Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

MIT CAACB Risk Assessment Case Study: Assessing Virus Cross-Contamination Risk between Two Simultaneous Processes in an Open Biomanufacturing Facility.

Some members of MIT's Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) previously published content on the "Quality Risk Management in the Context of Viral Contamination", which described tools, procedures, and methodologies for assessing and managing the risk of a potential virus contamination in cell culture processes. To address the growing industry interest in moving manufacturing toward open ballrooms with functionally closed systems and to demonstrate how the ideas of risk management can be leveraged to perform a risk assessment, CAACB conducted a case study exercise of these new manufacturing modalities. In the case study exercise, a cross-functional team composed of personnel from many of CAACB's industry membership collaboratively assessed the risks of viral cross-contamination between a human and non-human host cell system in an open manufacturing facility. This open manufacturing facility had no walls to provide architectural separation of two processes occurring simultaneously, specifically a recombinant protein perfusion cell culture process using the human cell line, HEK-293 (Process 1) and a downstream postviral filtration unit operation (Process 2) of a recombinant protein produced in CHO cells. This viral risk assessment focused on cross-contamination of the Process 2 filtration unit operation after the Process 1 perfusion bioreactor was contaminated with a virus that went undetected. The workflow for quality risk management that is recommended by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) was followed, which included identifying and mapping the manufacturing process, defining the risk question, risk evaluation, and risk control. The case study includes a completed Failure Mode and Effects Analysis (FMEA) to provide descriptions of the specific risks and corresponding recommended risk reduction actions.

Large-scale cultured meat production: Trends, challenges and promising biomanufacturing technologies.

Food systems of the future will need to face an increasingly clear reality - that a protein-rich diet is essential for good health, but traditional meat products will not suffice to ensure safety, sustainability, and equity of food supply chains at a global scale. This paper provides an in-depth analysis of bioprocess technologies needed for cell-based meat production and challenges in reaching commercial scale. Specifically, it reviews state-of-the-art bioprocess technologies, current limitations, and opportunities for research across four domains: cell line development, cell culture media, scaffolding, and bioreactors. This also includes exploring innovations to make cultured meat a viable protein alternative across numerous key performance indicators and for specific applications where traditional livestock is not an option (e.g., local production, space exploration). The paper explores tradeoffs between production scale, product quality, production cost, and footprint over different time horizons. Finally, a discussion explores various factors that may impact the ability to successfully scale and market cultured meat products: social acceptance, environmental tradeoffs, regulatory guidance, and public health benefits. While the exact nature of the transition from traditional livestock to alternative protein products is uncertain, it has already started and will likely continue to build momentum in the next decade.

Conserved Outer Tegument Component UL11 from Herpes Simplex Virus 1 Is an Intrinsically Disordered, RNA-Binding Protein.

A distinguishing morphological feature of all herpesviruses is the multiprotein tegument layer located between the nucleocapsid and lipid envelope of the virion. Tegument proteins play multiple roles in viral replication, including viral assembly, but we do not yet understand their individual functions or how the tegument is assembled and organized. UL11, the smallest tegument protein, is important for several distinct processes in replication, including efficient virion morphogenesis and cell-cell spread. However, the mechanistic understanding of its role in these and other processes is limited in part by the scant knowledge of its biochemical and structural properties. Here, we report that UL11 from herpes simplex virus 1 (HSV-1) is an intrinsically disordered, conformationally dynamic protein that undergoes liquid-liquid phase separation (LLPS) in vitro Intrinsic disorder may underlie the ability of UL11 to exert multiple functions and bind multiple partners. Sequence analysis suggests that not only all UL11 homologs but also all HSV-1 tegument proteins contain intrinsically disordered regions of different lengths. The presence of intrinsic disorder, and potentially, the ability to form LLPS, may thus be a common feature of the tegument proteins. We hypothesize that tegument assembly may involve the formation of a biomolecular condensate, driven by the heterogeneous mixture of intrinsically disordered tegument proteins.IMPORTANCE Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate.

Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay.

Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and resources used for protein purification and further studies. This protocol describes the use of the Thermofluor assay, in combination with a custom 24-condition screen, to identify buffer conditions that increase protein thermostability, using the conserved herpesviral protein UL37 as an example. Detailed instructions on screen conditions, running the Thermofluor MATLAB script, and analyzing the data are provided. In comparison to circular dichroism (CD), the buffer screen in combination with Thermofluor assay provides a faster and more informative method to analyze protein thermostability.

The dynamic nature of the conserved tegument protein UL37 of herpesviruses.

In all herpesviruses, the space between the capsid shell and the lipid envelope is occupied by the unique tegument layer composed of proteins that, in addition to structural roles, play many other roles in the viral replication. UL37 is a highly conserved tegument protein that has activities ranging from virion morphogenesis to directional capsid trafficking to manipulation of the host innate immune response and binds multiple partners. The N-terminal half of UL37 (UL37N) has a compact bean-shaped α-helical structure that contains a surface region essential for neuroinvasion. However, no biochemical or structural information is currently available for the C-terminal half of UL37 (UL37C) that mediates most of its interactions with multiple binding partners. Here, we show that the C-terminal half of UL37 from pseudorabies virus UL37C is a conformationally flexible monomer composed of an elongated folded core and an unstructured C-terminal tail. This elongated structure, along with that of its binding partner UL36, explains the nature of filamentous tegument structures bridging the capsid and the envelope. We propose that the dynamic nature of UL37 underlies its ability to perform diverse roles during viral replication.

Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1.

Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37.IMPORTANCE The ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires detailed navigational charts in the form of their three-dimensional structures. Here, we determined the crystal structure of the N-terminal half of a conserved tegument protein, UL37, from HSV-1. This structure, along with our previously reported structure of the UL37 homolog from PRV, provides a much needed 3-dimensional template for the dissection of both conserved and virus-specific functions of UL37 in intracellular capsid trafficking.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.

Identification and characterization of a minisatellite contained within a novel miniature inverted-repeat transposable element (MITE) of Porphyromonas gingivalis.

BACKGROUND: Repetitive regions of DNA and transposable elements have been found to constitute large percentages of eukaryotic and prokaryotic genomes. Such elements are known to be involved in transcriptional regulation, host-pathogen interactions and genome evolution. RESULTS: We identified a minisatellite contained within a miniature inverted-repeat transposable element (MITE) in Porphyromonas gingivalis. The P. gingivalis minisatellite and associated MITE, named 'BrickBuilt', comprises a tandemly repeating twenty-three nucleotide DNA sequence lacking spacer regions between repeats, and with flanking 'leader' and 'tail' subunits that include small inverted-repeat ends. Forms of the BrickBuilt MITE are found 19 times in the genome of P. gingivalis strain ATCC 33277, and also multiple times within the strains W83, TDC60, HG66 and JCVI SC001. BrickBuilt is always located intergenically ranging between 49 and 591 nucleotides from the nearest upstream and downstream coding sequences. Segments of BrickBuilt contain promoter elements with bidirectional transcription capabilities. CONCLUSIONS: We performed a bioinformatic analysis of BrickBuilt utilizing existing whole genome sequencing, microarray and RNAseq data, as well as performing in vitro promoter probe assays to determine potential roles, mechanisms and regulation of the expression of these elements and their affect on surrounding loci. The multiplicity, localization and limited host range nature of MITEs and MITE-like elements in P. gingivalis suggest that these elements may play an important role in facilitating genome evolution as well as modulating the transcriptional regulatory system.