SARS-CoV-2 diverges from other betacoronaviruses in only partially activating the IRE1α/XBP1 ER stress pathway in human lung-derived cells

Despite the efficacy of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 5 million individuals worldwide and continues to spread in countries where the vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the activation status and requirement of the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and the murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α whereby it autophosphorylates, but its RNase fails to splice XBP1. Moreover, IRE1α was dispensable for optimal replication in human cells for all coronaviruses tested. Our findings demonstrate that IRE1α activation status differs upon infection with distinct betacoronaviruses and is not essential for efficient replication of any of them. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α through an unknown mechanism, perhaps as a strategy to eliminate detection by the host immune system.

SARS-CoV-2 variants of concern are dependent on IFITM2 for efficient replication in human lung cells

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human cells. To date, several "Variants of Concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the four SARS-CoV-2 VOCs (Alpha, Beta, Gamma and Delta) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all four VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than four orders of magnitude. In addition, an antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominating Delta variant.

Aggregated Mycobacterium tuberculosis Enhances the Inflammatory Response

Mycobacterium tuberculosis (Mtb) bacilli readily aggregate. We previously reported that Mtb aggregates lead to phagocyte death and subsequent efficient replication in the dead infected cells. Here, we examined the transcriptional response of human monocyte derived macrophages to phagocytosis of aggregated Mtb relative to phagocytosis of non-aggregated single or multiple bacilli. Infection with aggregated Mtb led to an early upregulation of pro-inflammatory associated genes and enhanced TNFα signaling via the NFκB pathway. These pathways were significantly more upregulated relative to infection with single or multiple non-aggregated bacilli per cell. Phagocytosis of aggregates led to a decreased phagosome acidification on a per bacillus basis and increased phagocyte cell death, which was not observed when Mtb aggregates were heat killed prior to phagocytosis. Mtb aggregates, observed in a granuloma from a patient, were found surrounding a lesion cavity. These observations suggest that TB aggregation may be a mechanism for pathogenesis. They raise the possibility that aggregated Mtb, if spread from individual to individual, could facilitate increased inflammation, Mtb growth, and macrophage cell death, potentially leading to active disease, cell necrosis, and additional cycles of transmission.

IFITM dependency of SARS-CoV-2 variants of concern

We have recently shown that a SARS-CoV-2 strain isolated in the Netherlands in February 2020 (NL-02-2020) hijacks interferon-induced transmembrane proteins, especially IFITM2, as entry cofactors for efficient infection. Here, we examined whether SARS-CoV-2 'variants of concern' (VOCs), including the currently dominating delta variant, maintained the dependency on IFITMs for efficient replication. Depletion of IFITM2 reduced viral RNA production from 31- (B.1.1.7) to 755-fold (P.1). In comparison, silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, silencing of IFITM2 generally reduced infectious virus production in Calu-3 cells to near background levels. An antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are important cofactors for replication of genuine SARS-CoV-2 VOCs, including the Delta variant.

Content Replication and Placement Schemes for Wireless Mesh Networks

<p>Recently, Wireless Mesh Networks (WMNs) have attracted much of interest from both academia and industry, due to their potential to provide an alternative broadband wireless Internet connectivity. However, due to different reasons such as multi-hop forwarding and the dynamic wireless link characteristics, the performance of current WMNs is rather low when clients are soliciting Web contents. Due to the evolution of advanced mobile computing devices; it is anticipated that the demand for bandwidth-onerous popular content (especially multimedia content) in WMNs will dramatically increase in the coming future. Content replication is a popular approach for outsourcing content on behalf of the origin content provider. This area has been well explored in the context of the wired Internet, but has received comparatively less attention from the research community when it comes to WMNs. There are a number of replica placement algorithms that are specifically designed for the Internet. But they do not consider the special features of wireless networks such as insufficient bandwidth, low server capacity, contention to access the wireless medium, etc. This thesis studies the technical challenges encountered when transforming the traditional model of multi-hop WMNs from an access network into a content network. We advance the thesis that support from packet relaying mesh routers to act as replica servers for popular content such as media streaming, results in significant performance improvement. Such support from infrastructure mesh routers benefits from knowledge of the underlying network topology (i.e., information about the physical connections between network nodes is available at mesh routers). The utilization of cross-layer information from lower layers opens the door to developing efficient replication schemes that account for the specific features of WMNs (e.g., contention between the nodes to access the wireless medium and traffic interference). Moreover, this can benefit from the underutilized resources (e.g., storage and bandwidth) at mesh routers. This utilization enables those infrastructure nodes to participate in content distribution and play the role of replica servers. In this thesis, our main contribution is the design of two lightweight, distributed, and scalable object replication schemes for WMNs. The first scheme follows a hierarchical approach, while the second scheme follows a flat one. The challenge is to replicate content as close as possible to the requesting clients and thus, reduce the access latency per object, while minimizing the number of replicas. The two schemes aim to address the questions of where and how many replicas should be placed in the WMN. In our schemes, we consider the underlying topology joint with link-quality metrics to improve the quality of experience. We show using simulation tests that the schemes significantly enhance the performance of a WMN in terms of reducing the access cost, bandwidth consumption and computation/communication cost.</p>

Content Replication and Placement Schemes for Wireless Mesh Networks

<p>Recently, Wireless Mesh Networks (WMNs) have attracted much of interest from both academia and industry, due to their potential to provide an alternative broadband wireless Internet connectivity. However, due to different reasons such as multi-hop forwarding and the dynamic wireless link characteristics, the performance of current WMNs is rather low when clients are soliciting Web contents. Due to the evolution of advanced mobile computing devices; it is anticipated that the demand for bandwidth-onerous popular content (especially multimedia content) in WMNs will dramatically increase in the coming future. Content replication is a popular approach for outsourcing content on behalf of the origin content provider. This area has been well explored in the context of the wired Internet, but has received comparatively less attention from the research community when it comes to WMNs. There are a number of replica placement algorithms that are specifically designed for the Internet. But they do not consider the special features of wireless networks such as insufficient bandwidth, low server capacity, contention to access the wireless medium, etc. This thesis studies the technical challenges encountered when transforming the traditional model of multi-hop WMNs from an access network into a content network. We advance the thesis that support from packet relaying mesh routers to act as replica servers for popular content such as media streaming, results in significant performance improvement. Such support from infrastructure mesh routers benefits from knowledge of the underlying network topology (i.e., information about the physical connections between network nodes is available at mesh routers). The utilization of cross-layer information from lower layers opens the door to developing efficient replication schemes that account for the specific features of WMNs (e.g., contention between the nodes to access the wireless medium and traffic interference). Moreover, this can benefit from the underutilized resources (e.g., storage and bandwidth) at mesh routers. This utilization enables those infrastructure nodes to participate in content distribution and play the role of replica servers. In this thesis, our main contribution is the design of two lightweight, distributed, and scalable object replication schemes for WMNs. The first scheme follows a hierarchical approach, while the second scheme follows a flat one. The challenge is to replicate content as close as possible to the requesting clients and thus, reduce the access latency per object, while minimizing the number of replicas. The two schemes aim to address the questions of where and how many replicas should be placed in the WMN. In our schemes, we consider the underlying topology joint with link-quality metrics to improve the quality of experience. We show using simulation tests that the schemes significantly enhance the performance of a WMN in terms of reducing the access cost, bandwidth consumption and computation/communication cost.</p>

SARS-CoV-2 exploits host DGAT and ADRP for efficient replication

Coronavirus Disease 2019 (COVID-19) is predominantly a respiratory tract infection that significantly rewires the host metabolism. Here, we monitored a cohort of COVID-19 patients’ plasma lipidome over the disease course and identified triacylglycerol (TG) as the dominant lipid class present in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced metabolic dysregulation. In particular, we pinpointed the lipid droplet (LD)-formation enzyme diacylglycerol acyltransferase (DGAT) and the LD stabilizer adipocyte differentiation-related protein (ADRP) to be essential host factors for SARS-CoV-2 replication. Mechanistically, viral nucleo capsid protein drives DGAT1/2 gene expression to facilitate LD formation and associates with ADRP on the LD surface to complete the viral replication cycle. DGAT gene depletion reduces SARS-CoV-2 protein synthesis without compromising viral genome replication/transcription. Importantly, a cheap and orally available DGAT inhibitor, xanthohumol, was found to suppress SARS-CoV-2 replication and the associated pulmonary inflammation in a hamster model. Our findings not only uncovered the mechanistic role of SARS-CoV-2 nucleocapsid protein to exploit LDs-oriented network for heightened metabolic demand, but also the potential to target the LDs-synthetase DGAT and LDs-stabilizer ADRP for COVID-19 treatment.

Metabolic Modifications by Common Respiratory Viruses and Their Potential as New Antiviral Targets

Respiratory viruses are known to be the most frequent causative mediators of lung infections in humans, bearing significant impact on the host cell signaling machinery due to their host-dependency for efficient replication. Certain cellular functions are actively induced by respiratory viruses for their own benefit. This includes metabolic pathways such as glycolysis, fatty acid synthesis (FAS) and the tricarboxylic acid (TCA) cycle, among others, which are modified during viral infections. Here, we summarize the current knowledge of metabolic pathway modifications mediated by the acute respiratory viruses respiratory syncytial virus (RSV), rhinovirus (RV), influenza virus (IV), parainfluenza virus (PIV), coronavirus (CoV) and adenovirus (AdV), and highlight potential targets and compounds for therapeutic approaches.

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.