Targeting Host PIM Protein Kinases Reduces Mayaro Virus Replication

Mayaro virus (MAYV) manipulates cell machinery to successfully replicate. Thus, identifying host proteins implicated in MAYV replication, represents an opportunity to discover potential antiviral targets. PIM kinases are enzymes that regulate essential cell functions and also appear to be critical factors in the replication of certain viruses. In this study we explored the consequences of PIM kinase inhibition in the replication of MAYV and other arboviruses. Cytopathic effects or viral titers in samples from MAYV-, Chikungunya-, Una- or Zika-infected cells treated with PIM kinase inhibitors were evaluated using an inverted microscope or plaque-forming assays. The expression of viral proteins E1 and nsP1 in MAYV-infected cells was assessed using an immunofluorescence confocal microscope or Western blot. Our results revealed that PIM kinase inhibition partially prevented MAYV-induced cell damage and also promoted a decrease in viral titers for MAYV, UNAV and ZIKV. The inhibitory effect of PIM kinase blocking was observed for each of the MAYV strains tested and also occurred as late as 8 hpi. Finally, PIM kinase inhibition suppressed the expression of MAYV E1 and nsP1 proteins. Taken together, these findings suggest that PIM kinases could represent an antiviral target for MAYV and other arboviruses.

Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.

HSP27 Protein Dampens Encephalomyocarditis Virus Replication by Stabilizing Melanoma Differentiation-Associated Gene 5

Heat shock proteins (HSPs) are a protein family that respond to physiological stress, such as heat, starvation, and infection. As cellular protein chaperones, they play an important role in protein folding, assembly, and degradation. Though it is well known that HSP27 is involved in a range of viral infections, its role during an encephalomyocarditis virus (EMCV) infection is not known. Here, we report that EMCV degrades HSP27 and that EMCV proteins 2Cpro and 3Apro are primarily responsible for its degradation. Consequently, loss of cellular HSP27 augmented EMCV proliferation, an effect that could be reversed upon HSP27 overexpression. Importantly, we found that HSP27 positively regulated EMCV-triggered type I interferon (IFN) production. Moreover, overexpression of 2Cpro and 3Apro significantly blocked type I IFN production. We also found for the first time that HSP27, as a molecular chaperone, can specifically interact with MDA5 and stabilize the expression of MDA5. Collectively, this study shows that HSP27 dampens EMCV infectivity by positively regulating EMCV-triggered retinoic acid-inducible gene (RIG)-I-like receptor (RLR)/melanoma differentiation-associated gene 5 (MDA5) signal pathway, while EMCV proteins 2Cpro and 3Apro interact with HSP27 and degrade HSP27 protein expression to allow EMCV proliferation. Our findings provide further mechanistic evidence for EMCV partaking in immune escape mechanisms, and that 2Cpro and 3Apro could serve as potential antiviral targets.

Genome-wide characterization of SARS-CoV-2 cytopathogenic proteins in the search of antiviral targets

Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease-2019 (COVID-19). We sought to identify antiviral targets through genome-wide characterization of SARS-CoV-2 proteins that are crucial for viral pathogenesis and that cause harmful cytopathic effects. All twenty-nine viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins including eight non-structural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14 and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a and ORF7b) were identified that altered cellular proliferation and integrity, and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the twelve proteins, ORF3a was chosen for further study in mammalian cells. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis, and caused activation of pro-inflammatory response with production of the cytokines TNF-alpha, IL-6, and IFN-beta1, possibly through the activation of NF-kappa B. To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, deltaG188. Compared to wild type ORF3a, the delataG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19.

An integrated method for optimized identification of effective natural inhibitors against SARS-CoV-2 3CLpro

The current severe situation of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reversed and posed great threats to global health. Therefore, there is an urgent need to find out effective antiviral drugs. The 3-chymotrypsin-like protease (3CLpro) in SARS-CoV-2 serve as a promising anti-virus target due to its essential role in the regulation of virus reproduction. Here, we report an improved integrated approach to identify effective 3CLpro inhibitors from effective Chinese herbal formulas. With this approach, we identified the 5 natural products (NPs) including narcissoside, kaempferol-3-O-gentiobioside, rutin, vicenin-2 and isoschaftoside as potential anti-SARS-CoV-2 candidates. Subsequent molecular dynamics simulation additionally revealed that these molecules can be tightly bound to 3CLpro and confirmed effectiveness against COVID-19. Moreover, kaempferol-3-o-gentiobioside, vicenin-2 and isoschaftoside were first reported to have SARS-CoV-2 3CLpro inhibitory activity. In summary, this optimized integrated strategy for drug screening can be utilized in the discovery of antiviral drugs to achieve rapid acquisition of drugs with specific effects on antiviral targets.

Intracellular Low Iron Exerts Anti-BK Polyomavirus Effect by Inhibiting the Protein Synthesis of Exogenous Genes

BKPyV poses a serious threat to the health of immunocompromised patients, and there are currently no curative drugs. Understanding the relationship between the virus and intracellular environment contributes to the discovery of antiviral targets.

Metabolic Modifications by Common Respiratory Viruses and Their Potential as New Antiviral Targets

Respiratory viruses are known to be the most frequent causative mediators of lung infections in humans, bearing significant impact on the host cell signaling machinery due to their host-dependency for efficient replication. Certain cellular functions are actively induced by respiratory viruses for their own benefit. This includes metabolic pathways such as glycolysis, fatty acid synthesis (FAS) and the tricarboxylic acid (TCA) cycle, among others, which are modified during viral infections. Here, we summarize the current knowledge of metabolic pathway modifications mediated by the acute respiratory viruses respiratory syncytial virus (RSV), rhinovirus (RV), influenza virus (IV), parainfluenza virus (PIV), coronavirus (CoV) and adenovirus (AdV), and highlight potential targets and compounds for therapeutic approaches.