Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein

Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the μ1 ϕ Domain

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein μ1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of μ1 indicates that the μ1 ϕ domain is sufficient to elicit a cell death response. To evaluate the contribution of the μ1 ϕ domain to the induction of apoptosis following reovirus infection, ϕ mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in ϕ diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in ϕ affect the apoptotic potential of reovirus, suggesting that ϕ initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective ϕ mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the ϕ domain of μ1 plays an important regulatory role in reovirus-induced apoptosis and disease

Bid Regulates the Pathogenesis of Neurotropic Reovirus

Reovirus infection leads to apoptosis in both cultured cells and the murine central nervous system (CNS). NF-κB-driven transcription of proapoptotic cellular genes is required for the effector phase of the apoptotic response. Although both extrinsic death-receptor signaling pathways and intrinsic pathways involving mitochondrial injury are implicated in reovirus-induced apoptosis, mechanisms by which either of these pathways are activated and their relationship to NF-κB signaling following reovirus infection are unknown. The proapoptotic Bcl-2 family member, Bid, is activated by proteolytic cleavage following reovirus infection. To understand how reovirus integrates host signaling circuits to induce apoptosis, we examined proapoptotic signaling following infection of Bid-deficient cells. Although reovirus growth was not affected by the absence of Bid, cells lacking Bid failed to undergo apoptosis. Furthermore, we found that NF-κB activation is required for Bid cleavage and subsequent proapoptotic signaling. To examine the functional significance of Bid-dependent apoptosis in reovirus disease, we monitored fatal encephalitis caused by reovirus in the presence and absence of Bid. Survival of Bid-deficient mice was significantly enhanced in comparison to wild-type mice following either peroral or intracranial inoculation of reovirus. Decreased reovirus virulence in Bid-null mice was accompanied by a reduction in viral yield. These findings define a role for NF-κB-dependent cleavage of Bid in the cell death program initiated by viral infection and link Bid to viral virulence

A workshop on leadership for senior MD–PhD students

Leadership skills are essential for a successful career as a physician-scientist, yet many MD–PhD training programs do not offer formal training in leadership. The Vanderbilt Medical Scientist Training Program (MSTP) previously established a 2-day leadership workshop that has been held biennially since 2006 for students in the first and second years of the graduate school portion of combined MD and PhD training (G1/G2 students). Workshop attendees have consistently rated this workshop as a highly effective experience. However, opportunities for structured training in leadership competencies during the subsequent 3–5 years of MD–PhD training are limited. Given the success of the G1/G2 leadership workshop and the need for continuity in this model of leadership training, we developed a half-day workshop for MSTP students in the clinical years of medical school (M3/M4 students) to foster continued training in leadership. Our workshop curriculum, based in part on original cases drafted by Vanderbilt MSTP students, provides concrete strategies to manage conflict and navigate leadership transitions in the physician-scientist career path. The curriculum emphasizes both short-term competencies, such as effective participation as a member of a clinical team, and long-term competencies, such as leadership of a research team, division, or department. Our inaugural senior leadership workshop, held in August, 2015, was judged by student participants to be well organized and highly relevant to leadership concepts and skills. It will be offered biennially in our training curriculum for M3 and M4 MSTP students

A Single Mutation in the Carboxy Terminus of Reovirus Outer-Capsid Protein σ3 Confers Enhanced Kinetics of σ3 Proteolysis, Resistance to Inhibitors of Viral Disassembly, and Alterations in σ3 Structure

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein σ3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in σ3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the σ3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant σ3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced σ3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in σ3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant σ3 proteins revealed structural alterations in σ3 that correlate with the Y354H mutation. These results indicate that a single mutation in σ3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of σ3 cleavage during reovirus disassembly

IκB Kinase Subunits α and γ Are Required for Activation of NF-κB and Induction of Apoptosis by Mammalian Reovirus

Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-κB and downstream cellular genes. To better understand the mechanism of NF-κB activation by reovirus, NF-κB signaling intermediates under reovirus control were investigated at the level of Rel, IκB, and IκB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This biphasic pattern of Rel protein activation is associated with the degradation of the NF-κB inhibitor IκBα but not the structurally related inhibitors IκBβ or IκBɛ. Using IKK subunit-specific small interfering RNAs and cells deficient in individual IKK subunits, we demonstrate that IKKα but not IKKβ is required for reovirus-induced NF-κB activation and apoptosis. Despite the preferential usage of IKKα, both NF-κB activation and apoptosis were attenuated in cells lacking IKKγ/Nemo, an essential regulatory subunit of IKKβ. Moreover, deletion of the gene encoding NF-κB-inducing kinase, which is known to modulate IKKα function, had no inhibitory effect on either response in reovirus-infected cells. Collectively, these findings indicate a novel pathway of NF-κB/Rel activation involving IKKα and IKKγ/Nemo, which together mediate the expression of downstream proapoptotic genes in reovirus-infected cells

Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

El objetivo es presentar categorías de la Teoría Histórico-Cultural de la Actividad como caja de herramientas para construir indagaciones e intervenciones vinculadas al trabajo de Psicólogos y otros Agentes en escenarios educativos. En el marco de problemas cruciales de agenda psicoeducativa, como el de las unidades de análisis, se introduce la Teoría de la Actividad en su primera, segunda y tercera generación y “más allá”, como Engestrom caracterizó su desarrollo histórico. Se presentan similaridades y diferencias con conceptualizaciones de autores de perspectivas socio-cultural e histórico–cultural, originadas en el pensamiento de Vygotsky, como Cole, Rogoff, Wertsch, Chaiklin, Daniels y Edwards, tanto en relación a la Psicología del Desarrollo como al giro relacional en la experticia. Se despliega la Teoría como artefacto mediador para construir investigaciones psicoeducativas en curso sobre violencias en escuelas desde la perspectiva de los actores y participación de psicólogos en configuraciones de apoyo entre Educación Especial y Escuela Común. Finalmente, conceptualizaciones y figuras diseñadas por Engestrom y Yamazumi, en la post-generación de la Teoría, permiten analizar procesos de vinculación e intercambio entre Universidad y Escuelas para la inclusión y calidad educativas, en los que participan psicólogos en formación y formadores de la Universidad Nacional de La Plata.The aim is to present categories of Cultural-Historic-Activity-Theory as a tools cage for the building of research and interventions related to Psychologists and other Agents´ work in educational stages. In the frame of central problems of the psycho-educational agenda, as the units of analysis, the Activity Theory is introduced in its first, second and third generation and beyond, as Engestrom depicted its historical development. Similarities and differences are presented between Activity Theory and conceptualizations of different authors of socio-cultural and cultural-historic perspectives, both founded on Vygotsky thought, linked to Development Psychology and to the relational shift in expertice, as Cole, Rogoff, Wertsch, Chaiklin, Daniels and Edwards. The Activity Theory is displayed as a mediating artifact for building current psycho-educational research about violences at school from the perspective of social actors and about psychologists and other agents´ participation in supportive configurations between Special Education and Common Schools. At last, figures and conceptualizations drawn by Engestrom and Yamazumi in the post-generation of the Theory, allow to analyse processes of linking and exchange between University and Schools for educational enhancement and inclusion, in which psychologists in modeling and tutors of La Plata National University are participating.Facultad de Psicologí