The role of reactive oxygen species (ROS) in bladder cancer progression remains an unexplored field. Expression levels of enzymes regulating ROS levels are often altered in cancer. Search of publicly available micro-array data reveals that expression of mitochondrial manganese superoxide dismutase (Sod2), responsible for the conversion of superoxide (O 2 -. ) to hydrogen peroxide (H 2 O 2 ), is consistently increased in high grade and advanced stage bladder tumors. Here we aim to identify the role of Sod2 expression and ROS in bladder cancer. Using an in vitro human bladder tumor model we monitored the redox state of both non-metastatic (253J) and highly metastatic (253J B-V) bladder tumor cell lines. 253J B-V cells displayed significantly higher Sod2 protein and activity levels compared to their parental 253J cell line. The increase in Sod2 expression was accompanied by a significant decrease in catalase activity, resulting in a net increase in H 2 O 2 production in the 253J B-V line. Expression of pro-metastatic and -angiogenic factors, matrix metalloproteinase 9 (MMP-9) and vascular endothelial derived growth factor (VEGF), respectively, were similarly upregulated in the metastatic line. Expression of both MMP-9 and VEGF were shown to be H 2 O 2 -dependent, as removal of H 2 O 2 by overexpression of catalase attenuated their expression. Similarly, expression of catalase effectively reduced the clonogenic activity of 253J B-V cells. These findings indicate that metastatic bladder cancer cells display an altered antioxidant expression profile, resulting in a net increase in ROS production, which leads to the induction of redox-sensitive protumorigenic and pro-metastatic genes such as VEGF and MMP-9.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS. IMPORTANCEHuman cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

BackgroundHuman cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection.MethodsViral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay.ResultsAbundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33.ConclusionsViral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency.

The emerging SARS-CoV-2 infection associated with the outbreak of viral pneumonia in China is ongoing worldwide. There are no approved antiviral therapies to treat this viral disease. Here we examined the antiviral abilities of three broad-spectrum antiviral compounds gemcitabine, lycorine and oxysophoridine against SARS-CoV-2 in cell culture. We found that all three tested compounds inhibited viral replication in Vero-E6 cells at noncytotoxic concentrations. The antiviral effect of gemcitabine was suppressed efficiently by the cytidine nucleosides. Additionally, combination of gemcitabine with oxysophoridine had an additive antiviral effect against SARS-CoV-2. Our results demonstrate that broad-spectrum antiviral compounds may have a priority for the screening of antiviral compounds against newly emerging viruses to control viral infection.

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f Congenital human cytomegalovirus (HCMV) infection is the most frequent infectious cause of birth defects, primarily neurological disorders. Neural progenitor/stem cells (NPCs) are the major cell type in the subventricular zone and are susceptible to HCMV infection. In culture, the differentiation status of NPCs may change with passage, which in turn may alter susceptibility to virus infection. Previously, only early-passage (i.e., prior to passage 9) NPCs were studied and shown to be permissive to HCMV infection. In this study, NPC cultures derived at different gestational ages were evaluated after short (passages 3 to 6) and extended (passages 11 to 20) in vitro passages for biological and virological parameters (i.e., cell morphology, expression of NPC markers and HCMV receptors, viral entry efficiency, viral gene expression, virus-induced cytopathic effect, and release of infectious progeny). These parameters were not significantly influenced by the gestational age of the source tissues. However, extended-passage cultures showed evidence of initiation of differentiation, increased viral entry, and more efficient production of infectious progeny. These results confirm that NPCs are fully permissive for HCMV infection and that extended-passage NPCs initiate differentiation and are more permissive for HCMV infection. Later-passage NPCs being differentiated and more permissive for HCMV infection suggest that HCMV infection in fetal brain may cause more neural cell loss and give rise to severe neurological disabilities with advancing brain development.

The flaviviruses pose serious threats to human health. Being a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP), NS5 is the most conserved flavivirus protein and an important antiviral target. Previously reported NS5 structures represented by those from the Japanese encephalitis virus (JEV) and Dengue virus serotype 3 (DENV3) exhibit two apparently different global conformations, defining two sets of intra-molecular MTase-RdRP interactions. However, whether these NS5 conformations are conserved in flaviviruses and their specific functions remain elusive. Here we report two forms of DENV serotype 2 (DENV2) NS5 crystal structures representing two conformational states with defined analogies to the JEV-mode and DENV3-mode conformations, respectively, demonstrating the conservation of both conformation modes and providing clues for how different conformational states may be interconnected. Data from in vitro polymerase assays further demonstrate that perturbing the JEV-mode but not the DENV3-mode intramolecular interactions inhibits catalysis only at initiation, while the cell-based virological analysis suggests that both modes of interactions are important for virus proliferation. Our work highlights the role of MTase as a unique intra-molecular initiation factor specifically only through the JEV-mode conformation, providing an example of conformation-based crosstalk between naturally fused protein functional modules.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed a significant public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional nonstructural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-β production, most likely through suppression of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-β promoter after Sendai virus infection or poly(I·C) treatment. NS1 also suppressed the activation of the IFN-β promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by the proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response. WNV Nile virus (WNV) has received increased attention since its introduction to the United States. However, the pathogenesis of this virus is poorly understood. This study demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes the induction of interferon beta (IFN-β) by interacting with and degrading retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are crucial viral sensors in the host innate immune system. Further experiments suggested that NS1-mediated inhibition of the RIG-I-like receptor (RLR) signaling pathway involves inhibition of RIG-I K63-linked polyubiquitination and that the proteasome plays a role in RIG-I degradation. This study provides new insights into the regulation of WNV NS1 in the RLR signaling pathway and reveals a novel mechanism by which WNV evades the host innate immune response. The novel findings may guide us to discover new therapeutic targets and develop effective vaccines for WNV infections.