Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity.

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Safety and Efficacy of NVX-CoV2373 Covid-19 Vaccine.

BACKGROUND: Early clinical data from studies of the NVX-CoV2373 vaccine (Novavax), a recombinant nanoparticle vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that contains the full-length spike glycoprotein of the prototype strain plus Matrix-M adjuvant, showed that the vaccine was safe and associated with a robust immune response in healthy adult participants. Additional data were needed regarding the efficacy, immunogenicity, and safety of this vaccine in a larger population. METHODS: In this phase 3, randomized, observer-blinded, placebo-controlled trial conducted at 33 sites in the United Kingdom, we assigned adults between the ages of 18 and 84 years in a 1:1 ratio to receive two intramuscular 5-µg doses of NVX-CoV2373 or placebo administered 21 days apart. The primary efficacy end point was virologically confirmed mild, moderate, or severe SARS-CoV-2 infection with an onset at least 7 days after the second injection in participants who were serologically negative at baseline. RESULTS: A total of 15,187 participants underwent randomization, and 14,039 were included in the per-protocol efficacy population. Of the participants, 27.9% were 65 years of age or older, and 44.6% had coexisting illnesses. Infections were reported in 10 participants in the vaccine group and in 96 in the placebo group, with a symptom onset of at least 7 days after the second injection, for a vaccine efficacy of 89.7% (95% confidence interval [CI], 80.2 to 94.6). No hospitalizations or deaths were reported among the 10 cases in the vaccine group. Five cases of severe infection were reported, all of which were in the placebo group. A post hoc analysis showed an efficacy of 86.3% (95% CI, 71.3 to 93.5) against the B.1.1.7 (or alpha) variant and 96.4% (95% CI, 73.8 to 99.5) against non-B.1.1.7 variants. Reactogenicity was generally mild and transient. The incidence of serious adverse events was low and similar in the two groups. CONCLUSIONS: A two-dose regimen of the NVX-CoV2373 vaccine administered to adult participants conferred 89.7% protection against SARS-CoV-2 infection and showed high efficacy against the B.1.1.7 variant. (Funded by Novavax; EudraCT number, 2020-004123-16.).

The predictive role of symptoms in COVID-19 diagnostic models: A longitudinal insight.

To investigate the symptoms of SARS-CoV-2 infection, their dynamics and their discriminatory power for the disease using longitudinally, prospectively collected information reported at the time of their occurrence. We have analysed data from a large phase 3 clinical UK COVID-19 vaccine trial. The alpha variant was the predominant strain. Participants were assessed for SARS-CoV-2 infection via nasal/throat PCR at recruitment, vaccination appointments, and when symptomatic. Statistical techniques were implemented to infer estimates representative of the UK population, accounting for multiple symptomatic episodes associated with one individual. An optimal diagnostic model for SARS-CoV-2 infection was derived. The 4-month prevalence of SARS-CoV-2 was 2.1%; increasing to 19.4% (16.0%-22.7%) in participants reporting loss of appetite and 31.9% (27.1%-36.8%) in those with anosmia/ageusia. The model identified anosmia and/or ageusia, fever, congestion, and cough to be significantly associated with SARS-CoV-2 infection. Symptoms' dynamics were vastly different in the two groups; after a slow start peaking later and lasting longer in PCR+ participants, whilst exhibiting a consistent decline in PCR- participants, with, on average, fewer than 3 days of symptoms reported. Anosmia/ageusia peaked late in confirmed SARS-CoV-2 infection (day 12), indicating a low discrimination power for early disease diagnosis.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

Safety and Efficacy of the NVX-CoV2373 Coronavirus Disease 2019 Vaccine at Completion of the Placebo-Controlled Phase of a Randomized Controlled Trial.

BACKGROUND: The recombinant protein-based vaccine, NVX-CoV2373, demonstrated 89.7% efficacy against coronavirus disease 2019 (COVID-19) in a phase 3, randomized, observer-blinded, placebo-controlled trial in the United Kingdom. The protocol was amended to include a blinded crossover. Data to the end of the placebo-controlled phase are reported. METHODS: Adults aged 18-84 years received 2 doses of NVX-CoV2373 or placebo (1:1) and were monitored for virologically confirmed mild, moderate, or severe COVID-19 (onset from 7 days after second vaccination). Participants who developed immunoglobulin G (IgG) against nucleocapsid protein but did not show symptomatic COVID-19 were considered asymptomatic. Secondary outcomes included anti-spike (S) IgG responses, wild-type virus neutralization, and T-cell responses. RESULTS: Of 15 185 participants, 13 989 remained in the per-protocol efficacy population (6989 NVX-CoV2373, 7000 placebo). At a maximum of 7.5 months (median, 4.5) postvaccination, there were 24 cases of COVID-19 among NVX-CoV2373 recipients and 134 cases among placebo recipients, a vaccine efficacy of 82.7% (95% confidence interval [CI], 73.3%-88.8%). Vaccine efficacy was 100% (95% CI, 17.9%-100.0%) against severe disease and 76.3% (95% CI, 57.4%-86.8%) against asymptomatic disease. High anti-S and neutralization responses to vaccination were evident, together with S-protein-specific induction of interferon-γ secretion in peripheral blood T cells. Incidence of serious adverse events and adverse events of special interest were similar between groups. CONCLUSIONS: A 2-dose regimen of NVX-CoV2373 conferred a high level of ongoing protection against asymptomatic, symptomatic, and severe COVID-19 through >6 months postvaccination. A gradual decrease of protection suggests that a booster may be indicated. CLINICAL TRIALS REGISTRATION: EudraCT, 2020-004123-16.

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium.

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp).

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

Safety and immunogenicity of seven COVID-19 vaccines as a third dose (booster) following two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK (COV-BOOST): a blinded, multicentre, randomised, controlled, phase 2 trial.

BACKGROUND: Few data exist on the comparative safety and immunogenicity of different COVID-19 vaccines given as a third (booster) dose. To generate data to optimise selection of booster vaccines, we investigated the reactogenicity and immunogenicity of seven different COVID-19 vaccines as a third dose after two doses of ChAdOx1 nCov-19 (Oxford-AstraZeneca; hereafter referred to as ChAd) or BNT162b2 (Pfizer-BioNtech, hearafter referred to as BNT). METHODS: COV-BOOST is a multicentre, randomised, controlled, phase 2 trial of third dose booster vaccination against COVID-19. Participants were aged older than 30 years, and were at least 70 days post two doses of ChAd or at least 84 days post two doses of BNT primary COVID-19 immunisation course, with no history of laboratory-confirmed SARS-CoV-2 infection. 18 sites were split into three groups (A, B, and C). Within each site group (A, B, or C), participants were randomly assigned to an experimental vaccine or control. Group A received NVX-CoV2373 (Novavax; hereafter referred to as NVX), a half dose of NVX, ChAd, or quadrivalent meningococcal conjugate vaccine (MenACWY)control (1:1:1:1). Group B received BNT, VLA2001 (Valneva; hereafter referred to as VLA), a half dose of VLA, Ad26.COV2.S (Janssen; hereafter referred to as Ad26) or MenACWY (1:1:1:1:1). Group C received mRNA1273 (Moderna; hereafter referred to as m1273), CVnCov (CureVac; hereafter referred to as CVn), a half dose of BNT, or MenACWY (1:1:1:1). Participants and all investigatory staff were blinded to treatment allocation. Coprimary outcomes were safety and reactogenicity and immunogenicity of anti-spike IgG measured by ELISA. The primary analysis for immunogenicity was on a modified intention-to-treat basis; safety and reactogenicity were assessed in the intention-to-treat population. Secondary outcomes included assessment of viral neutralisation and cellular responses. This trial is registered with ISRCTN, number 73765130. FINDINGS: Between June 1 and June 30, 2021, 3498 people were screened. 2878 participants met eligibility criteria and received COVID-19 vaccine or control. The median ages of ChAd/ChAd-primed participants were 53 years (IQR 44-61) in the younger age group and 76 years (73-78) in the older age group. In the BNT/BNT-primed participants, the median ages were 51 years (41-59) in the younger age group and 78 years (75-82) in the older age group. In the ChAd/ChAD-primed group, 676 (46·7%) participants were female and 1380 (95·4%) were White, and in the BNT/BNT-primed group 770 (53·6%) participants were female and 1321 (91·9%) were White. Three vaccines showed overall increased reactogenicity: m1273 after ChAd/ChAd or BNT/BNT; and ChAd and Ad26 after BNT/BNT. For ChAd/ChAd-primed individuals, spike IgG geometric mean ratios (GMRs) between study vaccines and controls ranged from 1·8 (99% CI 1·5-2·3) in the half VLA group to 32·3 (24·8-42·0) in the m1273 group. GMRs for wild-type cellular responses compared with controls ranged from 1·1 (95% CI 0·7-1·6) for ChAd to 3·6 (2·4-5·5) for m1273. For BNT/BNT-primed individuals, spike IgG GMRs ranged from 1·3 (99% CI 1·0-1·5) in the half VLA group to 11·5 (9·4-14·1) in the m1273 group. GMRs for wild-type cellular responses compared with controls ranged from 1·0 (95% CI 0·7-1·6) for half VLA to 4·7 (3·1-7·1) for m1273. The results were similar between those aged 30-69 years and those aged 70 years and older. Fatigue and pain were the most common solicited local and systemic adverse events, experienced more in people aged 30-69 years than those aged 70 years or older. Serious adverse events were uncommon, similar in active vaccine and control groups. In total, there were 24 serious adverse events: five in the control group (two in control group A, three in control group B, and zero in control group C), two in Ad26, five in VLA, one in VLA-half, one in BNT, two in BNT-half, two in ChAd, one in CVn, two in NVX, two in NVX-half, and one in m1273. INTERPRETATION: All study vaccines boosted antibody and neutralising responses after ChAd/ChAd initial course and all except one after BNT/BNT, with no safety concerns. Substantial differences in humoral and cellular responses, and vaccine availability will influence policy choices for booster vaccination. FUNDING: UK Vaccine Taskforce and National Institute for Health Research.

Construction and characterization of two SARS-CoV-2 minigenome replicon systems.

The ongoing COVID-19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virology and antiviral discovery, a noninfectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS-CoV-2 minigenome replicon systems. First, we constructed the IVT-CoV2-Rep complementary DNA template to generate a replicon messenger RNA (mRNA) with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT-CoV2-Rep in a variety of cell lines, which could be completely abolished by known SARS-CoV-2 replication inhibitors. Our data also suggest that the transient phenotype of IVT-CoV2-Rep is not due to host innate antiviral responses. In addition, we have developed a DNA-launched replicon BAC-CoV2-Rep, which supports the in-cell transcription of a replicon mRNA as initial replication template. The BAC-CoV2-Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT-CoV2-Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC-CoV2-Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS-CoV-2 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization.

Australian experience with robot-assisted Roux-en-Y gastric bypass with comparison to a conventional laparoscopic series.

BACKGROUND: Robotic surgery is a novel approach to abdominal surgery. In Australia, the uptake of robotic assistance for bariatric surgery has been relatively slow compared to many other countries. The aim of this study is to report the first high volume experience of robotic-assisted Roux-en-Y gastric bypass surgery in Australia (RRYGB) and compare outcomes with a similar laparoscopic group (LRYGB). METHODS: Retrospective analysis of 100 RRYGB versus 100 LRYGB was carried out over a period of seven years performed by two surgeons. These groups were matched by revisional status. Outcomes recorded included operative times, conversion rate, hospital stay, short-term (30 days) complication rates, and long-term complication rates. Baseline comorbidities of patients were also recorded. RESULTS: Baseline characteristics of the two groups were similar except for comorbidity rates (higher in LRYGB group). The mean age was 43 (RRYGB) and 44(LRYGB) years, respectively. The mean pre-op BMI was 44.3 in the RRYGB group and 44.7 in the LRYGB group. Mean operating time in the RRYGB group was 208 min compared to 175 min in the LRYGB group. The number of patients with major complications was 1 in the robotic group versus 5 in the laparoscopic group (P: 0.2166). Minor complications were higher in the robotic group (17 vs. 5, P: 0.0054). Median length of stay of patients with RRYGB was 4 days compared to 5 days for the LRYGB group. CONCLUSION: RRYGB has been successfully implemented in Australia with low complication rates compared to conventional laparoscopic RYGB. Operating times are longer compared to LRYGB which is consistent with most published literature. To justify increased costs generally associated with robotic surgery, better quality studies are needed to accurately assess potential cost savings with length of stay and safety benefits to patients and institutions.

Hypercoagulability of COVID-19 and Neurological Complications: A Review.

The SARS-CoV-2 virus, which causes Coronavirus disease 2019 (COVID-19), has resulted in millions of worldwide deaths. When the SARS-CoV-2 virus emerged from Wuhan, China in December 2019, reports of patients with COVID-19 revealed that hospitalized patients had acute changes in mental status, cognition, and encephalopathy. Neurologic complications can be a consequence from overall severity of the systemic infection, direct viral invasion of the SARS-CoV-2 virus in the central nervous system, and possible immune mediated mechanisms. We will examine the landscape regarding this topic in this review in addition to current understandings of COVID-19 and hemostasis, treatment, and prevention, as well as vaccination.

Sirolimus and Other Mechanistic Target of Rapamycin Inhibitors Directly Activate Latent Pathogenic Human Polyomavirus Replication.

BACKGROUND: Human polyomaviruses can reactivate in transplant patients, causing nephropathy, progressive multifocal leukoencephalopathy, Merkel cell carcinoma, pruritic, rash or trichodysplasia spinulosa. Sirolimus and related mechanistic target of rapamycin (mTOR) inhibitors are transplant immunosuppressants. It is unknown if they directly reactivate polyomavirus replication from latency beyond their general effects on immunosuppression. METHODS: In vitro expression and turnover of large T (LT) proteins from BK virus, JC virus (JCV), Merkel cell polyomavirus (MCV), human polyomavirus 7 (HPyV7), and trichodysplasia spinulosa polyomavirus (TSV) after drug treatment were determined by immunoblotting, proximity ligation, replicon DNA replication, and whole virus immunofluorescence assays. RESULTS: mTOR inhibition increased LT protein expression for all 5 pathogenic polyomaviruses tested. This correlated with LT stabilization, decrease in the S-phase kinase-associated protein 2 (Skp2) E3 ligase targeting these LT proteins for degradation, and increase in virus replication for JCV, MCV, TSV, and HPyV7. Treatment with sirolimus, but not the calcineurin inhibitor tacrolimus, at levels routinely achieved in patients, resulted in a dose-dependent increase in viral DNA replication for BKV, MCV, and HPyV7. CONCLUSIONS: mTOR inhibitors, at therapeutic levels, directly activate polyomavirus replication through a Skp2-dependent mechanism, revealing a proteostatic latency mechanism common to polyomaviruses. Modifying existing drug regimens for transplant patients with polyomavirus-associated diseases may reduce symptomatic polyomavirus replication while maintaining allograft-sparing immunosuppression.

Nanodroplet Oligomers (NanDOs) of Aß40.

Using atomic force microscopy (AFM) and nuclear magnetic resonance (NMR), we describe small Aß40 oligomers, termed nanodroplet oligomers (NanDOs), which form rapidly and at Aß40 concentrations too low for fibril formation. NanDOs were observed in putatively monomeric solutions of Aß40 (e.g., by size exclusion chromatography). Video-rate scanning AFM shows rapid fusion and dissolution of small oligomer-sized particles, of which the median size increases with peptide concentration. In NMR (13C HSQC), a small number of chemical shifts changed with a change in peptide concentration. Paramagnetic relaxation enhancement NMR experiments also support the formation of NanDOs and suggest prominent interactions in hydrophobic domains of Aß40. Addition of Zn2+ to Aß40 solutions caused flocculation of NanDO-containing solutions, and selective loss of signal intensity in NMR spectra from residues in the N-terminal domain of Aß40. NanDOs may represent the earliest aggregated form of Aß40 in the aggregation pathway and are akin to premicelles in solutions of amphiphilies.

SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1, and NRP-1 in human cells from the respiratory, urinary, digestive, reproductive, and immune systems.

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide spectrum of syndromes involving multiple organ systems and is primarily mediated by viral spike (S) glycoprotein through the receptor-binding domain (RBD) and numerous cellular proteins including ACE2, transmembrane serine protease 2 (TMPRSS2), kidney injury molecule-1 (Kim-1), and neuropilin-1 (NRP-1). In this study, we examined the entry tropism of SARS-CoV-2 and SARS-CoV using S protein-based pseudoviruses to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, digestive, reproductive, and immune systems. At least one cell line or type of primary cell from each organ system was infected by both pseudoviruses. Infection by pseudoviruses is effectively blocked by S1, RBD, and ACE2 recombinant proteins, and more weakly by Kim-1 and NRP-1 recombinant proteins. Furthermore, cells with robust SARS-CoV-2 pseudovirus infection had strong expression of either ACE2 or Kim-1 and NRP-1 proteins. ACE2 glycosylation appeared to be critical for the infections of both viruses as there was a positive correlation between infectivity of either SARS-CoV-2 or SARS-CoV pseudovirus with the level of glycosylated ACE2 (gly-ACE2). These results reveal that SARS-CoV-2 cell entry could be mediated by either an ACE2-dependent or -independent mechanism, thus providing a likely molecular basis for its broad tropism for a wide variety of cell types.

Attitudes of optometrists in the UK and Ireland to Digital Eye Strain and approaches to assessment and management.

PURPOSE: To investigate the attitudes and understanding of optometrists in the UK and Ireland towards Digital Eye Strain (DES), and to examine related practice patterns. METHODS: An anonymous online questionnaire was developed, covering attitude and understanding of DES, examination of patients who may be experiencing DES and approaches to management options. The questionnaire was promoted to UK and Ireland optometrists via professional bodies and local and area optometric committees. RESULTS: 406 responses were included in the analysis. Most respondents agreed that DES was an important concern for optometrists (88.9%). 91.4% reported they felt confident in discussing possible symptoms of DES and management options; this was weakly and negatively associated with number of years qualified (rs  = -0.198, p ≤ 0.001). Estimations of the proportion of patients affected by DES were lower than reports in the literature (median 25%, IQR 10%-50%). Most respondents always (60.6%) or frequently (21.9%) inquired about device usage in routine case history taking, and also asked follow-up questions, although 29.3% only asked about the presence of symptoms half the time or less. Advising on regular breaks (84.0%), lubricants (55.7%) and environment/set up (69.2%) were felt to be extremely or very important by most respondents. Advising on specialist spectacle lenses, specifically blue filtering designs, was considered extremely or very important by 34.2% and 15.2%, respectively. CONCLUSION: Given the agreement that DES is a significant issue causing frequent and persistent symptoms, and practitioners reported high levels of confidence in discussing DES, patients can expect to receive advice on symptoms and management from their optometrist. Simple management strategies were felt to be most important to advise on, with more uncertainty linked to specialist spectacle lenses.

Circular DNA tumor viruses make circular RNAs.

Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) cause ∼2% of all human cancers. RNase R-resistant RNA sequencing revealed that both gammaherpesviruses encode multiple, uniquely stable, circular RNAs (circRNA). EBV abundantly expressed both exon-only and exon-intron circRNAs from the BamHI A rightward transcript (BART) locus (circBARTs) formed from a spliced BART transcript and excluding the EBV miRNA region. The circBARTs were expressed in all verified EBV latency types, including EBV-positive posttransplant lymphoproliferative disease, Burkitt lymphoma, nasopharyngeal carcinoma, and AIDS-associated lymphoma tissues and cell lines. Only cells infected with the B95-8 EBV strain, with a 12-kb BART locus deletion, were negative for EBV circBARTs. Less abundant levels of EBV circRNAs originating from LMP2- and BHLF1-encoding genes were also identified. The circRNA sequencing of KSHV-infected primary effusion lymphoma cells revealed a KSHV-encoded circRNA from the vIRF4 locus (circvIRF4) that was constitutively expressed. In addition, KSHV polyadenylated nuclear (PAN) RNA locus generated a swarm (>100) of multiply backspliced, low-abundance RNase R-resistant circRNAs originating in both sense and antisense directions consistent with a novel hyperbacksplicing mechanism. In EBV and KSHV coinfected cells, exon-only EBV circBARTs were located more in the cytoplasm, whereas the intron-retaining circBARTs were found in the nuclear fraction. KSHV circvIRF4 and circPANs were detected in both nuclear and cytoplasmic fractions. Among viral circRNAs tested, none were found in polysome fractions from KSHV-EBV coinfected BC1 cells, although low-abundance protein translation from viral circRNAs could not be excluded. The circRNAs are a new class of viral transcripts expressed in gammaherpesvirus-related tumors that might contribute to viral oncogenesis.

Mitosis-related phosphorylation of the eukaryotic translation suppressor 4E-BP1 and its interaction with eukaryotic translation initiation factor 4E (eIF4E).

Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) inhibits cap-dependent translation in eukaryotes by competing with eIF4G for an interaction with eIF4E. Phosphorylation at Ser-83 of 4E-BP1 occurs during mitosis through the activity of cyclin-dependent kinase 1 (CDK1)/cyclin B rather than through canonical mTOR kinase activity. Here, we investigated the interaction of eIF4E with 4E-BP1 or eIF4G during interphase and mitosis. We observed that 4E-BP1 and eIF4G bind eIF4E at similar levels during interphase and mitosis. The most highly phosphorylated mitotic 4E-BP1 isoform (δ) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-γ, phosphorylated at Thr-70, Ser-83, and Ser-101, bound to eIF4E during mitosis. Two-dimensional gel electrophoretic analysis corroborated the identity of the phosphorylation marks on the eIF4E-bound 4E-BP1 isoforms and uncovered a population of phosphorylated 4E-BP1 molecules lacking Thr-37/Thr-46-priming phosphorylation. Moreover, proximity ligation assays for phospho-4E-BP1 and eIF4E revealed different in situ interactions during interphase and mitosis. The eIF4E:eIF4G interaction was not inhibited but rather increased in mitotic cells, consistent with active translation initiation during mitosis. Phosphodefective substitution of 4E-BP1 at Ser-83 did not change global translation or individual mRNA translation profiles as measured by single-cell nascent protein synthesis and eIF4G RNA immunoprecipitation sequencing. Mitotic 5'-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis.