Differences in the anatomy and physiology of the human and rat respiratory tracts and impact on toxicological assessments.

Inhalation is a critical route through which substances can exert adverse effects in humans; therefore, it is important to characterize the potential effects that inhaled substances may have on the human respiratory tract by using fit for purpose, reliable, and human relevant testing tools. In regulatory toxicology testing, rats have primarily been used to assess the effects of inhaled substances as they-being mammals-share similarities in structure and function of the respiratory tract with humans. However, questions about inter-species differences impacting the predictability of human effects have surfaced. Disparities in macroscopic anatomy, microscopic anatomy, or physiology, such as breathing mode (e.g., nose-only versus oronasal breathing), airway structure (e.g., complexity of the nasal turbinates), cell types and location within the respiratory tract, and local metabolism may impact inhalation toxicity testing results. This review shows that these key differences describe uncertainty in the use of rat data to predict human effects and supports an opportunity to harness modern toxicology tools and a detailed understanding of the human respiratory tract to develop testing approaches grounded in human biology. Ultimately, as the regulatory purpose is protecting human health, there is a need for testing approaches based on human biology and mechanisms of toxicity.

Progress and Remaining Opportunities to Increase the Use of Animal-free Antibodies in the USA.

The scientific and ethical issues associated with the use of animal-derived antibodies in research can be overcome by the use of animal-free, sequence-defined recombinant antibodies, whose benefits are well documented. Here, we describe progress made following a 2019 expert meeting focused on improving the quality and reproducibility of biomedical research by accelerating the production and use of animal-free recombinant antibodies in the USA. In the five intervening years since the meeting, participants have established multifaceted initiatives to tackle the next steps outlined during the meeting. These initiatives include: prioritising the replacement of ascites-derived and polyclonal antibodies; distributing educational materials describing recombinant antibodies; fostering public-private partnerships to increase access to recombinant antibodies; and increasing the availability of funding for recombinant antibody development. Given the widescale use of antibodies across scientific disciplines, a transition to modern antibody production methods relies on a commitment from government agencies, universities, industry and funding organisations, to initiatives such as those outlined here.

Defined approaches to classify agrochemical formulations into EPA hazard categories developed using EpiOcularTM reconstructed human corneal epithelium and bovine corneal opacity and permeability assays.

Many sectors have seen complete replacement of the in vivo rabbit eye test with reproducible and relevant in vitro and ex vivo methods to assess the eye corrosion/irritation potential of chemicals. However, the in vivo rabbit eye test remains the standard test used for agrochemical formulations in some countries. Therefore, two defined approaches (DAs) for assessing conventional agrochemical formulations were developed, using the EpiOcularTM Eye Irritation Test (EIT) [Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 492] and the Bovine Corneal Opacity and Permeability (OECD TG 437; BCOP) test with histopathology. Presented here are the results from testing 29 agrochemical formulations, which were evaluated against the United States Environmental Protection Agency's (EPA) pesticide classification system, and assessed using orthogonal validation, rather than direct concordance analysis with the historical in vivo rabbit eye data. Scientific confidence was established by evaluating the methods and testing results using an established framework that considers fitness for purpose, human biological relevance, technical characterisation, data integrity and transparency, and independent review. The in vitro and ex vivo methods used in the DAs were demonstrated to be as or more fit for purpose, reliable and relevant than the in vivo rabbit eye test. Overall, there is high scientific confidence in the use of these DAs for assessing the eye corrosion/irritation potential of agrochemical formulations.

A framework for establishing scientific confidence in new approach methodologies.

Robust and efficient processes are needed to establish scientific confidence in new approach methodologies (NAMs) if they are to be considered for regulatory applications. NAMs need to be fit for purpose, reliable and, for the assessment of human health effects, provide information relevant to human biology. They must also be independently reviewed and transparently communicated. Ideally, NAM developers should communicate with stakeholders such as regulators and industry to identify the question(s), and specified purpose that the NAM is intended to address, and the context in which it will be used. Assessment of the biological relevance of the NAM should focus on its alignment with human biology, mechanistic understanding, and ability to provide information that leads to health protective decisions, rather than solely comparing NAM-based chemical testing results with those from traditional animal test methods. However, when NAM results are compared to historical animal test results, the variability observed within animal test method results should be used to inform performance benchmarks. Building on previous efforts, this paper proposes a framework comprising five essential elements to establish scientific confidence in NAMs for regulatory use: fitness for purpose, human biological relevance, technical characterization, data integrity and transparency, and independent review. Universal uptake of this framework would facilitate the timely development and use of NAMs by the international community. While this paper focuses on NAMs for assessing human health effects of pesticides and industrial chemicals, many of the suggested elements are expected to apply to other types of chemicals and to ecotoxicological effect assessments.

Replacement of in vivo leptospirosis vaccine potency testing in the United States.

The U.S. Department of Agriculture (USDA) regulates the potency testing of leptospirosis vaccines, which are administered to animals to protect against infection by Leptospira bacteria. Despite the long-term availability of in vitro test methods for assessing batch potency, the use of hamsters in lethal in vivo batch potency testing persists to varying degrees across leptospirosis vaccine manufacturers. For all manufacturers of these products, data collected from public USDA records show an estimated 40% decline in the annual use of hamsters from 2014 to 2020, with an estimated 55% decrease in the number of hamsters expected to have been used in leptospirosis vaccine potency tests (i.e., those in USDA Category E). An estimated 49,000 hamsters were used in 2020, with about 15,000 hamsters in Category E specifically. Based on this assessment, additional efforts are needed to fully implement in vitro batch potency testing as a replacement for the in vivo batch potency test. We propose steps that can be taken collaboratively by the USDA Center for Veterinary Biologics (CVB), manufacturers of leptospirosis vaccines, government agencies, and non-governmental organizations to accelerate broader use of the in vitro approach.

Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

Use of Cause-and-Effect Analysis to Optimize the Reliability of In Vitro Inhalation Toxicity Measurements Using an Air-Liquid Interface.

In vitro inhalation toxicology methods are increasingly being used for research and regulatory purposes. Although the opportunity for increased human relevance of in vitro inhalation methods compared to in vivo tests has been established and discussed, how to systematically account for variability and maximize the reliability of these in vitro methods, especially for assays that use cells cultured at an air-liquid interface (ALI), has received less attention. One tool that has been used to evaluate the robustness of in vitro test methods is cause-and-effect (C&E) analysis, a conceptual approach to analyze key sources of potential variability in a test method. These sources of variability can then be evaluated using robustness testing and potentially incorporated into in-process control measurements in the assay protocol. There are many differences among in vitro inhalation test methods including the use of different types of biological test systems, exposure platforms/conditions, substances tested, and end points, which represent a major challenge for use in regulatory testing. In this manuscript, we describe how C&E analysis can be applied using a modular approach based on the idea that shared components of different test methods (e.g., the same exposure system is used) have similar sources of variability even though other components may differ. C&E analyses of different in vitro inhalation methods revealed a common set of recommended exposure systems and biological in-process control measurements. The approach described here, when applied in conjunction with Good Laboratory Practices (GLP) criteria, should help improve the inter- and intralaboratory agreement of in vitro inhalation test results, leading to increased confidence in these methods for regulatory and research purposes.

Human-relevant approaches to assess eye corrosion/irritation potential of agrochemical formulations.

There are multiple in vitro and ex vivo eye irritation and corrosion test methods that are available as internationally harmonized test guidelines for regulatory use. Despite their demonstrated usefulness to a broad range of substances through inter-laboratory validation studies, they have not been widely adopted for testing agrochemical formulations due to a lack of concordance with parallel results from the traditional regulatory test method for this endpoint, the rabbit eye test. The inherent variability of the rabbit test, differences in the anatomy of the rabbit and human eyes, and differences in modelling exposures in rabbit eyes relative to human eyes contribute to this lack of concordance. Ultimately, the regulatory purpose for these tests is protection of human health, and, thus, there is a need for a testing approach based on human biology. This paper reviews the available in vivo, in vitro and ex vivo test methods with respect to their relevance to human ocular anatomy, anticipated exposure scenarios, and the mechanisms of eye irritation/corrosion in humans. Each of the in vitro and ex vivo methods described is generally appropriate for identifying non-irritants. To discriminate among eye irritants, the human three-dimensional epithelial and full thickness corneal models provide the most detailed information about the severity of irritation. Consideration of the mechanisms of eye irritation, and the strengths and limitations of the in vivo, in vitro and ex vivo test methods, show that the in vitro/ex vivo methods are as or more reflective of human biology and less variable than the currently used rabbit approach. Suggestions are made for further optimizing the most promising methods to distinguish between severe (corrosive), moderate, mild and non-irritants and provide information about the reversibility of effects. Also considered is the utility of including additional information (e.g. physical chemical properties), consistent with the Organization for Economic Cooperation and Development's guidance document on an integrated approach to testing and assessment of potential eye irritation. Combining structural and functional information about a test substance with test results from human-relevant methods will ensure the best protection of humans following accidental eye exposure to agrochemicals.

Dynein mediates the localization and activation of mTOR in normal and human cytomegalovirus-infected cells.

Activation of stress signaling pathways normally leads to inhibition of the mammalian target of rapamycin complex 1 (mTORC1); however, human cytomegalovirus (HCMV) infection maintains mTORC1 activity in the presence of numerous types of stress. We previously demonstrated that HCMV infection maintains mTORC1 activity during amino acid deprivation through a Ras-related GTP-binding (Rag) protein-independent mechanism. This depends on the colocalization of mTOR and its activator, Rheb (Ras homology enriched in brain)-GTP, to a perinuclear position that corresponds to the viral cytoplasmic assembly compartment (AC). The data presented here show that the HCMV-induced, amino acid depletion-resistant perinuclear localization and activation of mTORC1 occurs as early as 8 h post-infection, prior to AC formation. We show that the molecular motor dynein is required for perinuclear localization of mTORC1 in both uninfected and HCMV-infected cells. Association between dynein and mTOR is shown by coimmunoprecipitation, and inhibition of dynein function using RNAi or the small molecule inhibitor ciliobrevin A inhibits mTORC1 activity in both uninfected and HCMV-infected cells. The data suggest that mTORC1 activation requires dynein-dependent transport to a position in the cell where it can be activated. Thus, the HCMV commandeers a cellular dynein-dependent mTORC1 activation mechanism to maintain stress-resistant mTORC1 activity during infection and to form the AC.

Evaluation of the avian acute oral and sub-acute dietary toxicity test for pesticide registration.

The United States Environmental Protection Agency (USEPA), as well as other international regulatory agencies, require pesticide registrants to submit toxicity data that are used to conduct ecological risk assessments. While the USEPA has required both an acute oral and sub-acute dietary test in birds, trends in the use of data from these tests over the past 20 years have suggested that the avian sub-acute dietary test generally does not contribute to risk assessment conclusions. To address this question, a retrospective analysis was conducted to evaluate 119 pesticides with publicly available ecological risk assessments that were registered into commerce between 1998 and 2017. New pesticides (i.e., registered in the United States within the past 20 years) were chosen for the retrospective analysis to show utility of these tests for modern pesticide chemistries. Risk quotient (RQ) values (a point estimate of exposure divided by a deterministic toxicity endpoint) from the avian acute oral and dietary tests, as well as risk assessment conclusions, were compared to determine which test(s) drove the risk assessment findings. The RQ values were chosen as the data point for comparison in order to assess total risk (i.e., exposure and toxicity). After comparing RQ values from avian acute oral versus sub-acute dietary tests, there was only one case in which an avian sub-acute dietary RQ was greater than the acute oral RQ. Thus, the sub-acute dietary test did not identify risk in greater than 99% (118 out of 119) of chemicals based on results that either the acute oral RQ was higher than the sub-acute dietary RQ, or both the acute oral and the subacute dietary tests did not generate an RQ value of concern. For the one exception, both the oral and sub-acute RQ values were greater than the USEPA's level of concern for endangered species. Based on the results of the retrospective analysis, it is concluded that in most cases avian risk can confidently be assessed without conducting the sub-acute dietary test.

Status of acute systemic toxicity testing requirements and data uses by U.S. regulatory agencies.

Acute systemic toxicity data are used by a number of U.S. federal agencies, most commonly for hazard classification and labeling and/or risk assessment for acute chemical exposures. To identify opportunities for the implementation of non-animal approaches to produce these data, the regulatory needs and uses for acute systemic toxicity information must first be clarified. Thus, we reviewed acute systemic toxicity testing requirements for six U.S. agencies (Consumer Product Safety Commission, Department of Defense, Department of Transportation, Environmental Protection Agency, Food and Drug Administration, Occupational Safety and Health Administration) and noted whether there is flexibility in satisfying data needs with methods that replace or reduce animal use. Understanding the current regulatory use and acceptance of non-animal data is a necessary starting point for future method development, optimization, and validation efforts. The current review will inform the development of a national strategy and roadmap for implementing non-animal approaches to assess potential hazards associated with acute exposures to industrial chemicals and medical products. The Acute Toxicity Workgroup of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), U.S. agencies, non-governmental organizations, and other stakeholders will work to execute this strategy.

Aerosol generation and characterization of multi-walled carbon nanotubes exposed to cells cultured at the air-liquid interface.

Aerosol generation and characterization are critical components in the assessment of the inhalation hazards of engineered nanomaterials (NMs). An extensive review was conducted on aerosol generation and exposure apparatus as part of an international expert workshop convened to discuss the design of an in vitro testing strategy to assess pulmonary toxicity following exposure to aerosolized particles. More specifically, this workshop focused on the design of an in vitro method to predict the development of pulmonary fibrosis in humans following exposure to multi-walled carbon nanotubes (MWCNTs). Aerosol generators, for dry or liquid particle suspension aerosolization, and exposure chambers, including both commercially available systems and those developed by independent researchers, were evaluated. Additionally, characterization methods that can be used and the time points at which characterization can be conducted in order to interpret in vitro exposure results were assessed. Summarized below is the information presented and discussed regarding the relevance of various aerosol generation and characterization techniques specific to aerosolized MWCNTs exposed to cells cultured at the air-liquid interface (ALI). The generation of MWCNT aerosols relevant to human exposures and their characterization throughout exposure in an ALI system is critical for extrapolation of in vitro results to toxicological outcomes in humans.

Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs).

The increased production and use of multi-walled carbon nanotubes (MWCNTs) in a diverse array of consumer, medical, and industrial applications have raised concerns about potential human exposure to these materials in the workplace and ambient environments. Inhalation is a primary route of exposure to MWCNTs, and the existing data indicate that they are potentially hazardous to human health. While a 90-day rodent inhalation test (e.g., OECD Test No. 413: subchronic inhalation toxicity: 90-day study or EPA Health Effects Test Guidelines OPPTS 870.3465 90-day inhalation toxicity) is recommended by the U.S. Environmental Protection Agency Office of Pollution Prevention and Toxics for MWCNTs (and other CNTs) if they are to be commercially produced (Godwin et al. in ACS Nano 9:3409-3417, 2015), this test is time and cost-intensive and subject to scientific and ethical concerns. As a result, there has been much interest in transitioning away from studies on animals and moving toward human-based in vitro and in silico models. However, given the multiple mechanisms of toxicity associated with subchronic exposure to inhaled MWCNTs, a battery of non-animal tests will likely be needed to evaluate the key endpoints assessed by the 90-day rodent study. Pulmonary fibrosis is an important adverse outcome related to inhalation exposure to MWCNTs and one that the non-animal approach should be able to assess. This review summarizes the state-of-the-science regarding in vivo and in vitro toxicological methods for predicting MWCNT-induced pulmonary fibrosis.

Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterials.

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs.

Framework to Evaluate Exposure Relevance and Data Needs for Risk Assessment of Nanomaterials using in Vitro Testing Strategies.

This article presents a multistage framework for evaluating the strength of evidence of nanomaterial (NM) exposure characterization data to optimize the utility of in vitro testing strategies for human health risk assessment. This framework is intended to aid risk assessors in evaluating the relevance of data from in vitro tests and to optimize the development of new in vitro testing strategies. The initial stage frames the exposure scenarios of interest in advance of testing to incorporate aspects such as release points, route of exposure, biological and environmental transformations, dose metrics, and biological targets in subsequent stages. The second stage considers characterization in the context of a realistic exposure and the third stage involves designing a testing strategy based on expected exposure conditions. For the fourth and final stage, we propose a matrix approach to evaluate the strength of evidence obtained in the first three stages as a basis for determining the best combination of test conditions and analytical methods available to characterize and measure exposure based on the NM type. This approach can also be used to evaluate existing data for their relevance to the expected exposure scenario and to further develop and optimize in vitro testing strategies. Implementation of the proposed strategy will generate meaningful information on NM properties and their interaction with biological systems, based on realistic exposure scenarios, which will be cost effective and can be applied for assessing risk and making intelligent regulatory decisions regarding the use and disposal of NMs.

Advancing Risk Analysis for Nanoscale Materials: Report from an International Workshop on the Role of Alternative Testing Strategies for Advancement.

The Society for Risk Analysis (SRA) has a history of bringing thought leadership to topics of emerging risk. In September 2014, the SRA Emerging Nanoscale Materials Specialty Group convened an international workshop to examine the use of alternative testing strategies (ATS) for manufactured nanomaterials (NM) from a risk analysis perspective. Experts in NM environmental health and safety, human health, ecotoxicology, regulatory compliance, risk analysis, and ATS evaluated and discussed the state of the science for in vitro and other alternatives to traditional toxicology testing for NM. Based on this review, experts recommended immediate and near-term actions that would advance ATS use in NM risk assessment. Three focal areas-human health, ecological health, and exposure considerations-shaped deliberations about information needs, priorities, and the next steps required to increase confidence in and use of ATS in NM risk assessment. The deliberations revealed that ATS are now being used for screening, and that, in the near term, ATS could be developed for use in read-across or categorization decision making within certain regulatory frameworks. Participants recognized that leadership is required from within the scientific community to address basic challenges, including standardizing materials, protocols, techniques and reporting, and designing experiments relevant to real-world conditions, as well as coordination and sharing of large-scale collaborations and data. Experts agreed that it will be critical to include experimental parameters that can support the development of adverse outcome pathways. Numerous other insightful ideas for investment in ATS emerged throughout the discussions and are further highlighted in this article.

Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from 1 lung, cryopreserved, and used "on demand" by applying slicing and cryopreservation methodology improvements. Fresh and cryopreserved (∼7 and ∼34 weeks; F&C) hPCLS from 1 donor lung were cultured for up to 29 days and evaluated for biomass, viability, tissue integrity, and inflammatory markers in response to lipopolysaccharide (LPS; 5 µg/ml) and Triton X-100 (TX100; 0.1%) challenge (24 h) at days 1, 8, 15, 22, and 29 following culture initiation. The F&C hPCLS retained biomass, viability, and tissue integrity throughout the 29 days and demonstrated immune responsiveness with up to ∼30-fold LPS-induced cytokine increases. Histologically, more than 70% of normal cytomorphological features were preserved in all groups through day 29. Similar retention of tissue viability and immune responsiveness post cryopreservation (4-6 weeks) and culture (up to 14 days) was observed in hPCLS from additional 3 donor lungs. Banking cryopreserved hPCLS from various donors (and disease states) provides a critical element in researching human-derived pulmonary tissue. The retention of viability and functional responsiveness (≥4 weeks) allows evaluation of long-term, complex endpoints reflecting key events in Adverse Outcome Pathways and positions hPCLS as a valuable human-relevant model for use in regulatory applications.

Human cell-based in vitro systems to assess respiratory toxicity: a case study using silanes.

Inhalation is a major route by which human exposure to substances can occur. Resources have therefore been dedicated to optimize human-relevant in vitro approaches that can accurately and efficiently predict the toxicity of inhaled chemicals for robust risk assessment and management. In this study-the IN vitro Systems to PredIct REspiratory toxicity Initiative-2 cell-based systems were used to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. A human bronchial epithelial cell line (BEAS-2B) and a reconstructed human tissue model (MucilAir, Epithelix) were exposed to triethoxysilane (TES) and trimethoxysilane (TMS) as vapor (mixed with N2 gas) at the air-liquid interface. Cell viability, cytotoxicity, and secretion of inflammatory markers were assessed in both cell systems and, for MucilAir tissues, morphology, barrier integrity, cilia beating frequency, and recovery after 7 days were also examined. The results show that both cell systems provide valuable information; the BEAS-2B cells were more sensitive in terms of cell viability and inflammatory markers, whereas MucilAir tissues allowed for the assessment of additional cellular effects and time points. As a proof of concept, the data were also used to calculate human equivalent concentrations. As expected, based on chemical properties and existing data, the silanes demonstrated toxicity in both systems with TMS being generally more toxic than TES. Overall, the results demonstrate that these in vitro test systems can provide valuable information relevant to predicting the likelihood of toxicity following inhalation exposure to chemicals in humans.

Human cytomegalovirus infection maintains mTOR activity and its perinuclear localization during amino acid deprivation.

The mammalian target of rapamycin (mTOR) kinase is present in 2 functionally distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). Active mTORC1 mediates phosphorylation of eIF4E-binding protein (4E-BP) and p70 S6 kinase (S6K), which is important for maintaining translation. During human cytomegalovirus (HCMV) infection, cellular stress responses are activated that normally inhibit mTORC1; however, previous data show that HCMV infection circumvents stress responses and maintains mTOR kinase activity. Amino acid deprivation is a stress response that normally inhibits mTORC1 activity. Amino acids can signal to mTORC1 through the Rag proteins, which promote the colocalization of mTORC1 with its activator Rheb-GTP in a perinuclear region, thereby inducing 4E-BP and S6K phosphorylation. As expected, our results show that amino acid depletion in mock-infected cells caused loss of mTORC1 activity and loss of the perinuclear localization; however, there was no loss of activity or perinuclear localization in HCMV-infected cells where the perinuclear localization of Rheb-GTP and mTOR coincided with the perinuclear assembly compartment (AC). This suggested that HCMV infection bypasses normal Rag-dependent amino acid signaling. This was demonstrated by short hairpin RNA (shRNA) depletion of Rag proteins, which had little effect on mTORC1 activity in infected cells but inhibited activity in mock-infected cells. Our data show that HCMV maintains mTORC1 activity in an amino acid- and Rag-independent manner through the colocalization of mTOR and Rheb-GTP, which occurs in association with the formation of the AC, thus bypassing inhibition that may result from lowered amino acid levels.

The changing role of mTOR kinase in the maintenance of protein synthesis during human cytomegalovirus infection.

The mammalian target of rapamycin (mTOR) kinase occurs in mTOR complex 1 (mTORC1) and complex 2 (mTORC2), primarily differing by the substrate specificity factors raptor (in mTORC1) and rictor (in mTORC2). Both complexes are activated during human cytomegalovirus (HCMV) infection. mTORC1 phosphorylates eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) and p70S6 kinase (S6K) in uninfected cells, and this activity is lost upon raptor depletion. In infected cells, 4E-BP1 and S6K phosphorylation is maintained when raptor or rictor is depleted, suggesting that either mTOR complex can phosphorylate 4E-BP1 and S6K. Studies using the mTOR inhibitor Torin1 show that phosphorylation of 4E-BP1 and S6K in infected cells depends on mTOR kinase. The total levels of 4E-BP1 and viral proteins representative of all temporal classes were lowered by Torin1 treatment and by raptor, but not rictor, depletion, suggesting that mTORC1 is involved in the production of all classes of HCMV proteins. We also show that Torin1 inhibition of mTOR kinase is rapid and most deleterious at early times of infection. While Torin1 treatment from the beginning of infection significantly inhibited translation of viral proteins, its addition at later time points had far less effect. Thus, with respect to mTOR's role in translational control, HCMV depends on it early in infection but can bypass it at later times of infection. Depletion of 4E-BP1 by use of short hairpin RNAs (shRNAs) did not rescue HCMV growth in Torin1-treated human fibroblasts as it has been shown to in murine cytomegalovirus (MCMV)-infected 4E-BP1(-/-) mouse embryo fibroblasts (MEFs), suggesting that during HCMV infection mTOR kinase has additional roles other than phosphorylating and inactivating 4E-BP1. Overall, our data suggest a dynamic relationship between HCMV and mTOR kinase which changes during the course of infection.