) and Mycobacterium tuberculosis (MtuIPMS) results in enzymes that are unable to catalyze the formation of α-IPM, although truncated NmeIPMS was still able to slowly hydrolyze AcCoA. The lack of catalytic activity of these truncation variants was confirmed by complementation studies with Escherichia coli cells lacking the α-IPMS gene, where transformation with the plasmids encoding the truncated α-IPMS enzymes was not able to rescue α-IPMS activity. X-ray crystal structures of both truncation variants reveal that both proteins are dimeric and that the catalytic sites of the proteins are intact, although the divalent metal ion that is thought to be responsible for activating substrate α-KIV is displaced slightly relative to its position in the substrate-bound, wild-type structure. Isothermal titration calorimetry and WaterLOGSY nuclear magnetic resonance experiments demonstrate that although these truncation variants are not able to catalyze the reaction between α-KIV and AcCoA, they are still able to bind the substrate α-KIV. It is proposed that the regulatory domain is crucial for ensuring protein dynamics necessary for competent catalysis.     

Heat shock protein 27 is increased in cyanotic tetralogy of Fallot myocardium and is associated with improved cardiac output and contraction

Tetralogy of Fallot (TOF) is a congenital heart condition in which the right ventricle is exposed to cyanosis and pressure overload. Patients have an increased risk of right ventricle dysfunction following corrective surgery. Whether the cyanotic myocardium is less tolerant of injury compared to non-cyanotic is unclear. Heat shock proteins (HSPs) protect against cellular stresses. The aim of this study was to examine HSP 27 expression in the right ventricle resected from TOF patients and determine its relationship with right ventricle function and clinical outcome. Ten cyanotic and ten non-cyanotic patients were studied. Western blotting was used to quantify HSP 27 in resected myocardium at (1) baseline (first 15 min of aortic cross clamp and closest representation of pre-operative status) and (2) after 15 min during ischemia until surgery was complete. The cyanotic group had significantly increased haematocrit, lower O₂ saturation, thicker interventricular septal wall thickness and released more troponin-I on post-operative day 1 (p?<?0.05). HSP 27 expression was significantly increased in the <15 min cyanotic compared to the <15 min non-cyanotic group (p?=?0.03). In the cyanotic group, baseline HSP 27 expression also significantly correlated with oxygen extraction ratio (p?=?0.028), post-operative basal septal velocity (p?=?0.036) and mixed venous oxygen saturation (p?=?0.02), markers of improved cardiac output/contraction. Increased HSP 27 expression and associated improved right ventricle function and systemic perfusion supports a cardio-protective effect of HSP 27 in cyanotic TOF.     

Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.     

B cells regulate murine gammaherpesvirus 68 latency

The dynamics of the establishment of, and reactivation from, gammaherpesviruses latency has not been quantitatively analyzed in the natural host. Gammaherpesvirus 68 (gammaHV68) is a murine gammaherpesvirus genetically related to primate gammaherpesviruses that establishes a latent infection in infected mice. We used limiting dilution reactivation (frequency of cells reactivating gammaHV68 in vitro) and limiting dilution PCR (frequency of cells carrying gammaHV68 genome) assays to compare gammaHV68 latency in normal (C57BL/6) and B-cell-deficient (MuMT) mice. After intraperitoneal (i.p.) inoculation, latent gammaHV68 was detected in the spleen, bone marrow, and peritoneal cells. Both B-cell-deficient and C57BL/6 mice established latent infection in peritoneal cells after either i.p. or intranasal (i.n.) inoculation. In contrast, establishment of splenic latency was less efficient in B-cell-deficient than in C57BL/6 mice after i.n. inoculation. Analysis of reactivation efficiency (reactivation frequency compared to frequency of cells carrying gammaHV68 genome) revealed that (i) regardless of route or mouse strain, splenic cells reactivated gammaHV68 less efficiently than peritoneal cells, (ii) the frequency of cells carrying gammaHV68 genome was generally comparable over the course of infection between C57BL/6 and B-cell-deficient mice, (iii) between 28 and 250 days after infection, cells from B-cell-deficient mice reactivated gammaHV68 10- to 100-fold more efficiently than cells from C57BL/6 mice, (iv) at 7 weeks postinfection, B-cell-deficient mice had more genome-positive peritoneal cells than C57BL/6 mice, and (v) mixing cells (ratio of 3 to 1) that reactivated inefficiently with cells that reactivated efficiently did not significantly decrease reactivation efficiency. Consistent with a failure to normally regulate chronic gammaHV68 infection, the majority of infected B-cell-deficient mice died between 100 and 200 days postinfection. We conclude that (i) B cells are not required for establishment of gammaHV68 latency, (ii) there are organ-specific differences in the efficiency of gammaHV68 reactivation, (iii) B cells play a crucial role in regulating reactivation of gammaHV68 from latency, and (iv) B cells are important for controlling chronic gammaHV68 infection.     

An RNAi-based approach identifies molecules required for glutamatergic and GABAergic synapse development

We report the results of a genetic screen to identify molecules important for synapse formation and/or maintenance. siRNAs were used to decrease the expression of candidate genes in neurons, and synapse development was assessed. We surveyed 22 cadherin family members and demonstrated distinct roles for cadherin-11 and cadherin-13 in synapse development. Our screen also revealed roles for the class 4 Semaphorins Sema4B and Sema4D in the development of glutamatergic and/or GABAergic synapses. We found that Sema4D affects the formation of GABAergic, but not glutamatergic, synapses. Our screen also identified the activity-regulated small GTPase Rem2 as a regulator of synapse development. A known calcium channel modulator, Rem2 may function as part of a homeostatic mechanism that controls synapse number. These experiments establish the feasibility of RNAi screens to characterize the mechanisms that control mammalian neuronal development and to identify components of the genetic program that regulate synapse formation and/or maintenance.