Mutational analysis of residues involved in nucleotide and divalent cation stabilization in the rotavirus RNA-dependent RNA polymerase catalytic pocket

AbstractThe rotavirus RNA-dependent RNA polymerase (RdRp), VP1, contains canonical RdRp motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential import to nucleotide and divalent cation stabilization, we aligned VP1 of divergent rotaviruses and the structural homolog reovirus λ3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRp motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRp activity to particle assembly

Continental-scale homogenization of residential lawn plant communities

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Landscape and Urban Planning 165 (2017): 54-63, doi:10.1016/j.landurbplan.2017.05.004.Residential lawns are highly managed ecosystems that occur in urbanized landscapes across the United States. Because they are ubiquitous, lawns are good systems in which to study the potential homogenizing effects of urban land use and management together with the continental-scale effects of climate on ecosystem structure and functioning. We hypothesized that similar homeowner preferences and management in residential areas across the United States would lead to low plant species diversity in lawns and relatively homogeneous vegetation across broad geographical regions. We also hypothesized that lawn plant species richness would increase with regional temperature and precipitation due to the presence of spontaneous, weedy vegetation, but would decrease with household income and fertilizer use. To test these predictions, we compared plant species composition and richness in residential lawns in seven U.S. metropolitan regions. We also compared species composition in lawns with understory vegetation in minimally-managed reference areas in each city. As expected, the composition of cultivated turfgrasses was more similar among lawns than among reference areas, but this pattern also held among spontaneous species. Plant species richness and diversity varied more among lawns than among reference areas, and more diverse lawns occurred in metropolitan areas with higher precipitation. Native forb diversity increased with precipitation and decreased with income, driving overall lawn diversity trends with these predictors as well. Our results showed that both management and regional climate shaped lawn species composition, but the overall homogeneity of species regardless of regional context strongly suggested that management was a more important driver.This research was supported by the Macrosystems Biology Program in the Emerging Frontiers Division of the Biological Sciences Directorate at the National Science Foundation (NSF) under grants EF-1065548, 1065737, 1065740, 1065741, 1065772, 1065785, 1065831, and 121238320

Ecological Homogenization of Urban USA

A visually apparent but scientifically untested outcome of land-use change is homogenization across urban areas, where neighborhoods in different parts of the country have similar patterns of roads, residential lots, commercial areas, and aquatic features. We hypothesize that this homogenization extends to ecological structure and also to ecosystem functions such as carbon dynamics and microclimate, with continental-scale implications. Further, we suggest that understanding urban homogenization will provide the basis for understanding the impacts of urban land-use change from local to continental scales. Here, we show how multi-scale, multi-disciplinary datasets from six metropolitan areas that cover the major climatic regions of the US (Phoenix, AZ; Miami, FL; Baltimore, MD; Boston, MA; Minneapolis–St Paul, MN; and Los Angeles, CA) can be used to determine how household and neighborhood characteristics correlate with land-management practices, land-cover composition, and landscape structure and ecosystem functions at local, regional, and continental scales

Residential household yard care practices along urban-exurban gradients in six climatically-diverse U.S. metropolitan areas

Residential land is expanding in the United States, and lawn now covers more area than the country’s leading irrigated crop by area. Given that lawns are widespread across diverse climatic regions and there is rising concern about the environmental impacts associated with their management, there is a clear need to understand the geographic variation, drivers, and outcomes of common yard care practices. We hypothesized that 1) income, age, and the number of neighbors known by name will be positively associated with the odds of having irrigated, fertilized, or applied pesticides in the last year, 2) irrigation, fertilization, and pesticide application will vary quadratically with population density, with the highest odds in suburban areas, and 3) the odds of irrigating will vary by climate, but fertilization and pesticide application will not. We used multi-level models to systematically address nested spatial scales within and across six U.S. metropolitan areas—Boston, Baltimore, Miami, Minneapolis-St. Paul, Phoenix, and Los Angeles. We found significant variation in yard care practices at the household (the relationship with income was positive), urban-exurban gradient (the relationship with population density was an inverted U), and regional scales (city-tocity variation). A multi-level modeling framework was useful for discerning these scaledependent outcomes because this approach controls for autocorrelation at multiple spatial scales. Our findings may guide policies or programs seeking to mitigate the potentially deleterious outcomes associated with water use and chemical application, by identifying the subpopulations most likely to irrigate, fertilize, and/or apply pesticides

Psychosocial impact of undergoing prostate cancer screening for men with BRCA1 or BRCA2 mutations.

OBJECTIVES: To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study, a multi-national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes. PARTICPANTS AND METHODS: Men enrolled in the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36-item short-form health survey (SF-36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale-Revised, risk perception and knowledge. The results of the baseline questionnaire are presented. RESULTS: A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1 and BRCA2 genes, respectively, and 174 were controls (familial mutation negative). Participants' perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF-36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status. CONCLUSION: This is the first study to report the psychosocial profile of men with BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer-specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening

Residues of the Rotavirus RNA-Dependent RNA Polymerase Template Entry Tunnel That Mediate RNA Recognition and Genome Replication▿

To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (+RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3′ end of rotavirus +RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral +RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with +RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of +RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with +RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates

Mammalian orthoreovirus can exit cells in extracellular vesicles.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization

Reovirus protein co-fractionates with EV-enriched fractions released from cells regardless of plasma membrane integrity phenotype.

L cells were adsorbed with three individual clones of T1L or T3D reovirus at an MOI of 1 PFU/cell. (A) Every 24 h, cell lysates were collected, and virus in the supernatant was quantified by FFA. Error bars indicate SD. n = 3. *, P n = 3. *, P Biorender.com. (D-G) Infected-cell supernatants were collected every 24 h for 96 h. Mock-infected supernatant was collected at 96 h, but reovirus protein was not detected. Reovirus protein association with large EV, medium EV, and small EV/free virus fractions was quantified following SDS-PAGE and immunoblotting. Representative immunoblots probed using reovirus antiserum for T1L (D) and T3D (F) and graphs showing results quantified from three independent immunoblots for T1L (E) and T3D (G) are shown. Asterisk denotes the reovirus λ3 protein band used for quantitation. Error bars indicate SD. n = 3. *, P n = 3. **, P < 0.01 by two-way ANOVA with Tukey’s multiple comparisons.</p

Little reovirus spontaneously associates with EVs.

(A-F) L cells were adsorbed with three individual clones of T1L or T3D reovirus at an MOI of 1 PFU/cell. In parallel, triple the amount of L cells were adsorbed with medium (mock). After 72 h, large and medium EVs were harvested via centrifugation from reovirus-infected cells to constitute the “virus-infected EV” samples and from mock-infected cells. 1 x 109 total PFU of free reovirus particles were mixed and incubated with large or medium EVs from mock-infected cells (mock EVs) or with EV storage buffer (buffer), then re-pelleted at respective centrifugation speeds. Equal volumes of all T1L (A, C, D) and T3D (B, E, F) samples were resolved by SDS-PAGE and Coomassie staining (A-B) or by SDS-PAGE with immunoblotting using anti-reovirus serum (C-F). The spontaneous association of free reovirus with mock large and medium EVs was quantified and compared to free T1L virus input (D) or free T3D virus input (F). Error bars indicate SD. n = 3. ****, P (TIF)</p

Reovirus plasma membrane disruption is strain-specific in Caco-2 cells.

Caco-2 cells were adsorbed with medium (mock) or with three individual clones of T1L or T3D reovirus at an MOI of 5 PFU/cell. Cell membrane disruption was quantified for T1L-, T3D-, and mock-infected cells every 24 h for 96 h using an LDH assay. A medium-only negative control and a kit-specific positive control quantified in triplicate at 96 h are shown. Error bars indicate SD. n = 3. ***, P (TIF)</p