Human Cytomegalovirus pUL37x1 is Important to Remodeling of Host Lipid Metabolism [Virus-Cell Interactions]

Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential to infection. While multiple host factors, including kinases and transcription factors, important to metabolic changes that occur following HCMV infection have been identified, little is known about the viral factors required to alter metabolism. In this study, we tested the hypothesis that pUL37x1 is important to the metabolic remodeling that is necessary for HCMV replication using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had similar levels of metabolites as those infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that relative to WT-infected cells subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infection—fatty acid elongase 7 (ELOVL7) and ER-stress related kinase PERK. Moreover, we observed that HCMV infection results in an increase in phospholipids with very long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells compared to WT-infected cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes associated with HCMV-infection, it is important to the remodeling of a subset of metabolic changes that occur during infection.

Importance

Human cytomegalovirus (HCMV) is a common pathogen that asymptomatically infects most people and establishes a lifelong infection. However, HCMV can cause end-organ disease that results in death in the immunosuppressed and is a leading cause of birth defects. HCMV infection depends on host metabolism, including lipid metabolism. However, the viral mechanisms for remodeling metabolism are poorly understood. In this study, we demonstrate that the viral UL37x1 protein (pUL37x1) is important for infection-associated increases in lipid metabolism, including fatty acid elongation to produce very long-chain fatty acids (VLCFAs). Further, we found that HCMV infection results in a significant increase in phospholipids, particularly those with VLCFA tails (PL-VLCFAs). We found that pUL37x1 was important for the high levels of fatty acid elongation and PL-VLCFAs accumulation that occurs in HCMV-infected cells. Our findings identify a viral protein that is important for changes to lipid metabolism that occurs following HCMV infection.

Contributions of Lower Body Strength Parameters to Critical Power and Anaerobic Work Capacity

This study examined the contribution of lower body strength and isokinetic peak torque measures to the prediction of critical power (CP) and anaerobic work capacity (AWC). Fourteen recreationally trained males (mean ± SD age: 22.4 ± 2.5 yrs; height: 177.9 ± 7.7 cm; body mass: 84.2 ± 12.4 kg) with anaerobic training experience completed this study. The lower body strength measures included one repetition max (1RM) bilateral back squat [BSq], isokinetic peak torque at 30°·sec-1 [PT30] and isokinetic peak torque at 240°·sec-1 [PT240]) of the dominant leg. The CP and AWC were determined from the 3-min all-out CP cycle ergometer test (CP3MT), with the resistance set at 4.5% of the total body mass. The CP was defined as the mean power output over the final 30s of the test and the AWC was calculated using the equation, AWC = 150s (P150 – CP), where P150 equals the mean power output for the first 150s. Stepwise regression analyses indicated that only BSq contributed significantly to the prediction of AWC (AWC = 0.0527[BSq] + 8.094 [SEE = 2.151 kJ; p = 0.012]), with a correlation of r2 = 0.423. None of the strength parameters significantly predicted CP. These findings indicated that BSq strength accounted for 42% of the variance in AWC, but lower body strength was not related to CP. The current results indirectly supports the unique metabolic characteristics of both CP and AWC in providing separate measures of an individual’s aerobic and anaerobic capabilities, respectively.
Corresponding Author: M. Travis Byrd, Department of Kinesiology and Health Promotion, University of Kentucky, 100 Seaton Building, Lexington, KY 40506-0219, Phone: 859-200-6859, Fax: 859-323-1090, Email: mark.travis.byrd@uky.edu
Copyright © 2019 by the National Strength & Conditioning Association.

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Attenuation of equine lentivirus alters mitochondrial protein expression profile from inflammation to apoptosis [Cellular Response to Infection]

Equine infectious anemia virus (EIAV) is an equine lentivirus similar to HIV-1, targets to host immune cells and causes life-long infection in horses. The Chinese live EIAV vaccine is attenuated from long-term passaging of a high virulent strain in vitro. The parent pathogenic strain (EIAVDLV34) induces a host inflammatory storm to cause severe pathological injury of animals. However, the vaccine strain (EIAVDLV121) induces a high level of apoptosis to eliminate the infected cells. To investigate how these processes are regulated, we performed a comparative proteomics analysis and functional study in equine monocyte-derived macrophages (eMDMs), and found that divergent mitochondrial protein expression profiles caused by EIAV strains with different virulence lead to disparate mitochondrial function, morphology and metabolism. This in turn promoted distinct transformation of macrophage inflammatory polarization and intrinsic apoptosis. In EIAVDLV34 infected cells, a high level of glycolysis and increased mitochondrial fragmentation were induced, resulting in M1-polarized pro-inflammatory type transformation of macrophages and subsequently producing a strong inflammatory response. Following infection with EIAVDLV121, the infected cells were transformed into M2-polarized anti-inflammatory macrophages by inhibition of glycolysis. In this case, decrease of mitochondrial membrane potential and impairment of electronic respiratory chain led to increased levels of apoptosis and ROS. These results are correlated with the viral pathogenicity loss and may help to understand the key mechanism of lentiviral attenuation.

IMPORTANCE

Following viral infection, the working pattern and function of the cell can be transformed through the impact on mitochondria. It still unknown how mitochondrial response changes in the cells infected with viruses in the process of virulence attenuation. EIAVDLV121 is the only effective lentiviral vaccine for large-scale use in world. EIAVDLV34 is a parent pathogenic strain. Unlike EIAVDLV34-induced inflammation storms, EIAVDLV121 can induce high levels of apoptosis. For the first time, we found that, after altering mitochondrial protein expression profile, EIAVDLV34 infected cells are transformed into M1-polarized type macrophages to cause inflammatory injury and the intrinsic apoptosis pathway is activated in EIAVDLV121 infected cells. These studies shed light on how the mitochondrial protein expression profile change from cells infected by pathogenic or attenuated lentivirus strains to drive different cellular response, especially from inflammation to apoptosis.

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TMPRSS2 is the major activating protease of influenza A virus in primary human airway cells and influenza B virus in human type II pneumocytes [Virus-Cell Interactions]

Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A and B virus (IAV/IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by as-yet undetermined protease(s).

Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII) and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9 and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited activation and spread of IBV in AECII, but did not affect IBV activation in HBEC and Calu-3 cells.

This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.

Importance

Influenza A and B viruses (IAV/IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site and a number of proteases have been shown to cleave HA in vitro. This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoire. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.

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Resistance exercise order does not affect the magnitude and duration of post-exercise blood pressure in older women

Abstract:
The aim of this study was to compare the effects of two resistance exercise order on post-exercise blood pressure (BP) in trained, non-hypertensive older women. Sixteen women (68.3 ± 3.3 years, 63.5 ± 11.6 kg, 157.5 ± 5.1 cm) performed two sessions with
eight exercises (3 sets of 8-12 repetitions) in distinct orders (from multi- to single-joint exercises [MS] or from single- to multi-joint exercises [SM]) and a control session, without exercise. Blood pressure and heart rate were obtained pre and post-sessions (60 min). Post-exercise hypotension was observed for systolic and mean BP in both the MS session (systolic BP: -6.9 mmHg, mean BP: -3.3 mmHg, P< 0.05) and SM session (systolic BP: -4.6 mmHg; mean BP: -1.1 mmHg). Post-exercise heart rate was higher than pre-session values until 30 min of recovery in both training sessions. Furthermore, systolic and mean blood pressure, and heart rate were lower than the values obtained in the control session (30 to 60 min and 0 min, respectively; P<0.05). There were no differences between the SM and MS sessions in any variable or at any moment. In conclusion, resistance exercise order does not interfere in the magnitude and duration of post-exercise hypotension in trained, non-hypertensive older women.
Corresponding author at: Crisieli Maria Tomeleri. João Garla street, 300. Cambé, Paraná, Brazil. ZIPCode: 86192-180. Phone +55 (43) 9917-7079; e-mail: crisieli@uol.com.br
There were no external funding sources for this study.
Copyright © 2019 by the National Strength & Conditioning Association.

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Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication [Structure and Assembly]

During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) UL34 and UL31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins in vitro. Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein UL25, but not to the other capsid proteins tested (i.e., VP5, VP23 and UL17) and that HSV-1 NEC binding nucleocapsids was mediated by NEC interaction with UL25. UL31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and UL25. We also showed that alanine substitution of UL31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles, that were similar in size and morphology to primary envelopes, in the perinuclear space. These results suggested that NEC binding via UL31 R281 and D282 to nucleocapsids, probably to UL25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment.

IMPORTANCE

Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis has not been reported thus far. In this study, we have presented data showing that two amino acids in the membrane distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.

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Cell Line Models for Human Cytomegalovirus Latency Faithfully Mimic Viral Entry by Macropinocytosis and Endocytosis [Virus-Cell Interactions]

Human cytomegalovirus (HCMV) enters primary CD34+ hematopoietic progenitor cells by macropinocytosis where it establishes latency in part because its tegument transactivating protein, pp71, remains associated with endosomes and is therefore unable to initiate productive, lytic replication. Here we show that multiple HCMV strains also enter cell line models used to study latency by macropinocytosis and endocytosis. In all latency models tested, tegument-delivered pp71 was found co-localized with endosomal markers and not associated with the seven other cytoplasmic localization markers tested. Like the capsid-associated pp150 tegument protein, we detected capsid proteins initially associated with endosomes but later in the nucleus. Inhibitors of macropinocytosis and endocytosis reduced latent viral gene expression and precluded reactivation. Importantly, we utilized electron microscopy to observe entry by macropinocytosis and endocytosis, providing additional visual corroboration to our functional studies. Our demonstration that HCMV enters cell line models for latency in a manner indistinguishable from its entry into primary cells illustrates the utility of these cell lines for probing the mechanisms, host genetics, and small molecule-mediated inhibition of HCMV entry into the cell types where it establishes latency.

Importance

Primary cells cultured in vitro currently provide the highest available relevance for examining molecular and genetic requirements for the establishment, maintenance, and reactivation of HCMV latency. However, their expense, heterogeneity, and intransigence to both long-term culture and molecular or genetic modification create rigor and reproducibility challenges for HCMV latency studies. There are several cell line models for latency not obstructed by deficiencies inherent in primary cells. However, many researchers view cell line studies of latency as physiologically irrelevant because of the perception that these models display numerous and significant differences from primary cells. Here we show that the very first step in a latent HCMV infection, entry of the virus into cells, occurs in cell line models indistinguishably from how it occurs in primary CD34+ hematopoietic progenitor cells. Our data argue that experimental HCMV latency in cell lines and primary cells is much more similar than it is different.

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Sol Belén Colqui: 3D Printing Is My Job And My Hobby

Sol Belén Colqui [Source: Women in 3D Printing]

Sol Belén Colqui is a young enthusiast, passionate about self-learning and entrepreneurship.

Her passion for knowledge has led her to train in the area of psychology, accounting, and programming. She met the world of additive manufacturing three years ago and since then she has perfected her 3D modeling and printing skills, investigating different tools, developments, and applications. She is the founder of #MujeresqueHacen, a makerspace oriented only to women where projects are developed combining different specializations (art, robotics, design, clothing, 3D printing).

Nora Toure: Sol, could you let us know about your background and what brought you to 3D printing in the first place?

Sol Belén Colqui: After finishing my studies in Economics and organizational management I specialized in accounting, then I started my degree in psychology. I met the 3d printing industry thanks to a meeting of a RepRap community that I attended out of curiosity.

Nora Toure: What was your very first experience with 3D Printing?

Sol Belén Colqui: My first approach to 3d printing was a keychain that was given to me, but the best experience was when I had my first 3d printer, I spent twenty minutes staring at a calibration cube.

Nora Toure: Could you explain furthermore what Design.S3D is and the services that you are providing?

Sol Belén Colqui: We provide design and modeling service, printing and training to companies. We have already worked for the police, the subway company and universities. Now, a large part of our work is about printing large scale pieces and complex designs, collaborating with designers, theater companies and artists.

Nora Toure: How did you come to build the company?

Sol Belén Colqui: Design.S3D was my second step in 3D printing, encouraging me to apply my knowledge and facilitate additive manufacturing to those who need it. This project allowed me to face new challenges and to know new technologies.

Nora Toure: You are also Women in 3D Printing Buenos Aires ambassador. What can you tell us about Buenos Aires’s community?

Sol Belén Colqui: Argentina has a collaborative community that is always willing to share their knowledge, support and help others. These months working as an ambassador for Wi3DP has allowed me to meet wonderful, intelligent and inspiring people.

As a community, we define ourselves as being enthusiastic and innovative. We share our experiences, projects and help each other.

Read the rest at Women in 3D Printing

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Comparative evaluation of the vaccine efficacy of three adenovirus-based vector types in the Friend retrovirus infection model [Vaccines and Antiviral Agents]

Adenovirus (AdV)-based vectors are popular experimental vaccine vectors, but despite their ability to induce strong immune responses, their application is impeded by wide-spread pre-existing immunity against many AdV types that can impair or even abrogate induction of transgene-specific immune responses. Therefore, the development of vectors based on AdV types with low seroprevalence is important for effective AdV-based immunization in humans.

We investigated the immunization efficacy of vectors based on AdV types 48 and 50 in the ovalbumin (ova) model as well as the Friend retrovirus (FV) model, which allows testing the protective effect of vaccine-induced immunity. Using ova-encoding vectors, we found a significantly lower induction of ova-specific CD8+ T cells and antibody responses by Ad48- and Ad50-based vectors compared to Ad5. Similarly, we found a reduced induction of FV-specific CD8+ T cell responses in Ad48- and Ad50.Leader-Gag-immunized mice compared to Ad5; however, some of those mice were able to control the FV infection, and protection correlated with the level of neutralizing antibodies 10 days after FV challenge. Analyses of AdV-specific antibodies and CD8+ T cells induced by the individual AdV types revealed a high level of cross-reactivity, and the efficacy of Ad48-based immunization was impaired in Ad5 pre-immune mice.

Our results show that immunity induced by Ad48- and Ad50-based vectors is reduced compared to Ad5, and is sufficient only in some of the immunized mice to control FV infection. A high level of cross-reactivity suggests that AdV pre-immunity must be considered even when applying rare AdV based vectors.

Importance

AdV-based vectors are important tools for the development of vaccines against a wide range of pathogens. While AdV vectors are generally considered safe and highly effective, their application can be severely impaired by pre-existing immunity due to wide-spread seroprevalence of some AdV types. The characterization of different AdV types with regard to immunogenicity and efficacy in challenge models is of great importance for the development of improved AdV-based vectors that allow for efficient immunization despite anti-AdV immunity. We show that immunity induced by an Ad48-based vector is inferior to Ad5, but can still mediate control of an FV infection in highly FV-susceptible mice. However, the efficacy of Ad48-based immunization was impaired in Ad5 pre-immune mice. Importantly, we found cross-reactivity of both humoral and cellular immune responses raised by the individual AdV types, suggesting that switching to a different AdV type may not be sufficient to circumvent pre-existing anti-AdV immunity.

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Vaccinia Virus Strain MVA can Induce Optimal CD8+ T cell responses to Directly Primed Antigens Depending on Vaccine Design [Pathogenesis and Immunity]

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. Whilst much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as Modified Vaccinia Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because it contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA.

Importance

Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here we find that the primacy of stable antigen is not generalisable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA, and so possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.

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