Differential Abundance of DNA Damage Sensors and Innate Immune Signaling Proteins in Inositol Polyphosphate 4-Phosphatase Type II-Negative Triple-Negative Breast Cancer Classified by Immunotype.

The influence of neoplastic cells on the tumor microenvironment is poorly understood. In this study, eight patient samples representing two immunotypes of triple-negative breast cancer (TNBC), defined by quantitative histologic criteria as T-cell desert and T-cell infiltrated (TCI), were compared via label-free quantitative protein mass spectrometry of material extracted directly from targeted regions of formalin-fixed, paraffin-embedded tissue sections. Of 2934 proteins quantitated, 439 were significantly differentially abundant, among which 361 were overabundant in TCI-TNBC. The 361-protein group included proteins involved in major histocompatibility complex-I antigen processing and presentation, viral defense, DNA damage response, and innate immune signaling. Immunohistochemical validation of selected proteins showed good positive correlation between neoplastic cell histoscores and label-free quantitation. Extension of immunohistochemical analysis to a total of 58 inositol polyphosphate 4-phosphatase type II-negative TNBC confirmed elevated levels of the DNA damage sensor interferon-γ-inducible protein 16, inflammasome adaptor ASC, and pore-forming protein gasdermin D in TCI-TNBC neoplastic cells. By contrast, cGMP-AMP synthase inhibitor BAF was elevated in the neoplastic cells of T-cell desert TNBC. These findings demonstrate a previously unknown correlation between the degree of T-cell infiltration in inositol polyphosphate 4-phosphatase type II-negative TNBC and the levels, in cognate neoplastic cells, of proteins that modulate innate immune signaling in response to DNA damage.

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, ß-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME).  Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.

Combination of deep XLMS with deep learning reveals an ordered rearrangement and assembly of a major protein component of the vaccinia virion.

IMPORTANCE: An outstanding problem in the understanding of poxvirus biology is the molecular structure of the mature virion. Via deep learning methods combined with chemical cross-linking mass spectrometry, we have addressed the structure and assembly pathway of P4a, a key poxvirus virion core component.

Deconstructing Allograft Adipose and Fascia Matrix: Fascia Matrix Improves Angiogenesis, Volume Retention, and Adipogenesis in a Rodent Model.

BACKGROUND: Autologous fat grafting is commonly used for soft-tissue repair (approximately 90,000 cases per year in the United States), but outcomes are limited by volume loss (20% to 80%) over time. Human allograft adipose matrix (AAM) stimulates de novo adipogenesis in vivo, but retention requires optimization. The extracellular matrix derived from superficial fascia, interstitial within the adipose layer, is typically removed during AAM processing. Thus, fascia, which contains numerous important proteins, might cooperate with AAM to stimulate de novo adipogenesis, improving long-term retention compared to AAM alone. METHODS: Human AAM and fascia matrix proteins (back and upper leg regions) were identified by mass spectrometry and annotated by gene ontology. A three-dimensional in vitro angiogenesis assay was performed. Finally, AAM and/or fascia (1 mL) was implanted into 6- to 8-week-old male Fischer rats. After 8 weeks, the authors assessed graft retention by gas pycnometry and angiogenesis (CD31) and adipocyte counts (hematoxylin and eosin) histologically. RESULTS: Gene ontology annotation revealed an angiogenic enrichment pattern unique to the fascia, including lactadherin, collagen alpha-3(V) chain, and tenascin-C. In vitro, AAM stimulated 1.0 ± 0.17 angiogenic sprouts per bead. The addition of fascia matrix increased sprouting by 88% (2.0 ± 0.12; P < 0.001). A similar angiogenic response (CD31) was observed in vivo. Graft retention volume was 25% (0.25 ± 0.13) for AAM, significantly increasing to 60% (0.60 ± 0.14) for AAM/fascia ( P < 0.05). De novo adipogenesis was 12% (12.4 ± 7.4) for AAM, significantly increasing to 51% (51.2 ± 8.0) for AAM/fascia ( P < 0.001) by means of adipocyte quantification. CONCLUSIONS: Combining fascia matrix with AAM improves angiogenesis and adipogenesis compared to AAM alone in rats. These preliminary in vitro and pilot animal studies should be further validated before definitive clinical adoption. CLINICAL RELEVANCE STATEMENT: When producing an off-the-shelf adipose inducing product by adding a connective tissue fascial component (that is normally discarded) to the mix of adipose matrix, vasculogenesis is increased and, thus, adipogenesis and graft survival is improved. This is a significant advance in this line of product.

Non-Apoptotic Caspase Activity Preferentially Targets a Novel Consensus Sequence Associated With Cytoskeletal Proteins in the Developing Auditory Brainstem.

The auditory brainstem relies on precise circuitry to facilitate sound source localization. In the chick, the development of this specialized circuitry requires non-apoptotic activity of caspase-3, for which we previously identified several hundred proteolytic substrates. Here we tested whether the sequence of the caspase cleavage site differentially encodes proteolytic preference in apoptotic and non-apoptotic contexts. We constructed a consensus sequence for caspase activity in the non-apoptotic chick auditory brainstem comprising the four residues N-terminal to the cleavage site: IX(G/R)D↓ where X represents no significant enrichment and ↓ represents the cleavage site. We identified GO terms significantly enriched among caspase substrates containing motifs found in the above consensus sequence. (G/R)D↓ was associated with the term "Structural Constituent of Cytoskeleton" (SCoC), suggesting that SCoC proteins may be specifically targeted by caspase activity during non-apoptotic developmental processes. To ascertain whether this consensus sequence was specific to the non-apoptotic auditory brainstem at embryonic day (E) 10, we used protein mass spectrometry of brainstems harvested at a time when auditory brainstem neurons undergo apoptotic cell death (E13). The apoptotic motif VD was significantly enriched among E13 cleavage sites, indicating that motif preference at the P2 subsite had shifted toward the canonical caspase consensus sequence. Additionally, Monte Carlo simulations revealed that only the GD motif was associated with SCoC substrates in the apoptotic auditory brainstem, indicating that GD encodes specificity for SCoC proteins in both non-apoptotic and apoptotic contexts, despite not being preferred in the latter. Finally, to identify candidate human non-apoptotic consensus sequences, we used Monte Carlo analyses to determine motifs and motif pairs associated with SCoC caspase substrates in the Degrabase, a database of cleavage sites in human apoptotic cell lines. We found 11 motifs significantly associated with SCoC proteolysis, including IXXD and GD. We employed a stepwise method to select motif pairs that optimized SCoC specificity for a given coverage of SCoC cleavage events, yielding 11 motif pairs likely to be preferred in SCoC-directed human non-apoptotic caspase consensus sequences. GD + IXXD was among these motif pairs, suggesting a conservation of non-apoptotic consensus sites among vertebrates.

Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development.

Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. These auditory brainstem caspase-3 substrates were enriched for proteins previously shown to be cleaved by caspase-3, especially in non-apoptotic contexts. Functional annotation analysis revealed that our caspase-3 substrates were also enriched for proteins associated with several protein categories, including proteins found in extracellular vesicles (EVs), membrane-bound nanoparticles that function in intercellular communication. The proteome of EVs isolated from the auditory brainstem was highly enriched for our caspase-3 substrates. Additionally, we identified two caspase-3 substrates with known functions in axon guidance, namely Neural Cell Adhesion Molecule (NCAM) and Neuronal-glial Cell Adhesion Molecule (Ng-CAM), that were found in auditory brainstem EVs and expressed in the auditory pathway alongside cleaved caspase-3. Taken together, these data suggest a novel developmental mechanism whereby caspase-3 influences auditory brainstem circuit formation through the proteolytic cleavage of extracellular vesicle (EV) proteins.

Structure-Based Deep Mining Reveals First-Time Annotations for 46 Percent of the Dark Annotation Space of the 9,671-Member Superproteome of the Nucleocytoplasmic Large DNA Viruses.

We conducted an exhaustive search for three-dimensional structural homologs to the proteins of 20 key phylogenetically distinct nucleocytoplasmic DNA viruses (NCLDV). Structural matches covered 429 known protein domain superfamilies, with the most highly represented being ankyrin repeat, P-loop NTPase, F-box, protein kinase, and membrane occupation and recognition nexus (MORN) repeat. Domain superfamily diversity correlated with genome size, but a diversity of around 200 superfamilies appeared to correlate with an abrupt switch to paralogization. Extensive structural homology was found across the range of eukaryotic RNA polymerase II subunits and their associated basal transcription factors, with the coordinated gain and loss of clusters of subunits on a virus-by-virus basis. The total number of predicted endonucleases across the 20 NCLDV was nearly quadrupled from 36 to 132, covering much of the structural and functional diversity of endonucleases throughout the biosphere in DNA restriction, repair, and homing. Unexpected findings included capsid protein-transcription factor chimeras; endonuclease chimeras; enzymes for detoxification; antimicrobial peptides and toxin-antitoxin systems associated with symbiosis, immunity, and addiction; and novel proteins for membrane abscission and protein turnover.IMPORTANCE We extended the known annotation space for the NCLDV by 46%, revealing high-probability structural matches for fully 45% of the 9,671 query proteins and confirming up to 98% of existing annotations per virus. The most prevalent protein families included ankyrin repeat- and MORN repeat-containing proteins, many of which included an F-box, suggesting extensive host cell modulation among the NCLDV. Regression suggested a minimum requirement for around 36 protein structural superfamilies for a viable NCLDV, and beyond around 200 superfamilies, genome expansion by the acquisition of new functions was abruptly replaced by paralogization. We found homologs to herpesvirus surface glycoprotein gB in cytoplasmic viruses. This study provided the first prediction of an endonuclease in 10 of the 20 viruses examined; the first report in a virus of a phenolic acid decarboxylase, proteasomal subunit, or cysteine knot (defensin) protein; and the first report of a prokaryotic-type ribosomal protein in a eukaryotic virus.

Induction of protection in mice against a respiratory challenge by a vaccine formulated with exosomes isolated from Chlamydia muridarum infected cells.

The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected HeLa cells (C. muridarum-exosomes), induce protective immune responses in mice following vaccination using CpG plus Montanide as adjuvants. Exosomes, collected from uninfected HeLa cells and PBS, formulated with the same adjuvants, were used as negative controls. Mass spectrometry analyses identified 113 C. muridarum proteins in the C. muridarum-exosome preparation including the major outer membrane protein and the polymorphic membrane proteins. Vaccination with C. muridarum-exosomes elicited robust humoral and cell-mediated immune responses to C. muridarum elementary bodies. Following vaccination, mice were challenged intranasally with C. muridarum. Compared to the negative controls, mice immunized with C. muridarum-exosomes were significantly protected as measured by changes in body weight, lungs' weight, and number of inclusion forming units recovered from lungs. This is the first report, of a vaccine formulated with Chlamydia exosomes, shown to elicit protection against a challenge.

Novel C1q receptor-mediated signaling controls neural stem cell behavior and neurorepair.

C1q plays a key role as a recognition molecule in the immune system, driving autocatalytic complement cascade activation and acting as an opsonin. We have previously reported a non-immune role of complement C1q modulating the migration and fate of human neural stem cells (hNSC); however, the mechanism underlying these effects has not yet been identified. Here, we show for the first time that C1q acts as a functional hNSC ligand, inducing intracellular signaling to control cell behavior. Using an unbiased screening strategy, we identified five transmembrane C1q signaling/receptor candidates in hNSC (CD44, GPR62, BAI1, c-MET, and ADCY5). We further investigated the interaction between C1q and CD44 , demonstrating that CD44 mediates C1q induced hNSC signaling and chemotaxis in vitro, and hNSC migration and functional repair in vivo after spinal cord injury. These results reveal a receptor-mediated mechanism for C1q modulation of NSC behavior and show that modification of C1q receptor expression can expand the therapeutic window for hNSC transplantation.

Vaccinia Virus Ankyrin-Repeat/F-Box Protein Targets Interferon-Induced IFITs for Proteasomal Degradation.

IFITs are interferon-induced proteins that can bind 5'-triphosphate or ribose-unmethylated capped ends of mRNA to inhibit translation. Although some viruses avoid IFITs by synthesizing RNAs with eukaryotic-like caps, no viral proteins were known to antagonize IFITs. We show that the N- and C-terminal portions of C9, a protein required for vaccinia virus to resist the human type I interferon-induced state, bind IFITs and ubiquitin regulatory complexes, respectively. Together, the two C9 domains target IFITs for proteasomal degradation, thereby providing interferon resistance similar to that also achieved by knockout of IFITs. Furthermore, ectopic expression of C9 rescues the interferon sensitivity of a vaccinia virus mutant with an inactivated cap 1-specific ribose-methyltransferase that is otherwise unable to express early proteins. In contrast, the C9-deletion mutant expresses early proteins but is blocked by IFITs at the subsequent genome uncoating/replication step. Thus, poxviruses use mRNA cap methylation and proteosomal degradation to defeat multiple antiviral activities of IFITs.

Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2.

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C-to-U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co-immunoprecipitation (co-IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size-exclusion chromatography (SEC) fractions ≥ 670 kDa, while fractions estimated to be approximately 413 kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts with RNase A abolished the relationship of editing activity with high-MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low-MW inactive fractions. Active editing factor complexes were affinity-purified using anti-RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co-IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 complexes were discovered to be highly associated with C-to-U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.

The Vaccinia virion: Filling the gap between atomic and ultrastructure.

We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an 'attachment protein' sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus.

Exploitation of nuclear functions by human rhinovirus, a cytoplasmic RNA virus.

Protein production, genomic RNA replication, and virion assembly during infection by picornaviruses like human rhinovirus and poliovirus take place in the cytoplasm of infected human cells, making them the quintessential cytoplasmic pathogens. However, a growing body of evidence suggests that picornavirus replication is promoted by a number of host proteins localized normally within the host cell nucleus. To systematically identify such nuclear proteins, we focused on those that appear to re-equilibrate from the nucleus to the cytoplasm during infection of HeLa cells with human rhinovirus via quantitative protein mass spectrometry. Our analysis revealed a highly selective re-equilibration of proteins with known mRNA splicing and transport-related functions over nuclear proteins of all other functional classes. The multifunctional splicing factor proline and glutamine rich (SFPQ) was identified as one such protein. We found that SFPQ is targeted for proteolysis within the nucleus by viral proteinase 3CD/3C, and a fragment of SFPQ was shown to migrate to the cytoplasm at mid-to-late times of infection. Cells knocked down for SFPQ expression showed significantly reduced rhinovirus titers, viral protein production, and viral RNA accumulation, consistent with SFPQ being a pro-viral factor. The SFPQ fragment that moved into the cytoplasm was able to bind rhinovirus RNA either directly or indirectly. We propose that the truncated form of SFPQ promotes viral RNA stability or replication, or virion morphogenesis. More broadly, our findings reveal dramatic changes in protein compartmentalization during human rhinovirus infection, allowing the virus to systematically hijack the functions of proteins not normally found at its cytoplasmic site of replication.

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size.

Recapitulating the human tumor microenvironment: Colon tumor-derived extracellular matrix promotes angiogenesis and tumor cell growth.

Extracellular matrix (ECM) is an essential and dynamic component of all tissues and directly affects cellular behavior by providing both mechanical and biochemical signaling cues. Changes in ECM can alter tissue homeostasis, potentially leading to promotion of cellular transformation and the generation of tumors. Therefore, understanding ECM compositional changes during cancer progression is vital to the development of targeted treatments. Previous efforts to reproduce the native 3D cellular microenvironment have utilized protein gels and scaffolds that incompletely recapitulate the complexity of native tissues. Here, we address this problem by extracting and comparing ECM from normal human colon and colon tumor that had metastasized to liver. We found differences in protein composition and stiffness, and observed significant differences in vascular network formation and tumor growth in each of the reconstituted matrices, both in vitro and in vivo. We studied free/bound ratios of NADH in the tumor and endothelial cells using Fluorescence Lifetime Imaging Microscopy as a surrogate for the metabolic state of the cells. We observed that cells seeded in tumor ECM had higher relative levels of free NADH, consistent with a higher glycolytic rate, than those seeded in normal ECM. These results demonstrate that the ECM plays an important role in the growth of cancer cells and their associated vasculature.

Migration of breast cancer cell lines in response to pulmonary laminin 332.

Because tumor cell motility is a requirement for metastasis, we hypothesized that lung tissue harbors substances that induce tumor cell migration. MCF-7 breast carcinoma cells exposed to small airway epithelial cells and conditioned medium exhibited dose-dependent tumor cell migration. Among the extracellular matrix proteins in the conditioned medium identified by mass spectrometry, laminin 332 (LM332) had the greatest contribution to the migration of MCF-7 cells. Immunoblotting and immunohistochemistry for LM332-specific chains identified LM332 in the lung and in pulmonary epithelial cells. Antibodies to either LM332 or its integrin receptor inhibited MCF-7 motility, and knockdown of LM332 chains also reduced its migration-inducing activity. Taken together, these findings implicate LM332 as a component of lung tissue that can induce motility in breast carcinoma cells that have been transported to lung during metastasis. Earlier studies on LM332 in tumor progression have examined LM332 expression in tumor cells. This investigation, in comparison, provides evidence that the tumor promoting potential of LM332 may originate in the lung microenvironment rather than in tumor cells alone. Furthermore, this study provides evidence that the motility-inducing properties of the microenvironment can reside in epithelial cells. The findings raise the possibility that LM332 plays a role in the pulmonary metastases of breast carcinoma and may provide a target for antimetastasis therapy.

Protein Primary Structure of the Vaccinia Virion at Increased Resolution

Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (J. Matson, W. Chou, T. Ngo, and P. D. Gershon, Virology 452-453:310-323, 2014, doi:http://dx.doi.org/10.1016/j.virol.2014.01.012) to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation "hot spots" within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of vaccinia virus proteins. IMPORTANCE: Poxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry.

Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5' and 3' Noncoding Regions of Genomic RNAs.

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3' or 5' noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication.

Single nucleoprotein residue modulates arenavirus replication complex formation.

UNLABELLED: The Arenaviridae are enveloped, negative-sense RNA viruses with several family members that cause hemorrhagic fevers. This work provides immunofluorescence evidence that, unlike those of New World arenaviruses, the replication and transcription complexes (RTC) of lymphocytic choriomeningitis virus (LCMV) colocalize with eukaryotic initiation factor 4E (eIF4E) and that eIF4E may participate in the translation of LCMV mRNA. Additionally, we identify two residues in the LCMV nucleoprotein (NP) that are conserved in every mammalian arenavirus and are required for recombinant LCMV recovery. One of these sites, Y125, was confirmed to be phosphorylated by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). NP Y125 is located in the N-terminal region of NP that is disordered when RNA is bound. The other site, NP T206, was predicted to be a phosphorylation site. Immunofluorescence analysis demonstrated that NP T206 is required for the formation of the punctate RTC that are typically observed during LCMV infection. A minigenome reporter assay using NP mutants, as well as Northern blot analysis, demonstrated that although NP T206A does not form punctate RTC, it can transcribe and replicate a minigenome. However, in the presence of matrix protein (Z) and glycoprotein (GP), translation of the minigenome message with NP T206A was inhibited, suggesting that punctate RTC formation is required to regulate viral replication. Together, these results highlight a significant difference between New and Old World arenaviruses and demonstrate the importance of RTC formation and translation priming in RTC for Old World arenaviruses. IMPORTANCE: Several members of the Arenaviridae cause hemorrhagic fevers and are classified as category A pathogens. Arenavirus replication-transcription complexes (RTC) are nucleated by the viral nucleoprotein. This study demonstrates that the formation of these complexes is required for virus viability and suggests that RTC nucleation is regulated by the phosphorylation of a single nucleoprotein residue. This work adds to the body of knowledge about how these key viral structures are formed and participate in virus replication. Additionally, the fact that Old World arenavirus complexes colocalize with the eukaryotic initiation factor 4E, while New World arenaviruses do not, is only the second notable difference observed between New and Old World arenaviruses, the first being the difference in the glycoprotein receptor.