Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Role of ARP2/3 Complex-Driven Actin Polymerization in RSV Infection

Respiratory syncytial virus (RSV) is the leading viral agent causing bronchiolitis and pneumonia in children under five years old worldwide. The RSV infection cycle starts with macropinocytosis-based entry into the host airway epithelial cell membrane, followed by virus transcription, replication, assembly, budding, and spread. It is not surprising that the host actin cytoskeleton contributes to different stages of the RSV replication cycle. RSV modulates actin-related protein 2/3 (ARP2/3) complex-driven actin polymerization for a robust filopodia induction on the infected lung epithelial A549 cells, which contributes to the virus’s budding, and cell-to-cell spread. Thus, a comprehensive understanding of RSV-induced cytoskeletal modulation and its role in lung pathobiology may identify novel intervention strategies. This review will focus on the role of the ARP2/3 complex in RSV’s pathogenesis and possible therapeutic targets to the ARP2/3 complex for RSV.

Release of HSV-1 Cell-Free Virions: Mechanisms, Regulation, and Likely Role in Human-Human Transmission

Herpes simplex virus type 1, or HSV-1, is a widespread human pathogen that replicates in epithelial cells of the body surface and then establishes latent infection in peripheral neurons. When HSV-1 replicates, viral progeny must be efficiently released to spread infection to new target cells. Viral spread occurs via two major routes. In cell-cell spread, progeny virions are delivered directly to cellular junctions, where they infect adjacent cells. In cell-free release, progeny virions are released into the extracellular milieu, potentially allowing the infection of distant cells. Cell-cell spread of HSV-1 has been well studied and is known to be important for in vivo infection and pathogenesis. In contrast, HSV-1 cell-free release has received less attention, and its significance to viral biology is unclear. Here, I review the mechanisms and regulation of HSV-1 cell-free virion release. Based on knowledge accrued in other herpesviral systems, I argue that HSV-1 cell-free release is likely to be tightly regulated in vivo. Specifically, I hypothesize that this process is generally suppressed as the virus replicates within the body, but activated to high levels at sites of viral reactivation, such as the oral mucosa and skin, in order to promote efficient transmission of HSV-1 to new human hosts.

Vitronectin acts as a key regulator of adhesion and migration in umbilical cord-derived MSCs under different stress conditions

Mesenchymal stem cell (MSC)-based cellular therapy gets compromised as adverse microenvironmental conditions like nutrient deprivation, ischemia, hypoxia affect migration and engraftment, in addition to viability, of MSCs at the target site post transplantation. To improve the treatment efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under such microenvironmental stress conditions. In our study, we observed that Wharton's jelly-MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion with reduction in cellular migration under serum starvation. The changes in adhesion and migration characteristics were accompanied by extensive stress fibre formation and altered ECM gene expression with notable induction in vitronectin (VTN) expression and reduction in MMP-1 expression. Molecular and phenotypic correlative studies advocated the possible role of VTN and not MMP-1, in regulating adhesion and migration of WJ-MSCs. NF-kb was found to be the positive regulator of VTN expression while ERK pathway regulated it negatively. Further investigation with inhibition of these signalling pathways or VTN knockdown studies under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in migration. VTN knockdown under serum starvation also led to reduction in actin stress fibre along with reversal in expression of several ECM genes. Additionally, VTN induction being absent in hypoxia-treated WJ-MSCs, the hypoxic cells showed no significant change in the adhesion and migration properties. However, when VTN expression was induced under hypoxia by ERK pathway inhibition, similar increase in cell spread area and adhesion was observed. Our study thus highlights VTN as a factor which is induced under serum starvation stress and possibly affects the adhesion and migration properties of WJ-MSCs.

ECG and Pacing Criteria for Differentiating Conduction System Pacing from Myocardial Pacing

During His-Purkinje conduction system (HPS) pacing, it is crucial to confirm capture of the His bundle or left bundle branch versus myocardialonly capture. For this, several methods and criteria for differentiation between non-selective (ns) capture – capture of the HPS and the adjacent myocardium – and myocardial-only capture were developed. HPS capture results in faster and more homogenous depolarisation of the left ventricle than right ventricular septal (RVS) myocardial-only capture. Specifically, the depolarisation of the left ventricle (LV) does not require slow cell-to-cell spread of activation from the right side to the left side of the interventricular septum but begins simultaneously with QRS onset as in native depolarisation. These phenomena greatly influence QRS complex morphology and form the basis of electrocardiographic differentiation between HPS and myocardial paced QRS. Moreover, the HPS and the working myocardium are different tissues within the heart muscle that vary not only in conduction velocities but also in refractoriness and capture thresholds. These last two differences can be exploited for the diagnosis of HPS capture using dynamic pacing manoeuvres, namely differential output pacing, programmed stimulation and burst pacing. This review summarises current knowledge of this subject.

The role of IFITM proteins in tick-borne encephalitis virus infection

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in endemic regions of northern Asia and central and northern Europe. Interferon induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as the West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show the most significant role being that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR–Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of co-culture assays suggest that TBEV might partially escape IFN- and IFITM-mediated suppression during high-density co-culture infection when the virus enters naïve cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. Importance: TBEV infection may result in encephalitis, chronic illness or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new endemic centers arise. Although effective TBEV vaccines have been approved, vaccination coverage is low, and, due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. The IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies regarding the role of IFITM proteins in the TBEV infection have been published so far. Understanding of antiviral innate immune responses is crucial for future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both IFN- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

Mutations in the Methyltransferase Motifs of L Protein Attenuate Newcastle Disease Virus by Regulating Viral Translation and Cell-to-Cell Spread

Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts.

The Molecular Tweezer CLR01 Inhibits Antibody-Resistant Cell-to-Cell Spread of Human Cytomegalovirus

Human cytomegalovirus (HCMV) uses two major ways for virus dissemination: infection by cell-free virus and direct cell-to-cell spread. Neutralizing antibodies can efficiently inhibit infection by cell-free virus but mostly fail to prevent cell-to-cell transmission. Here, we show that the ‘molecular tweezer’ CLR01, a broad-spectrum antiviral agent, is not only highly active against infection with cell-free virus but most remarkably inhibits antibody-resistant direct cell-to-cell spread of HCMV. The inhibition of cell-to-cell spread by CLR01 was not limited to HCMV but was also shown for the alphaherpesviruses herpes simplex viruses 1 and 2 (HSV-1, -2). CLR01 is a rapid acting small molecule that inhibits HCMV entry at the attachment and penetration steps. Electron microscopy of extracellular virus particles indicated damage of the viral envelope by CLR01, which likely impairs the infectivity of virus particles. The rapid inactivation of viral particles by CLR01, the viral envelope as the main target, and the inhibition of virus entry at different stages are presumably the key to inhibition of cell-free virus infection and cell-to-cell spread by CLR01. Importance: While cell-free spread enables the human cytomegalovirus (HCMV) and other herpesviruses to transmit between hosts, direct cell-to-cell spread is thought to be more relevant for in vivo dissemination within infected tissues. Cell-to-cell spread is resistant to neutralizing antibodies, thus contributing to the maintenance of virus infection and virus dissemination in the presence of an intact immune system. Therefore, it would be therapeutically interesting to target this mode of spread in order to treat severe HCMV infections and to prevent dissemination of virus within the infected host. The molecular tweezer CLR01 exhibits broad-spectrum antiviral activity against a number of enveloped viruses and efficiently blocks antibody-resistant cell-to-cell spread of HCMV, thus representing a novel class of small molecules with promising antiviral activity.