Flaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential for accurate folding and post-translational stability rather than translation efficiency. Specifically, we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts an aberrant topology in the absence of the EMC leading to degradation. Together, our data highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing challenging transmembrane proteins during synthesis.

bGroup A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy.

The stimulation of angiotensin II (Ang II), the effector peptide of renin–angiotensin system, has been reported to increase the expression of vascular endothelial growth factor (VEGF) through the activation of the Ang II type 1 receptor (AT1R). In this study, we investigated whether hyperglycemia (HG, 33 mM glucose) in ARPE-19 cells could promote the expression of VEGF independently of Ang II through prorenin receptor (PRR), via an NADPH oxidase (Nox)-dependent mechanism. ARPE-19 cells were treated with the angiotensin converting enzyme (ACE) inhibitor perindopril to block the synthesis of Ang II. Treatment with HG induced VEGF expression in ARPE-19 cells, which was attenuated by pretreatment with the inhibitors of Nox, but not those of nitric oxide synthase, xanthine oxidase and mitochondrial O2 synthesis. In addition, Nox-derived O2− and H2O2 signaling in the regulation of VEGF was determined by using both polyethylene glycol (PEG)-catalase (CAT) and PEG-superoxide dismutase (SOD). We demonstrated that small interfering RNA (siRNA)-mediated knockdown of PRR, Nox2 and Nox4 significantly reduced the HG-induced stimulation of VEGF. On the other hand, Nox4 overexpression significantly potentiated PRR-induced stimulation of VEGF under hyperglycemia in ARPE-19 cells. Furthermore, Nox4 was shown to be associated with enhanced activities of ERK1/2 and NF-κB (p65), indicating their involvement in PRR-induced activation of VEGF under HG in ARPE-19 cells. Our results support the hypothesis that Nox4-derived reactive oxygen species (ROS) signaling is implicated in the hyperglycemia-induced increase of VEGF expression through PRR in ARPE-19 cells. However, further work is needed to evaluate the role of PRR and Nox-s in HG-induced stimulation of VEGF in vivo.

11Flaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic 12 reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is 13 indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential 14 for accurate folding and post-translational stability rather than translation efficiency. Specifically, 15we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of 16 EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-17 synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we 18 showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts 19 an aberrant topology in the absence of the EMC leading to degradation. Together, our data 20 highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve 21 optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing 22 challenging transmembrane proteins during synthesis.Subsequently, the polyprotein is cleaved by the viral NS2B-NS3 protease and the cellular signal 27 peptidase into the individual structural (C, prM, E) and non-structural (NS1-5) proteins required 28 for replication and assembly into new virions (Neufeldt et al., 2018). Similar to cellular 29 transmembrane proteins, the viral polyprotein relies on the host cell machinery for targeting to the 30 ER, translocation across the membrane and insertion of transmembrane domains (TMDs) into 31 the lipid bilayer during its translation. These processes are facilitated by the signal recognition 32 particle (SRP), the translocon-associated protein (TRAP) complex (also known as signal-33 sequence receptor complex), the Sec61 translocon and the signal peptidase complex. These cell 34 components were all identified as essential host factors for flavivirus infection in genetic screens 35 underscoring their importance for virus replication (Krishnan et al., 2008; Marceau et al., 2016; 36 Sessions et al., 2009; Zhang et al., 2016). For most transmembrane proteins, recognition of a 37 hydrophobic TMD or signal sequence by the SRP followed by transfer to the Sec61 translocation 38 channel are sufficient for membrane targeting and accurate topogenesis (Shao and Hegde, 2011). 39Recently, it was shown that the ER membrane protein complex (EMC) plays a role in the insertion 40 and/or stabilization of certain transmembrane proteins (Chitwood et al., 2018; Guna et al., 2018; 41 Shurtleff et al., 2018). Intriguingly, several genome-wide CRISPR knockout (KO) screens for 42 flavivirus dependency factors showed strong enrichment of the EMC (Ma et al., 2015; Marceau 43 et al., 2016; Savidis et al., 2016; Zhang et al., 2016). However, its role in the virus life cycle is not 44 yet understood. 45The EMC was originally discovered in a genetic screen for yeast mutants modulating the unfolded 46 protein response (UPR) (Jonikas et al., 2009). Genetic interac...

Objective: The language used to describe people with substance use disorder impacts stigma and influences clinical decision making. This study evaluates the presence of stigmatizing language (SL) in clinical notes and detects patient-and provider-level differences. Methods: All free-text notes generated in a large health system for patients with substance-related diagnoses between December 2020 and November 2021 were included. A natural language processing algorithm using the National Institute on Drug Abuse's "Words Matter" list was developed to identify use of SL in context. Results: There were 546,309 notes for 30,391 patients, of which 100,792 (18.4%) contained SL. A total of 18,727 patients (61.6%) had at least one note with SL. The most common SLs used were "abuse" and "substance abuse." Nurses were least likely to use SL (4.1%) while physician assistants were most likely (46.9%). Male patients were more likely than female patients to have SL in their notes (adjusted odds ratio [aOR], 1.17; 95% confidence internal [CI], 1.11-1.23), younger patients aged 18 to 24 were less likely to have SL than patients 45 to 54 years (aOR, 0.55; 95% CI, 0.50-0.61), Asian patients were less likely to have SL than White patients (aOR, 0.45; 95% CI, 0.36-0.56), and Hispanic patients were less likely to have SL than non-Hispanic patients (aOR, 0.88; 95% CI, 0.80-0.98). Conclusions:The majority of patients with substance-related diagnoses had at least one note containing SL. There were also several patient characteristic disparities associated with patients having SL in their notes. The work suggests that more clinician interventions about use of SL are needed.