An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy.

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. The nonstructural small (NSs) protein is the primary virulence factor of RVFV that suppresses the host's antiviral innate immune response. Bioinformatic analysis and AlphaFold structural modeling identified four putative LC3-interacting regions (LIR) motifs (NSs 1-4) in the RVFV NSs protein, which suggest that NSs interacts with the host LC3-family proteins. Using, isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments, the C-terminal LIR motif (NSs4) was confirmed to interact with all six human LC3 proteins. Phenylalanine at position 261 (F261) within NSs4 was found to be critical for the interaction of NSs with LC3, retention of LC3 in the nucleus, as well as the inhibition of autophagy in RVFV infected cells. These results provide mechanistic insights into the ability of RVFV to overcome antiviral autophagy through the interaction of NSs with LC3 proteins.

Venezuelan equine encephalitis virus E1 protein interacts with PDIA6 and PDI inhibition reduces alphavirus production.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus transmitted by mosquitos that can cause a febrile illness and induce severe neurological complications in humans and equine populations. Currently there are no FDA approved vaccines or antiviral treatments to combat VEEV. Proteomic techniques were utilized to create an interactome of the E1 fusion glycoprotein of VEEV. VEEV E1 interacted with a number of cellular chaperone proteins including protein disulfide isomerase family A member 6 (PDIA6). PDI inhibition through LOC14 and/or nitazoxanide treatment effectively decreased production of VEEV and other alphaviruses in vitro, including eastern equine encephalitis virus, Sindbis virus, and chikungunya virus. Decreased oxidoreductive capabilities of PDIs through LOC14 or nitazoxanide treatment impacted both early and late events in viral replication, including the production of non-infectious virions and decreased VEEV E1 disulfide bond formation. Results from this study identified PDIs as critical regulators of alphavirus replication and potential therapeutic targets.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy toward them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3X (DDX3), a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented support the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

Phloretin Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia in Rats by Regulating the Inflammatory Response, Oxidative Stress and Apoptosis.

The inflammatory process is proposed to be one of the factors to benign prostatic enlargement (BPH), and this is the first study examining the anti-inflammatory ability of phloretin in treating rats with testosterone-induced BPH. BPH would be induced by testosterone (10 mg/kg/day testosterone subcutaneously for 28 days), and the other groups of rats were treated with phloretin 50 mg/kg/day or 100 mg/kg/day orally (phr50 or phr100 group) after induction. Prostate weight and prostate weight to body weight ratio were significantly reduced in the Phr100 group. Reduced dihydrotestosterone without interfering with 5α-reductase was observed in the phr100 group. In inflammatory proteins, reduced IL-6, IL-8, IL-17, NF-κB, and COX-2 were seen in the phr100 group. In reactive oxygen species, malondialdehyde was reduced, and superoxide dismutase and glutathione peroxidase were elevated in the phr100 group. In apoptotic assessment, elevated cleaved caspase-3 was observed in rats of the phr100 group. Enhanced pro-apoptotic Bax and reduced anti-apoptotic Bc1-2 could be seen in the phr100 group. In histological stains, markedly decreased glandular hyperplasia and proliferative cell nuclear antigen were observed with reduced expression in the phr100 group. Meanwhile, positive cells of terminal deoxynucleotidyl transferase dUTP nick end labeling were increased in the phr100 group. In conclusion, the treatment of phloretin 100 mg/kg/day could ameliorate testosterone-induced BPH.

Effect of the Chinese Herbal Medicine SS-1 on a Sjögren's Syndrome-Like Disease in Mice.

Sjögren's syndrome (SS) is an inflammatory autoimmune disease primarily affecting the exocrine glands; it has a major impact on patients' lives. The Chinese herbal formula SS-1 is composed of Gan Lu Yin, Sang Ju Yin, and Xuefu Zhuyu decoction, which exerts anti-inflammatory, immunomodulatory, and antifibrotic effects. Our previous study demonstrated that SS-1 alleviates clinical SS. This study aimed to evaluate the efficacy and mechanism of the Chinese herbal formula SS-1 for salivary gland protein-induced experimental Sjögren's syndrome (ESS). These results showed that ESS treatment with the Chinese herbal formula SS-1 (1500 mg/kg) significantly alleviated the severity of ESS. We found that SS-1 substantially improved saliva flow rates in SS mice and ameliorated lymphocytic infiltrations in submandibular glands. In addition, salivary gland protein-induced SS in mice treated with SS-1 significantly lowered proinflammatory cytokines (including IFN-γ, IL-6, and IL-17A) in mouse salivary glands and decreased serum anti-M3R autoantibody levels. In addition, we found that CD4+ T cells isolated from SS-1-treated SS mice significantly reduced the percentages of IFN-γ-producing CD4+ T cells (Th1) and IL-17A-producing CD4+ T cells (Th17). Our data show that SS-1 alleviates ESS through anti-inflammatory and immunomodulatory effects, which provides new insight into the clinical treatment of SS.

PERK Is Critical for Alphavirus Nonstructural Protein Translation.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4-5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.

Homoseongomycin, a compound isolated from marine actinomycete bacteria K3-1, is a potent inhibitor of encephalitic alphaviruses.

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 µM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 µM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 µM. It also inhibited EEEV replication with an EC50 of 1.2 µM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 µM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Galangin ameliorates experimental autoimmune encephalomyelitis in mice via modulation of cellular immunity.

Multiple sclerosis (MS) causes neurologic disabilities that effect musculature, sensory systems, and vision. This is largely due to demyelination of nerve fibers caused by chronic inflammation. Corticosteroid treatments ameliorate symptoms of MS, but do not successfully cure the disease itself. In the current study, the application of galangin, a phytochemical flavonoid extracted from the ginger family of Alpinis officinarum, on experimental autoimmune encephalomyelitis (EAE; mouse model for MS) was explored. This study investigated prophylactic and therapeutic activity of the drug and mechanisms by which it acts. The results revealed that galangin at 40 and 80 mg/kg could lower the incidence rate of MS, and alleviate clinical/pathological manifestations. Mice administered galangin presented with less limb paralysis, lower levels of inflammatory cell infiltrates, and decreased demyelination compared to vehicle controls. Levels of CD4+IFNγ+ (TH1) and CD4+IL-17A+ (TH17) cells in the spinal cords of EAE mice administered galangin were reduced and both cell types were not capable of expansion. More surprisingly, galangin inhibited antigen presentation and cytokine production by dendritic cells (DC). Formation of cytokines like IL-6, IL-12, and IL-23 were significantly decreased due to galangin in co-culture models of DC and T-cells. Taken together, the data lead one to conclude that galangin could potentially be used as a potent immunoregulatory agent to alleviate clinical symptoms and reduce the prevalence of MS.

Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway.

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

Clinical Utility of a Highly Sensitive Lateral Flow Immunoassay as determined by Titer Analysis for the Detection of anti-SARS-CoV-2 Antibodies at the Point-of-Care.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), became a pandemic in early 2020. Lateral flow immunoassays for antibody testing have been viewed as a cheap and rapidly deployable method for determining previous infection with SARS-CoV-2; however, these assays have shown unacceptably low sensitivity. We report on nine lateral flow immunoassays currently available and compare their titer sensitivity in serum to a best-practice enzyme-linked immunosorbent assay (ELISA) and viral neutralization assay. For a small group of PCR-positive, we found two lateral flow immunoassay devices with titer sensitivity roughly equal to the ELISA; these devices were positive for all PCR-positive patients harboring SARS-CoV-2 neutralizing antibodies. One of these devices was deployed in Northern Italy to test its sensitivity and specificity in a real-world clinical setting. Using the device with fingerstick blood on a cohort of 27 hospitalized PCR-positive patients and seven hospitalized controls, ROC curve analysis gave AUC values of 0.7646 for IgG. For comparison, this assay was also tested with saliva from the same patient population and showed reduced discrimination between cases and controls with AUC values of 0.6841 for IgG. Furthermore, during viral neutralization testing, one patient was discovered to harbor autoantibodies to ACE2, with implications for how immune responses are profiled. We show here through a proof-of-concept study that these lateral flow devices can be as analytically sensitive as ELISAs and adopted into hospital protocols; however, additional improvements to these devices remain necessary before their clinical deployment.