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Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 to 10 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization. Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus-host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds. [ABSTRACT FROM AUTHOR]

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. [ABSTRACT FROM AUTHOR]

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92pol replication protein and the DDX3- like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. [ABSTRACT FROM AUTHOR]

Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication. [ABSTRACT FROM AUTHOR]

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation. [ABSTRACT FROM AUTHOR]

RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses. [ABSTRACT FROM AUTHOR]

Similar to animal viruses, the abundant plant positive-strand RNA viruses replicate in infected cells by exploiting the vast resources of the host. This review focuses on virus–host interactions during tombusvirus replication. The multifunctional tombusvirus p33 replication protein not only interacts with itself, the viral p92pol polymerase, and viral RNA, but also with approximately 100 cellular proteins and subcellular membranes. Several negative regulatory host proteins, such as cyclophilins and WW motif containing proteins, also bind to p33 and interfere with p33''s functions. To explain how p33 can perform multiple functions, we propose that a variety of interactions involving p33 result in the commitment of p33 molecules to specific tasks. This facilitates tight spatial and temporal organization of viral replication in infected cells. [Copyright &y& Elsevier]

Abstract: Recruited host factors aid replication of plus-strand RNA viruses. In this paper, we show that Dbp2 DEAD-box helicase of yeast, which is a homolog of human p68 DEAD-box helicase, directly affects replication of Tomato bushy stunt virus (TBSV). We demonstrate that Dbp2 binds to the 3′-end of the viral minus-stranded RNA and enhances plus-strand synthesis by the viral replicase in a yeast-based cell-free TBSV replication assay. In vitro data with wt and an ATPase-deficient Dbp2 mutant indicate that Dbp2 unwinds local secondary structures at the 3′-end of the TBSV (−)RNA. We also show that Dbp2 complements the replication deficiency of TBSV in yeast containing reduced amount of Ded1 DEAD-box helicase, another host factor involved in TBSV replication, suggesting that Dbp2 and Ded1 helicases play redundant roles in TBSV replication. We also show that the orthologous AtRH20 DEAD-box helicase from Arabidopsis can increase tombusvirus replication in vitro and in yeast. [Copyright &y& Elsevier]

Abstract: To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, we are studying protein families, whose members have been identified during previous high throughput screening. In this paper, we have characterized the WW domain-containing protein family from yeast and plants. We find that, in addition to Rsp5 E3 ubiquitin ligase, yeast Wwm1 and Prp40 and three Arabidopsis WW domain-containing proteins are strong inhibitors of TBSV replication. The tombusvirus replicase complex isolated from yeast with down-regulated Wwm1 protein level was more active. Accumulation of viral p92pol was reduced when Wwm1 was over-expressed, suggesting that the stability of p92pol might be reduced, as observed with Rsp5. Moreover, replication of two insect RNA viruses is also inhibited by Wwm1 and Rsp5, suggesting that WW domain-containing proteins might have broad regulatory effects on RNA viruses. Thus, artificial antiviral proteins with WW domains could be useful antiviral strategy. [Copyright &y& Elsevier]

Abstract: Post-translational modifications of viral replication proteins could be widespread phenomena during the replication of plus-stranded RNA viruses. In this article, we identify two lysines in the tombusvirus p33 replication co-factor involved in ubiquitination and show that the same lysines are also important for the p33 to interact with the host Vps23p ESCRT-I factor. We find that the interaction of p33 with Vps23p is also affected by a “late-domain”-like sequence in p33. The combined mutations of the two lysines and the late-domain-like sequences in p33 reduced replication of a replicon RNA of Tomato bushy stunt virus in yeast model host, in plant protoplasts, and plant leaves, suggesting that p33-Vps23p ESCRT protein interaction affects tombusvirus replication. Using ubiquitin-mimicking p33 chimeras, we demonstrate that high level of p33 ubiquitination is inhibitory for TBSV replication. These findings argue that optimal level of p33 ubiquitination plays a regulatory role during tombusvirus infections. [Copyright &y& Elsevier]

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and hostcoded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery. [ABSTRACT FROM AUTHOR]

To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication. [ABSTRACT FROM AUTHOR]

In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3' end region of TRV. TRV siRNA biogenesis and antiviral silencing are strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2, and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities. [ABSTRACT FROM AUTHOR]

Recombinant adeno‐associated viral vectors (rAAV) are promising therapies for genetic diseases. Although current platforms for recombinant vector production can generate drug material for pre‐clinical and clinical studies, rAAV biomanufacturing will eventually face commercial supply challenges if per cell vector productivity and process scalability are not improved. Because considerable efforts have traditionally focused on optimizing rAAV plasmid design, herein we investigate the impact of host cell proteins on vector production to identify proteins that may enhance rAAV yield. Using a rAAV2‐GFP‐producing Saccharomyces cerevisiae model in combination with the yeast Tet Hughes Collection screening library, we identified 22 gene candidates that improved rAAV DNA replication (rAAV‐GFP/18s rDNA ratio) and vector yield (benzonase‐resistant rAAV DNA vector genome titer) as high as 6‐fold and 15‐fold relative to control, respectively. The candidate proteins participate in biological processes such as DNA replication, ribosome biogenesis, and RNA and protein processing. The best five candidates (PRE4, HEM4, TOP2, GPN3, and SDO1) were further screened by generating overexpression mutants in the YPH500 yeast strain. Subsequent clone evaluation was performed to confirm the rAAV‐promoting activity of selected candidates under plate‐based and bioreactor‐controlled fermentation conditions. Digital droplet PCR analysis of cell lysate and AVB resin‐purified material confirmed HEM4 and TOP2 overexpression mutants displayed the highest per cell total rAAV DNA productivity (1.6 and 1.7‐fold increase over control, respectively) and per cell vector productivity (3 and 4‐fold over control, respectively). This evaluation confirmed that overexpression of HEM4 and TOP2 proteins enhanced total and benzonase‐resistant rAAV DNA yield. Further studies are needed to understand their mechanism of action and to assess their potential application in molecular strategies for rAAV production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2725, 2019 [ABSTRACT FROM AUTHOR]

Recent in vitro proteomics screens revealed that many host proteins could interact with the replication proteins of Tomato bushy stunt virus (TBSV), which is a small, plus-stranded RNA virus (Z. Li, D. Barajas, T. Panavas, D. A. Herbst, and P. D. Nagy, J. Virol. 82:6911-6926, 2008). To further our understanding of the roles of host factors in TBSV replication, we have tested the effect of Rsp5p, which is a member of the Nedd4 family of E3 ubiquitin ligases. The full-length Rsp5p, via its WW domain, is shown to interact with p33 and the central portion of p92pol replication proteins. We find that overexpression of Rsp5p inhibits TBSV replication in Saccharomyces cerevisiae yeast, while downregulation of Rsp5p leads to increased TBSV accumulation. The inhibition is caused by Rsp5p-guided degradation of p92pol, while the negative effect on the p33 level is less pronounced. Interestingly, recombinant Rsp5p also inhibits TBSV RNA replication in a cell-free replication assay, likely due to its ability to bind to p33 and p92pol. We show that the WW domain of Rsp5p, which is involved in protein interactions, is responsible for inhibition of TBSV replication, whereas the HECT domain, involved in protein ubiquitination, is not necessary for Rsp5p-mediated inhibition of viral replication. Overall, our data suggest that direct binding between Rsp5p and p92pol reduces the stability of p92pol, with consequent inhibition of TBSV replicase activity. [ABSTRACT FROM AUTHOR]

Soft tissue giant cell tumor is a rare tumor. It occurs mainly in adults, in the lower extremities, and it has not gender predominance. Clinically it has a multinodular aspect and the best therapeutic option is a conservative surgical resection. We describe the case of a 55-year-old man with a multilobullated neoformation that affected the first toe of left foot, with a hystopathological diagnosis of giant cell tumor of soft tissues. The therapy consisted on partial amputation of the affected toe, to remove completely the tumor. [ABSTRACT FROM AUTHOR]

Abstract: To identify host proteins interacting with the membrane-bound replication proteins of tombusviruses, we performed membrane yeast two-hybrid (MYTH) screens based on yeast cDNA libraries. The screens led to the identification of 57 yeast proteins interacting with replication proteins of two tombusviruses. Results from a split ubiquitin assay with 12 full-length yeast proteins and the viral replication proteins suggested that the replication proteins of two tombusviruses interact with a similar set of host proteins. Follow-up experiments with the yeast Cpr1p cyclophilin, which has prolyl isomerase activity that catalyzes cis–trans isomerization of peptidyl–prolyl bonds, confirmed that Cpr1p interacted with the viral p33 replication protein in yeast and in vitro. Replication of Tomato bushy stunt virus replicon RNA increased in cpr1Δ yeast, while over-expression of Cpr1p decreased viral replication. We also show that the Ess1p parvulin prolyl isomerase partly complements Cpr1p function as an inhibitor of tombusvirus replication. [ABSTRACT FROM AUTHOR]

Abstract: Higher plants use RNA silencing as a defense mechanism against viral infections, but viruses may encode suppressor proteins that counteract these defenses. Several virus-encoded suppressors also exert an inhibitory effect on endogenous small RNA regulatory pathways. Here we characterized the Tobacco rattle virus-encoded 16-kDa (TRV-16K) protein as a suppressor that blocked local RNA silencing induced by single (s)- and double-stranded (ds) RNA, indicating that TRV-16K interfered with a step in the silencing pathway downstream of dsRNA formation. The suppressor activity of TRV-16K was severely compromised by moderate to high dosages of dsRNA inducer. When silencing was locally triggered by ssRNA or low levels of dsRNA, silencing suppression by TRV-16K was associated with reduced accumulation of silencing-related siRNAs. TRV-16K also prevented partially cell-to-cell movement and systemic propagation of silencing but not transitive amplification of RNA silencing. We showed that neither TRV nor TRV-16K caused a global deregulation of the microRNA-regulatory pathway in Arabidopsis, suggesting that interference with microRNA biology was not a prerequisite for TRV, and probably many other plant viruses, to develop systemic infections in plants. [Copyright &y& Elsevier]

Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae) vps23Δ bro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant- negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ~600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a "measuring string" during VRC assembly and spherule formation. [ABSTRACT FROM AUTHOR]

In recent years higher education has experienced rapid expansion due to, among other things, the imperative of preparing the tables of technical, professional and research that meet the requirements of economic development, political and social life. Based on this growth and because of the curricular issues facing higher education institutions, has created a need to rationalize and systematize the curriculum development efforts taking place in them. The concept of social need is part of the deficiencies that are identified with an ideal situation, beneficial to the individual and the community, and whose satisfaction requires the response of other members of society or its institutions. The aim of this study was to identify the demand for degree programs in humanities offered by the Faculty of Arts, in various regions of the state of Puebla, specifically in the regions of Chignahuapan-Zacatlán, Teziutlán and Tehuacan, in order that can be incorporated into the model of regionalization of the Autonomous University of Puebla. The method used for this study was descriptive and analytical to carry out a survey applied to 4075 students last semester of high school in the area of social sciences and humanities. Through this work we present the results obtained in each of the identified regions and a general comparison that shows the demand for each of the degrees offered at the Faculty of Arts. [ABSTRACT FROM AUTHOR]

Background: Nevus sebaceous of Jadassohn is a congenital hamartomatous lesion, which involves sebaceous gland, apocrine, and trichilemmal proliferative changes. It presents three clinical stages; the last one is characterized by epithelial tumors arising. Objective: To determine the frequency and associated tumors type, as well as the clinic and epidemiologic characteristics related with them. Material and method: A retrospective analysis at the Instituto Dermatológico de Jalisco Dr. José Barba Rubio during a five-year period was made. All histopathology reports of nevus sebaceous of Jadassohn were included and three different pathologists in a blind fashion reviewed the slides of tumors arising in nevus sebaceous. Results: We included 260 cases with diagnostic of nevus sebaceous of Jadassohn, mean age 19.7 years, female sex predominance (55%), the most frequent localization was scalp (61.5%). Eleven per cent of the 260 cases were in tumor stage, with predominance in 31 to 40 years old (27.5%). Benign tumors were the most frequent (97%), trichoblastoma predominated (50%); only one case of basal cell carcinoma was found. More than one tumor was associated to nevus sebaceous of Jadassohn in 13.7% of cases. Conclusions: Arising malignant tumors in nevus sebaceous of Jadassohn are uncommon, moreover in pediatric age. Prospective studies are necessary in order to determine the real association with nevus sebaceous of Jadassohn tumors. [ABSTRACT FROM AUTHOR]

The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation (HCL 134H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL134H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally. we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro. Additional keywords: posttranscriptional gene silencing. [ABSTRACT FROM AUTHOR]

We present our experience of adapting a rubric for peer feedback in our data visualization course and exploring the utilization of that rubric by students across two semesters. We first discuss the results of an automatable quantitative analysis of the rubric responses, and then compare those results to a qualitative analysis of summative survey responses from students regarding the rubric and peer-feedback process. We conclude with lessons learned about the visualization rubric we used, as well as what we learned more broadly about using quantitative analysis to explore this type of data. These lessons may be useful for other educators wanting to utilize the same data visualization rubric, or wanting to explore the utilization of rubrics already deployed for peer feedback.

Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus-host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds., (© 2017. Published by The Company of Biologists Ltd.)

Background and Objective: To assess repeatability and speed of a line-scan ophthalmoscope (LSO) image-based tracking system and compare to the point-scanning approach., Patients and Methods: Thirty-five eyes with retinal diseases underwent volume scans using two spectral-domain optical coherence tomography (OCT) devices: a line-scan tracking Cirrus HD-OCT (Carl Zeiss Meditec; Dublin, CA) and point-scan tracking Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany). Eyes were also imaged on the Cirrus HD-OCT with tracking disabled., Results: Mean difference in central subfield thickness (CST) between consecutive scans was 2.6 µm for the Cirrus without tracking, 1.7 µm with tracking, and 3.6 µm for the Spectralis. The repeatability standard deviation was 3.0 µm for the Cirrus without tracking, 1.5 µm with tracking, and 4.0 µm for the Spectralis. Coefficient of variation for the CST was 1.1% for the Cirrus without tracking, 0.5% with tracking, and 1.4% for the Spectralis. Mean scan acquisition time was 12.3 ± 6.2 seconds for the Spectralis, 7.8 ± 6.7 for the Cirrus with tracking, and 4.3 ± 0.6 for the Cirrus without tracking., Conclusion: Real-time LSO image-based retinal tracking appears to improve repeatability of OCT retinal thickness measurements., (Copyright 2015, SLACK Incorporated.)

Unlabelled: Replication of (+)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WW-domain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WW-domain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections., Importance: Replication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting tombusvirus replication in yeast cells and in vitro using purified components. The WW domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play proviral roles in replication. Altogether, Rsp5 is a CIRF against a tombusvirus, and it possibly has a regulatory function during viral replication in infected cells., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.

Our increasing understanding of virus-host interactions is revealing a complex role for host factors during virus replication. Besides the role of some host proteins in defense against viruses, it is becoming clear that viruses also hijack several host functions to utilize them for their multiplication. Genome-wide screens using high-throughput methods are being conducted to identify most of the host factors affecting virus replication in a number of host-virus systems. For selected plant viruses, such as bromo- and tombusviruses, yeast has been developed as a model host, thus greatly accelerating genome-wide systematic approaches to identify critical host factors of virus multiplication. In plants, gene knock out T-DNA libraries and virus-induced RNA silencing, among other strategies, can be utilized to identify and characterize host factors involved in virus replication. An additional strategy to study the role of host factors is the use of dominant-negative (DN) mutants, which are mutant versions of host proteins capable of interfering with the function of the wild-type protein without the need of knocking out the given gene from the chromosome. This method allows one to study the relevance of host factors for virus replication in wild-type plants and may overcome some limitations of other methods. Here, we provide guidelines to the use of a DN mutant strategy for the study of host factors and compare the advantages and limitations with other methods. The use of more diverse methods to study gene function in plants is increasing the probability of successfully identifying and characterizing host factors affecting virus replication in plant systems.

microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate mRNAs that result in translational inhibition, mRNA decay, or slicing within the region of complementarity. miRNA processing activity on complementary target mRNAs generates 3' end cleavage products that contain -ligation-competent, 5'-monophosphates. Precise mapping of miRNA-directed cleavage sites within target transcripts is, therefore, possible using RNA ligase-mediated 5' amplification of cDNA ends (RLM-RACE). Here, we provide a comprehensive RLM-RACE-based protocol for the amplification of 5' ends derived from cleaved transcripts resulting from miRNA-guided cleavage events. Novel strategies for high-throughput analysis of miRNA cleavage products have emerged as powerful tools for the novo identification of miRNA targets in a genomic perspective. In this work, we also describe a novel methodology for genome-wide identification of miRNA targets that exploits RLM-RACE for non-sequence-specific enrichment of cleaved transcripts, T7 RNA polymerase-mediated amplification of target products, and microarray hybridization.

To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication.

Despite the advantages of using internal ultrasound-assisted lipoplasty instead of the classic tumescent lipoplasty, such as reduced bleeding and tissue damage, the authors found no objective or comparative study of these techniques in humans. For this reason, they conducted a clinical study to determine the amount of bleeding and tissue damage caused by each of the techniques. A simple clinical assay was accomplished at the Jalisco Plastic Surgery Institute on seven female patients scheduled for abdominal lipectomy. Two similar sections of the surgical area were marked for lipoplasty techniques: classic tumescent lipoplasty on one side and internal ultrasound-assisted lipoplasty on the other. Both areas were treated simultaneously by surgeons experienced in each technique. Laboratory tests and histologic studies were performed on the aspirated material and the manipulated tissue, respectively. The fluids sent to the laboratory were analyzed to determine the amount of bleeding and tissue damage. In the laboratory, the degree of lesion and tissue damage was evaluated in the dermis, nerves, blood vessels, and adipose cells. With internal ultrasound-assisted lipoplasty, indicators of tissue damage such as glutamic oxalacetic transaminase, pyruvic oxalacetic transaminase, cholinesterase, and myoglobin showed higher values than with tumescent lipoplasty. The same was found for hemoglobin levels and in the histologic data indicative of tissue damage; both values were statistically significant at < 0.001. Internal ultrasound-assisted lipoplasty was not demonstrated to be more innocuous or to have a selective effect in adipose cells, and it generally resulted in more tissue damage and bleeding than the classic tumescent technique.