Transcription factor EB (TFEB) interaction with RagC is disrupted during enterovirus D68 infection.

Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is crucial for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus at glutamine 60 (Q60) immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC-binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells but not in autophagy-defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. IMPORTANCE: Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.

Enterovirus D68 capsid formation and stability requires acidic compartments.

IMPORTANCE: The respiratory picornavirus enterovirus D68 is a causative agent of acute flaccid myelitis, a childhood paralysis disease identified in the last decade. Poliovirus, another picornavirus associated with paralytic disease, is a fecal-oral virus that survives acidic environments when passing from host to host. Here, we follow up on our previous work showing a requirement for acidic intracellular compartments for maturation cleavage of poliovirus particles. Enterovirus D68 requires acidic vesicles for an earlier step, assembly, and maintenance of viral particles themselves. These data have strong implications for the use of acidification blocking treatments to combat enterovirus diseases.

SIRT-1 is required for release of enveloped enteroviruses.

Enterovirus D68 (EV-D68) is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1's proviral activity is, we demonstrate, mediated through the repression of endoplasmic reticulum stress (ER stress). Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.

Enterovirus D68 capsid formation and stability requires acidic compartments.

Enterovirus D68 (EV-D68), a picornavirus traditionally associated with respiratory infections, has recently been linked to a polio-like paralytic condition known as acute flaccid myelitis (AFM). EV-D68 is understudied, and much of the field's understanding of this virus is based on studies of poliovirus. For poliovirus, we previously showed that low pH promotes virus capsid maturation, but here we show that, for EV-D68, inhibition of compartment acidification during a specific window of infection causes a defect in capsid formation and maintenance. These phenotypes are accompanied by radical changes in the infected cell, with viral replication organelles clustering in a tight juxtanuclear grouping. Organelle acidification is critical during a narrow window from 3-4hpi, which we have termed the "transition point," separating translation and peak RNA replication from capsid formation, maturation and egress. Our findings highlight that acidification is crucial only when vesicles convert from RNA factories to virion crucibles.

Race-based differences in drug use prior to onset of opioid use disorder.

Rates of opioid use disorder (OUD) have increased dramatically over the past two decades, a rise that has been accompanied by changing demographics of those affected. Early exposure to drugs is a known risk factor for later development of opioid use disorder; but how and whether this risk factor may differ between racial groups is unknown. Our study seeks to identify race differences in self-report of current and past substance use in OUD-diagnosed treatment-seeking individuals. Patients (n = 157) presenting for methadone maintenance treatment at a racially diverse urban opioid treatment program were approached and consented for study involvement. Participants were administered substance use history questionnaires and urine drug screening at intake. Chi-square, t-tests, and rank-sum were used to assess race differences in demographic variables. Logistic and linear regressions assessed the relationship between race and substance use for binary and continuous variables, respectively. 61% of the population identified as Black and 39% as White. Black participants were significantly older; age was thus included as a covariate. Logistic regressions demonstrated that despite similar urine toxicology at intake, White participants were significantly more likely to report having used prescription opioids and psychedelic, stimulant, and sedative substance classes prior to their first use of non-pharmaceutical opioids. Compared to Black participants, White treatment-seeking OUD-diagnosed individuals reported using a wider range of substances ever and prior to first use of non-pharmaceutical opioids. There were no differences, however, in presentation for OUD treatment, suggesting different pathways to OUD, which may carry important clinical implications.

Starvation after infection restricts enterovirus D68 replication.

Enterovirus D68 (EV-D68) is a respiratory pathogen associated with acute flaccid myelitis, a childhood paralysis disease. No approved vaccine or antiviral treatment exists against EV-D68. Infection with this virus induces the formation of autophagosomes to enhance its replication but blocks the downstream autophagosome- lysosome fusion steps. Here, we examined the impact of autophagy induction through starvation, either before (starvation before infection, SBI) or after (starvation after infection, SAI) EV-D68 infection. We showed that SAI, but not SBI, attenuated EV-D68 replication in multiple cell lines and abrogated the viral-mediated cleavage of host autophagic flux-related proteins. Furthermore, SAI induced autophagic flux during EV-D68 replication and prevented production of virus-induced membranes, which are required for picornavirus replication. Pharmacological inhibition of autophagic flux during SAI did not rescue EV-D68 titers. SAI had the same effect in multiple cell types, and restricted the replication of several medically relevant picornaviruses. Our results highlight the significance of autophagosomes for picornavirus replication and identify SAI as an attractive broad-spectrum anti-picornavirus strategy.Abbreviations: BAF: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CQ: chloroquine; CVB3: coxsackievirus B3; EV-D68: enterovirus D68; hpi: hour post-infection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; NSP2B: nonstructural protein 2B; PV: poliovirus; RES: resveratrol; RV14: rhinovirus 14; SAI: starvation after infection; SBI: starvation before infection; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB.

SNAP23 is essential for germination of EV-D68 replication organelles.

Picornaviruses rearrange host cell membranes to facilitate their own replication. Here we investigate the Qbc SNARE, SNAP23, which is found at the plasma membrane and plays roles in exocytosis. We found that knockdown of SNAP23 expression inhibits virus replication but not release from cells. Knocking down SNAP23 inhibits viral RNA replication and synthesis of structural proteins. Normal cellular levels of SNAP23 are required for an early step in virus production, prior to or at the stage of virus RNA replication. We report that SNAP23 knockdown generates large, electron-light structures, and that infection of cells with these structures does not alter them, and those cells fail to generate viral RNA replication sites. We suggest that SNAP23 may play a role in maintaining membranes and lipids needed for generating virus replication organelles. Further investigation is needed to determine the precise role of this crucial SNARE protein in EV-D68 replication.

The JAK-STAT pathway in hypertrophic stress signaling and genomic stress response.

The JAK-STAT signaling pathway plays a central role in transducing stress and growth signals in the hypertrophic heart. Unlike most signal transducers, JAKs and STATs signal in a number of different ways, both within the JAK-STAT pathway and in collaboration with other signaling pathways. In this review, we discuss how IL-6 activates cells lacking IL-6 receptors through trans-signaling and examine JAK-STAT pathway interaction with GPCR-linked pathways both within and between cells. Finally, we discuss recent studies showing how the JAK-STAT pathway can intersect with a general transcriptional regulatory mechanism to effect transcription of STAT-dependent stress response genes.

Pharmacogenomics as an aspect of molecular autopsy for forensic pathology/toxicology: does genotyping CYP 2D6 serve as an adjunct for certifying methadone toxicity?

Pharmacogenomics, applied as an aspect of molecular autopsy, may be used as an adjunct for certifying methadone fatalities. Methadone is metabolized by cytochrome P-450 (CYP) 1A2, 3A4, and 2D6. We hypothesized that methadone toxicity may be partially due to CYP 2D6 *3, *4, and *5 variant alleles, resulting in poor drug metabolism. A retrospective analysis was performed on covariables and risk factors of 21 methadone cases from the Milwaukee County Medical Examiner's Office (1998-2000). PCR genotyping showed: one heterozygous for 2D6*3, two homozygous for 2D6*4, five heterozygous for 2D6*4, and one heterozygous for both 2D6*3 and *4. This limited number of cases showed that the prevalence of poor metabolizer was higher but not significantly different from that of a control group (n = 23) (P > 0.05, Fisher Exact Test). Thus, CYP 2D6 mutations may not yet be directly associated with methadone toxicity. However, pharmacogenomics, complementing other case findings, served as an adjunct in interpreting methadone toxicity of poor and intermediate metabolizers.